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https://openalex.org/W4394924176	https://www.qeios.com/read/Z5UVUM/pdf	English	null	Review of: "Examining the Ethical and Geopolitical Context of Global Food Security Policy"	null	2,024	cc-by	221	Qeios, CC-BY 4.0 · Review, April 18, 2024 Qeios ID: Z5UVUM · https://doi.org/10.32388/Z5UVUM Review of: "Examining the Ethical and Geopolitical Context of Global Food Security Policy" Akinboade Oludele Akinloye Potential competing interests: No potential competing interests to declare. Potential competing interests: No potential competing interests to declare. A very timely report, informative and comprehensively put together. However, I agree with the view that there are important microeconomic dimensions of food insecurity, which, though they operate at the individual level, cannot just be wished away by the wave of the hand. There are fundamental historical, cultural, as well as environmental factors that impact local level food security and which have to be attended to by the individual who is experiencing food insecurity. In respect of the multiplicity of the stakeholders who impact or are impacted by food insecurity, I also lean on the view that allows a wide plurality of free individual or societal initiatives and forms of human responsibility harmonized (at most) from the bottom up, and seeking an adequate response to a concrete problem in a concrete historical time. This approach that considers the cruciality of engaging even the small person in some remote location in Africa, Asia, and Latin America is important to ending what is generally believed to be a wicked problem. Qeios ID: Z5UVUM · https://doi.org/10.32388/Z5UVUM 1/1
https://openalex.org/W2606978122	https://aip.scitation.org/doi/pdf/10.1063/1.4982029	English	null	Role of hydrogen carrier gas on the growth of few layer hexagonal boron nitrides by metal-organic chemical vapor deposition	AIP advances	2,017	cc-by	5,408	RESEARCH ARTICLE \| APRIL 17 2017 Role of hydrogen carrier gas on the growth of few layer hexagonal boron nitrides by metal-organic chemical vapor deposition Dong Yeong Kim; Nam Han ; Hokyeong Jeong; Jaewon Kim; Sunyong Hwang; Jong Kyu Kim Dong Yeong Kim; Nam Han ; Hokyeong Jeong; Jaewon Kim; Sunyong Hwang; Jong Kyu Ki AIP Advances 7, 045116 (2017) https://doi.org/10.1063/1.4982029 AIP Advances 7, 045116 (2017) https://doi.org/10.1063/1.4982029 RESEARCH ARTICLE \| APRIL 17 2017 Role of hydrogen carrier gas on the growth of few layer hexagonal boron nitrides by metal-organic chemical vapor deposition RESEARCH ARTICLE \| APRIL 17 2017 Articles You May Be Interested In Articles You May Be Interested In Analysis of the reverse leakage current in AlGaN/GaN Schottky barrier diodes treated with fluorine plasma Appl. Phys. Lett. (March 2012) Promotion of hole injection enabled by GaInN/GaN light-emitting triodes and its effect on the efficiency droop Appl. Phys. Lett. (November 2011) High-resolution x-ray absorption studies of core excitons in hexagonal boron nitride Appl. Phys. Lett. (November 2012) Analysis of the reverse leakage current in AlGaN/GaN Schottky barrier diodes treated with fluorine plasma Appl. Phys. Lett. (March 2012) Promotion of hole injection enabled by GaInN/GaN light-emitting triodes and its effect on the efficiency droop Appl. Phys. Lett. (November 2011) 24 October 2024 04:48:39 High-resolution x-ray absorption studies of core excitons in hexagonal boron nitride Role of hydrogen carrier gas on the growth of few layer hexagonal boron nitrides by metal-organic chemical vapor deposition Dong Yeong Kim, Nam Han, Hokyeong Jeong, Jaewon Kim, Sunyong and Jong Kyu Kima Department of Materials Science and Engineering, Pohang University of Science and Technology (POSTECH), Pohang 37673, South Korea (Received 3 January 2017; accepted 9 April 2017; published online 17 April 2017) Dong Yeong Kim, Nam Han, Hokyeong Jeong, Jaewon Kim, Sunyong Hwang, and Jong Kyu Kima Department of Materials Science and Engineering, Pohang University of Science and Technology (POSTECH), Pohang 37673, South Korea Dong Yeong Kim, Nam Han, Hokyeong Jeong, Jaewon Kim, Sunyong and Jong Kyu Kima Department of Materials Science and Engineering, Pohang University of Science and Technology (POSTECH), Pohang 37673, South Korea (Received 3 January 2017; accepted 9 April 2017; published online 17 April 2017) Department of Materials Science and Engineering, Pohang University of Science and Technology (POSTECH), Pohang 37673, South Korea (Received 3 January 2017; accepted 9 April 2017; published online 17 April 2017 and echnology ( OS C ), ohang , South o ea (Received 3 January 2017; accepted 9 April 2017; published online 17 April 2017) Few layer hexagonal boron nitride (h-BN) ﬁlms were grown on 2-inch sapphire substrates by using metal-organic chemical vapor deposition (MOCVD) with two different carrier gases, hydrogen (H2) and nitrogen (N2). Structural, optical and elec- trical properties of the MOCVD-grown h-BN ﬁlms were systematically investigated by various spectroscopic analyses and electrical conduction measurement. Based on the experimental ﬁndings including narrower X-ray photoelectron spectra, reduced intensity of the shoulder peaks in near edge X-ray absorption ﬁne structure spec- tra, and decreased electrical conduction by more than three orders of magnitude when H2 carrier gas is employed, it was concluded that H2 has an advantage over N2 as the carrier gas for MOCVD growth of h-BN which is attributed to the heal- ing of crystalline defects by etching and regrowth processes occurring under the pulsed source-injection mode. © 2017 Author(s). All article content, except where otherwise noted, is licensed under a Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). AIP ADVANCES 7, 045116 (2017) aCorresponding author, e-mails: kimjk@postech.ac.kr 045116-2 Kim et al. 045116-2 Kim et al. AIP Advances 7, 045116 (2017) AIP Advances 7, 045116 (2017) AIP Advances 7, 045116 (2017) AIP Advances 7, 045116 (2017) Meanwhile, carrier gas is also an important factor determining properties of semiconductors grown by chemical vapor deposition (CVD) method including MOCVD growth, especially for atom- ically thin and ﬂat 2D materials since it strongly affects the gas phase chemical reaction inﬂuencing growth rate, surface morphology and crystallinity of the grown ﬁlm. There are several reports on the evolution of h-BN crystal under different gas ambient with and without nitrogen (N), in which the carrier gas is considered as a source of N atoms.20,21 M. Chubarov et al., claimed that pure hydro- gen gas (H2) ambient seems to be necessary for growth of rhombohedral BN layers by a hot wall MOCVD at the temperature of 1500 oC based on X-ray diffraction pattern.22 However, there is a lack of systematic studies on the effect of carrier gas on the structural properties of MOCVD-grown h-BN, and explanation why H2 carrier gas is more suitable for the growth of sp2-hybridized h-BN ﬁlm. In this work, we synthesized few-layer h-BN ﬁlms grown with two different carrier gases, H2 and nitrogen gas (N2) for understanding the role of carrier gas in the MOCVD growth of h-BN. Structural properties of two h-BN ﬁlms were investigated by the various spectroscopic techniques including X-ray photoelectron spectroscopy (XPS) and near-edge X-ray absorption ﬁne structure (NEXAFS) spectroscopy. In addition, electrical conduction in MOCVD-grown h-BN ﬁlms were also measured for comparing crystallinity because shallow defects of h-BN can inﬂuence the electrical conduction by generating charge carriers.23 We found that the h-BN ﬁlm grown with N2 carrier gas contains more defects comparing to the ﬁlm grown with H2 carrier gas resulting in the peak broadening in the XPS spectra, and defect-related shoulder peaks observed in the NEXAFS spectra. In addition, the electrical conduction was remarkably larger through the h-BN ﬁlm grown with N2 carrier gas. Based on the experimental observations, it was concluded that H2 has advantages over N2 as a carrier gas for h-BN MOCVD growth, and the possible reasons why the crystallinity of h-BN can be improved when H2 is used as a carrier gas are discussed. 045116-2 Kim et al. 24 October 2024 04:48:39 h-BN ﬁlms were grown on 2-inch sapphire substrates by a commercial MOCVD system with capability of 11×2-inch wafers at 1050 ◦C and reactor pressure of 30 mbar. A pulsed source-injection mode, in which TEB and NH3 were injected at different times with an interruption time, i.e., no injection of sources in between, was employed for alleviating undesired pre-reactions between gas- phase sources.24,25 A single pulse cycle was composed of 4 steps; injection of 10 sccm TEB for 5 seconds, interruption for 2 seconds, injection of 8,000 sccm NH3 for 4 seconds, and interruption for 2 seconds, and the total number of the pulse cycle was 100. In order to investigate the role of the carrier gas on MOCVD growth of h-BN ﬁlms, two h-BN ﬁlms were synthesized by using different carrier gases, H2 and N2, while all the other growth parameters were kept same. For the structural characterization, atomic force microscopy (AFM), Raman spectroscopy, X- ray reﬂectance (XRR), optical absorption spectroscopies were performed for the as-grown samples. XPS and NEXAFS measurements were carried out at 4D beam line in Pohang Accelerator Labo- ratory for the transferred h-BN ﬁlms onto a conductive silicon substrate by using PMMA-assisted transferring method. PMMA was spun on the grown h-BN ﬁlm, followed by the delamination of the h-BN ﬁlm from the sapphire substrate which was easily done by immersing it to the diluted hydroﬂuoric acid solution. For the electrical conduction measurement, rectangular 100 × 50 µm2 Ti/Au (20/150 nm) metal contacts with spacing of 3.5 µm were fabricated by using conventional photolithography and electron beam evaporation of Ti and Au, followed by rapid thermal annealing at 800 ◦C for 1 minute under N2 ambient. The current-voltage measurement was performed in the vac- uum condition (pressure < 10 mTorr) at the elevated temperature of 500 K in a chamber probe station system. Figure 1 shows the structural and optical properties of two h-BN ﬁlms grown with H2 and N2 carrier gas, respectively. Raman scattering at around 1370 cm-1 is observed from both ﬁlms which corresponds to the E2g vibration mode of h-BN7 as shown in Fig. 1(a). Figure 1(b) shows the optical absorption spectra from the ﬁlms. Role of hydrogen carrier gas on the growth of few layer hexagonal boron nitrides by metal-organic chemical vapor deposition [http://dx.doi.org/10.1063/1.4982029] 24 October 2024 04:48:39 Hexagonal boron nitride (h-BN) is a two-dimensional (2D) layered material with honeycomb lattice like graphene but composed of alternating boron and nitrogen atoms.1–3 Despite of the same atomic structure with graphene which has zero bandgap energy, it has wide bandgap energy of approximately 6 eV.4–7 Synthesis of h-BN has attracted a great attention for its promising potential applications in 2D (opto-) electronics as an ideal insulting substrate8,9 gate dielectrics,10 and tunneling barriers since it is an atomically thin layered insulator with clean surface without dangling bonds and charged defects. It can be also utilized as a surface passivation layer because of its chemical inertness and high oxidization resistance.11 In addition, h-BN was also proposed as an active material for an ultraviolet light emitter replacing the conventional Hg-vapor-based lamps or AlGaN-based ultraviolet light-emitting diodes (LEDs) due to its strong light-matter interaction originating from the 2D nature.4,5,12–14 In the industrial perspective, wafer-scale growth of 2D materials becomes one of the most critical issues. Metal-organic chemical vapor deposition (MOCVD) has been proposed as a very promising solution to achieve wafer-scale growth of h-BN on sapphire or other substrates, since Y. Kobayashi et al., demonstrated MOCVD-grown h-BN as a releasing layer for transferring the GaN-based LED epitaxial structure grown on the h-BN onto foreign ﬂexible substrates.15–17 Recently, Q. S. Paduano et al, reported self-terminating effect in MOCVD growth of h-BN18 and investigated effects of growth parameters including reactor pressure, and V/III ratio, deﬁned as molar ﬂow ratio between NH3 and triethylborane (TEB) which are source of nitrogen and boron respectively, on thickness of MOCVD-grown h-BN.19 X. Z. Du et al., studied effects of V/III ratio on the luminescence properties of MOCVD-grown h-BN and succeeded in synthesizing h-BN epitaxial layers without emission at around 300 nm which is known to be from impurities such as C and O.14 © Author(s) 2017 7, 045116-1 2158-3226/2017/7(4)/045116/7 045116-2 Kim et al. The optical bandgap energies obtained from the absorption edge are identical, 5.92 eV, for the two ﬁlms despite of a little difference in the absorbance peak position and a long-wavelength absorption tail observed from the h-BN ﬁlm grown with N2 carrier gas. Thickness of the grown ﬁlms is estimated to be 2.66 nm and 1.55 nm for the h-BN grown with H2 and N2 carrier gas, respectively, by ﬁtting the XRR spectrum shown in Fig. 1(c). The both h-BN ﬁlms consist of only a few atomic layers which is due to self-terminating effect occurring at low pressure and high V/III AIP Advances 7, 045116 (2017) , ( ) FIG. 1. (a) Raman spectrum, (b) optical absorption spectrum and the optical bandgap energy, (c) the measured and ﬁtted X-ray reﬂectance, and (d) surface morphology obtained by AFM of the h-BN ﬁlm grown with H2 carrier and that with N2 carrier gas. FIG. 1. (a) Raman spectrum, (b) optical absorption spectrum and the optical bandgap energy, (c) the measured and ﬁtted X-ray reﬂectance, and (d) surface morphology obtained by AFM of the h-BN ﬁlm grown with H2 carrier and that with N2 carrier gas. 24 October 2024 04:48:39 ratio growth conditions,18,19 but the ﬁlms have a different saturated thickness depending on the carrier gas. It is also experimentally observed that thickness of the sample is no longer increasing when the total pulse cycle number is increased further (not shown here). The surface roughness is investigated by AFM. The both ﬁlms have very smooth surface with root-mean-square(RMS) roughness smaller than 0.3 nm in the scanning area of 5 um × 5 um, but the h-BN ﬁlm grown with H2 carrier gas shows slightly smoother surface than that grown with N2 carrier gas as seen in Fig. 1(d). Figure 2(a) and (b) are the B 1s and N 1s core-level XPS spectra of the h-BN ﬁlms, respectively. Open circles indicate measured data and the red lines show the ﬁtted data. The binding energy of the B 1s core level is 190.9 eV for both ﬁlms. However, there is an additional peak in B 1s XPS spectrum from the h-BN ﬁlm grown with N2 carrier gas at 192.6 eV. 045116-2 Kim et al. This peak may originate from the sp3-hybridized boron atoms because their binding energy is typically higher than sp2-hydrydized boron atoms.26 In addition, the FWHM of the B 1s main peak becomes larger from 1.80 eV to 1.88 eV when N2 is used as the carrier gas instead of H2. Since peak broadening is related to the numbers of contributing chemical bonding,27 boron atoms in the h-BN ﬁlm grown with N2 carrier gas possess FIG. 2. Core level XPS spectrum of (a) B 1s and (b) N 1s for the h-BN ﬁlms grown with different carrier gas. FIG. 2. Core level XPS spectrum of (a) B 1s and (b) N 1s for the h-BN ﬁlms grown with different carrier gas. AIP Advances 7, 045116 (2017) FIG. 3. (a) B K-edge NEXAFS spectrum (inset) enlarged graph showing the vicinity of shoulder peak and (b) N K-edge NEXAFS spectrum of the MOCVD-grown h-BN ﬁlms with different carrier gases. FIG. 3. (a) B K-edge NEXAFS spectrum (inset) enlarged graph showing the vicinity of shoulder peak and (b) N K-edge NEXAFS spectrum of the MOCVD-grown h-BN ﬁlms with different carrier gases. more defective chemical bonding states comparing to that grown with H2 carrier gas. Meanwhile, the binding energy of the N 1s core level is 398.4 eV for the both h-BN ﬁlms and the FWHM is also almost identical. In order to further investigate structural properties of MOCVD-grown h-BN ﬁlms depending on the carrier gas, NEXAFS spectroscopy is carried out by using the synchrotron X-ray source. Figure 3(a) is the normalized B K-edge NEXAFS spectra of the two h-BN ﬁlms. Peaks at 192.0 eV originate from the transition between B 1s core level and π* anti-bonding orbital state of the sp2- hybridized boron atoms. Double peaks at around 199.0 eV are due to the transition between B 1s state and σ* anti-bonding states.28–30 The NEXAFS spectrum supports that both BN ﬁlms are composed of sp2-hybridized boron atoms, thus, h-BN phase. An important feature in the B K-edge spectra is the shoulders at both lower- and higher-energy of the π* peak as clearly shown in inset of the Fig. 3(a). 045116-2 Kim et al. For the sp2-hybridized layered materials, X-ray absorption from 1s core-level to π* state decreases as X-ray incident angle increases, and it is expressed by the following equation.32 I = C · P 3 ( 1 + 1 2 3cos2θ −1 3cos2α −1 ) + (1 −P) 2 sin2α (1) (1) (1) where, C is a constant, P is the degree of polarization, which is set to be 0.85 for the equipment used in this experiment, θ is the polarization angle of the incident beam with respect to the surface normal direction, and α is the average tilt angle of the planar geometry. The intensity of the π* peak in B K-edge spectrum decreases as θ increases for the both h-BN ﬁlms as expected. However, there is difference in intensity reduction ratio with increasing the X-ray incident angle between two ﬁlms due to the difference in the average tilt angle α that indicates how well the atomic planes are aligned in the in-plane direction. The average tilt angles obtained by using Eq. 1 are 21.2◦and 33.7◦for h-BN ﬁlms grown with H2 carrier gas and N2 carrier gas, respectively, as shown in Fig. 4(b). The average tilt angle is strongly related to the atomic defects existing in layered ﬁlms because defects can generate chemical bonding deviating from the in-plane direction, and anti-bonding orbitals formed not in the direction perpendicular to the in-plane but in an oblique direction. Therefore, the larger average tilt angle obtained from the h-BN grown with N2 carrier gas compared to that obtained from the h-BN grown with H2 carrier gas is consistent with the previously presented results showing improved crystallinity of the h-BN ﬁlm when H2 is used. 24 October 2024 04:48:39 Electrical conduction was measured for both samples because it can be strongly affected by defects in h-BN ﬁlms. 045116-2 Kim et al. The shoulders with higher energy (Shoulder II in inset) at around 193.0 eV is reported as the nitrogen vacancy-related peak and the shoulder with lower energy (Shoulder I in inset) at around 190.1 eV is related to the boron atoms bonded to the four neighbor nitrogen atoms.28 Consequently, both the shoulders indicate the existence of defects and atomic disorders in the h-BN layers. When N2 is used as the carrier gas instead of H2, both shoulder peaks in the B K-edge become more prominent, indicating that the crystallinity of grown h-BN ﬁlm becomes deteriorated. Figure 3(b) is the normalized N K-edge NEXAFS spectra of the h-BN ﬁlms, indicating that there is no signiﬁcant change in shape and position of peaks regardless of the carrier gas. Peak at 399.7 eV and 406.8 eV correspond to π* and σ* states, respectively, which also indicates existence of sp2-hydridized nitrogen atoms.28–30 24 October 2024 04:48:39 Figure 4(a) is the NEXAFS B K-edge spectra around the π* peak at various X-ray incident angles. Based on the transition theory, optical transition probability is determined by wavefunctions of initial and ﬁnal states and polarization of the incident light.31 In the case of NEXAFS B K-edge spectrum which originates from the transition from 1s state to π* anti-bonding state, the initial 1s FIG. 4. (a) Angle-dependent B K-edge NEXAF spectrum of the h-BN ﬁlms grown with different carrier gases. (b) Measured intensity of the π* peak as a function of X-ray incident angle (open red circles) and the ﬁtted data (blue line) for calculating average tilt angle. FIG. 4. (a) Angle-dependent B K-edge NEXAF spectrum of the h-BN ﬁlms grown with different carrier gases. (b) Measured intensity of the π* peak as a function of X-ray incident angle (open red circles) and the ﬁtted data (blue line) for calculating average tilt angle. 045116-5 Kim et al. 045116-5 AIP Advances 7, 045116 (2017) AIP Advances 7, 045116 (2017) Kim et al. state has even-symmetry wavefunction. Therefore, this optical transition is simply determined by the relation between the wavefunction of π* anti-bonding orbital, which is vertically aligned to the h-BN atomic plane, and the polarization direction of the incident X-ray controlled by the incident angle of the linearly polarized synchrotron source. 045116-2 Kim et al. The etching of the grown III-nitride semiconductors by H2 at an elevated temperature is well-known phenomenon.35,36 Defective regions with point defects or grain boundaries of the grown h-BN ﬁlm can be preferentially etched during interruption steps of source-injection cycle when only H2 carrier gas is injected to the reactor. Then, new h-BN crystal can be regrown at the etched region for TEB or NH3 injection step. Consequently, the crystallinity of h-BN can be improved by healing the defects through the iteration of the etching and regrowth process during the MOCVD growth when H2 carrier gas is used. 24 October 2024 04:48:39 In order to support the proposed process, the etching and regrowth, experimentally, the effect of the number of pulse cycle on the characteristics of the h-BN grown with H2 carrier gas was investigated. Increasing the number of pulse cycle does not result in a remarkable increase in the thickness of the grown ﬁlm, as shown in the XRR spectra in Fig. 6(a) due to the self-terminating effect.18,19 Despite similar thickness of the ﬁlms, however, there is a signiﬁcant change in optical absorption spectra, as shown in Fig. 6(b). When pulse cycle is 10, absorption peak is at around 198 nm which may be related to the turbostatic BN layers or relatively thinner h-BN layer formed at the early stage of the h-BN growth. In addition, there is a shoulder at around 210 nm which is suppressed by increasing the numbers of pulse cycle. Finally, the h-BN ﬁlm grown for 120 pulse cycles shows the same optical absorbance characteristics as discussed in Fig. 1(b). The improvement of the h-BN characteristics as the pulse cycle increases is in agreement with the prediction of the etching and regrowth process presented above. In summary, we have synthesized few-layer h-BN ﬁlms on sapphire substrate by MOCVD by using two different carrier gases, H2 and N2, in order to ﬁgure out the role of the carrier gas on the characteristics of the h-BN. We observed broader XPS spectra, more prominent shoulder peaks in NEXAFS spectrum, and much larger electrical conduction from the h-BN ﬁlm grown with N2 carrier gas in comparison with those from the h-BN grown with H2 carrier gas, which indicates that more defects exist in the h-BN ﬁlm when N2 carrier gas is used. 045116-2 Kim et al. Figure 5 shows the electrical conduction in the h-BN ﬁlms, and the measured data (symbols) is well matched with the calculated ones (solid line) following the Poole-Frenkel type conduction behavior expressed by, J = qµNCE · exp  −q φT − p qE/πεiε0 kT  (2) (2) where, q is element charge, µ is drift mobility, NC is the density of states in the conduction band, E is the electric ﬁeld, φT is trap-level, εi and ε0 are the dielectric constant in dielectric ﬁlm and vacuum, respectively, and kT is the thermal energy.33 The Poole-Frenkel type conduction behavior indicates that there are traps which can be ionized under a strong electric ﬁeld. Despite of the same Poole- Frenkel type conduction behavior for both the h-BN ﬁlms, there is huge difference in conduction level. The current density in the h-BN ﬁlm grown with N2 carrier gas is more than three orders of magnitude higher than that in the h-BN grown with H2 carrier gas. Such a signiﬁcant difference in the electrical conduction level is associated with the large amount of defects acting as donors or acceptors in the h-BN ﬁlm. Carrier type in MOCVD-grown h-BN ﬁlm was not experimentally determined, but FIG. 5. Poole-Frenkel type electrical conduction in h-BN ﬁlms grown with different carrier gases. FIG. 5. Poole-Frenkel type electrical conduction in h-BN ﬁlms grown with different carrier gases. AIP Advances 7, 045116 (2017) FIG. 6. (a) X-ray reﬂectance, and (b) optical absorption spectrum of the h-BN ﬁlms grown for different pulse cycles of 10, 50, 120 when H2 carrier gas is used. FIG. 6. (a) X-ray reﬂectance, and (b) optical absorption spectrum of the h-BN ﬁlms grown for different pulse cycles of 10, 50, 120 when H2 carrier gas is used. it may be electron because of nitrogen vacancy, which can act as a donor by making shallow defect levels,34 observed in the NEXAFS B K-edge spectrum. Based on the experimental observations, it can be concluded that H2 has advantages over N2 as a carrier gas for the growth of h-BN by MOCVD, which can be elucidated by etching and regrowth process occurring under the pulsed source-injection mode employed in this study. 045116-2 Kim et al. Therefore, it was concluded that H2 has an advantage over N2 as a carrier for h-BN MOCVD growth which is more effective etching and regrowth processes occurring under the pulsed source-injection mode. Defects in h-BN ﬁlm can be healed by such etching and regrowth processes, as a result, the crystallinity is improved during the MOCVD growth when H2 carrier gas is employed. 1 A. Pakdel, Y. Bando, and D. Golberg, Chem. Soc. Rev. 43, 934–959 (2014). 2 A. Lipp, K. A. Schwetz, and K. Hunold, J. Eur. Ceram. Soc. 5, 3–9 (1989). 3 A. Gupta, T. Sakthivel, and S. Seal, Prog, Mater Sci. 73, 44–126 (2015). 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https://openalex.org/W4391381107	https://amt.copernicus.org/preprints/amt-2023-148/amt-2023-148.pdf	English	null	Reply on RC1	null	2,024	cc-by	17,537	Time-resolved measurements of the densities of individual frozen hydrometeors and of fresh snowfall Dhiraj K. Singh1, Eric R. Pardyjak1, and Timothy J. Garrett2 1Department of Mechanical Engineering, University of Utah, Salt Lake City, UT, USA 2Department of Atmospheric Sciences, University of Utah, Salt Lake City, UT, USA Correspondence: Eric R. Pardyjak (pardyjak@eng.utah.edu) j g , yj , y 1Department of Mechanical Engineering, University of Utah, Salt Lake City, UT, USA 2Department of Atmospheric Sciences, University of Utah, Salt Lake City, UT, USA Correspondence: Eric R. Pardyjak (pardyjak@eng.utah.edu) Abstract. It is a challenge to obtain accurate measurements of the microphysical properties of delicate, structurally complex, frozen and semi-frozen hydrometeors. We present a new technique for the real-time measurement of the density of freshly fallen individual snowﬂakes. A new thermal-imaging instrument, the Differential Emissivity Imaging Disdrometer (DEID), is shown through laboratory and ﬁeld experiments to be capable of providing accurate estimates of individual snowﬂake and bulk-snow hydrometeor density (which can be interpreted as snow-to-liquid ratio or SLR). The method exploits the rate of heat 5 transfer during the melting of a hydrometeor on a heated metal plate, which is a function of the temperature difference between Abstract. It is a challenge to obtain accurate measurements of the microphysical properties of delicate, structurally complex, frozen and semi-frozen hydrometeors. We present a new technique for the real-time measurement of the density of freshly fallen individual snowﬂakes. A new thermal-imaging instrument, the Differential Emissivity Imaging Disdrometer (DEID), is shown through laboratory and ﬁeld experiments to be capable of providing accurate estimates of individual snowﬂake and Abstract. It is a challenge to obtain accurate measurements of the microphysical properties of delicate, structurally complex, frozen and semi-frozen hydrometeors. We present a new technique for the real-time measurement of the density of freshly fallen individual snowﬂakes. A new thermal-imaging instrument, the Differential Emissivity Imaging Disdrometer (DEID), is shown through laboratory and ﬁeld experiments to be capable of providing accurate estimates of individual snowﬂake and bulk-snow hydrometeor density (which can be interpreted as snow-to-liquid ratio or SLR). The method exploits the rate of heat 5 transfer during the melting of a hydrometeor on a heated metal plate, which is a function of the temperature difference between the hotplate surface and the top of the hydrometeor. The product of the melting speed and melting time yields an equivalent particle thickness normal to the hotplate surface, which can then be used in combination with the particle mass and area on the plate to determine a particle density. Time-resolved measurements of the densities of individual frozen hydrometeors and of fresh snowfall Dhiraj K. Singh1, Eric R. Pardyjak1, and Timothy J. Garrett2 1Department of Mechanical Engineering, University of Utah, Salt Lake City, UT, USA 2Department of Atmospheric Sciences, University of Utah, Salt Lake City, UT, USA Correspondence: Eric R. Pardyjak (pardyjak@eng.utah.edu) Uncertainties in estimates of particle density are approximately 4% based on calibrations ith l b t d d ti l d f t d f l ti f lt d t d f ﬁld i ith 10 bulk-snow hydrometeor density (which can be interpreted as snow-to-liquid ratio or SLR). The method exploits the rate of heat 5 transfer during the melting of a hydrometeor on a heated metal plate, which is a function of the temperature difference between the hotplate surface and the top of the hydrometeor. The product of the melting speed and melting time yields an equivalent particle thickness normal to the hotplate surface, which can then be used in combination with the particle mass and area on the plate to determine a particle density. Uncertainties in estimates of particle density are approximately 4% based on calibrations with laboratory-produced particles made from water and frozen solutions of salt and water, and from ﬁeld comparisons with 10 both high-resolution imagery of falling snow and traditional snowpack density measurements obtained at 12-hour intervals. For 17 storms, individual particle densities vary from 19 to 495 kg m−3 and storm-mean snow densities vary from 40 to 100 kg m−3. We observe probability distribution functions for hydrometeor density that are nearly Gaussian with kurtoses of ≈3 and skewnesses of ≈0.01. with laboratory-produced particles made from water and frozen solutions of salt and water, and from ﬁeld comparisons with 10 both high-resolution imagery of falling snow and traditional snowpack density measurements obtained at 12-hour intervals. For 17 storms, individual particle densities vary from 19 to 495 kg m−3 and storm-mean snow densities vary from 40 to 100 kg m−3. We observe probability distribution functions for hydrometeor density that are nearly Gaussian with kurtoses of ≈3 and skewnesses of ≈0.01. 10 https://doi.org/10.5194/amt-2023-148 Preprint. Discussion started: 18 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. 2 DEID measurement techniques for obtaining hydrometeor mass and density 40 The DEID consists of an infrared camera pointed at the surface of a low-emissivity aluminum hotplate. To quantify hydrometeor area on the hotplate, the DEID makes use of the contrasting thermal emissivities of water (ε > 0.95) and aluminum (ε < 0.1) at the same temperature. Owing to the high difference in emissivity, melted hydrometeors with nearly the same thermodynamic temperature as the heated plate have strongly contrasting radiative temperatures, such that droplets on the heated plate can be easily discriminated using a thermal camera. The hotplate surface is roughened, which prevents displacement of melted 45 snowﬂakes at high wind speeds as demonstrated in wind tunnel experiments with wind speeds varying from 2 to 12 m s−1. be easily discriminated using a thermal camera. The hotplate surface is roughened, which prevents displacement of melted 45 snowﬂakes at high wind speeds as demonstrated in wind tunnel experiments with wind speeds varying from 2 to 12 m s−1. 1 Introduction 15 While the spherical-particle approach offers the advantage of simplicity, it was found to lead to snowﬂake density estimates that were signiﬁcantly biased low relative to a method that required an added camera system, likely because snowﬂakes are not in fact spheres. 35 Here, we describe a new method for estimating particle-by-particle frozen hydrometeor density that, like the spherical- particle method, uses only the DEID to measure mass but infers particle volume instead from DEID measurements of melting time, particle area, and estimates of the rate of heat transfer from the hotplate to the hydrometeor to obtain a ‘melting-speed’ (MS). 1 Introduction 15 Avalanche forecasting in mountainous regions 5 depends, in part, on knowledge of the the vertical density structure of freshly fallen snow (Morrison et al., 2023), a parameter that is typically measured at sparse intervals (Schweizer et al., 2011; Proksch et al., 2016) using techniques such as micro- computed tomography (µCT), or more typically, with manual gravimetric methods (Proksch et al., 2016). Improvements to weather prediction are currently hampered by an inability to assimilate information about ongoing variabil- ity in frozen and semi-frozen precipitation particles (Rasmussen et al., 2011). Avalanche forecasting in mountainous regions Improvements to weather prediction are currently hampered by an inability to assimilate information about ongoing variabil- p p y p y y g g ity in frozen and semi-frozen precipitation particles (Rasmussen et al., 2011). Avalanche forecasting in mountainous regions 25 depends, in part, on knowledge of the the vertical density structure of freshly fallen snow (Morrison et al., 2023), a parameter that is typically measured at sparse intervals (Schweizer et al., 2011; Proksch et al., 2016) using techniques such as micro- computed tomography (µCT), or more typically, with manual gravimetric methods (Proksch et al., 2016). In our previous work, we showed that a new thermal-imaging instrument, the Differential Emissivity Imaging Disdrometer In our previous work, we showed that a new thermal-imaging instrument, the Differential Emissivity Imaging Disdrometer (DEID), can be used to measure individual hydrometeor density based on the ﬁrst automated direct measurements of particle 30 mass in combination with estimates of the particle spherical-equivalent effective diameter, or by using concurrent photographic imagery of the morphological characteristics of hydrometeors as they fall (Singh et al., 2021; Rees et al., 2021). While the spherical-particle approach offers the advantage of simplicity, it was found to lead to snowﬂake density estimates that were signiﬁcantly biased low relative to a method that required an added camera system, likely because snowﬂakes are not in fact spheres 35 (DEID), can be used to measure individual hydrometeor density based on the ﬁrst automated direct measurements of particle 30 mass in combination with estimates of the particle spherical-equivalent effective diameter, or by using concurrent photographic imagery of the morphological characteristics of hydrometeors as they fall (Singh et al., 2021; Rees et al., 2021). 1 Introduction 15 Frozen and semi-frozen hydrometeors have a very wide range of porosities (Dunnavan et al., 2019). Determining their partic- ulate densities and bulk snow-to-liquid ratios (SLR) once fallen on the ground is important to a wide range of ﬁelds including hydrology (Rango and Martinec, 1995; Sturm et al., 2010), climatology (Dickinson, 1983), remote sensing at wavelengths ranging from the visible to the microwave (Kendra et al., 1994; Kokhanovsky and Zege, 2004; Gergely et al., 2010), and the t i ti f ﬂk f ll d i th d li t d l (R tl d d H bb 1984 H t l 2004 F ll 20 Frozen and semi-frozen hydrometeors have a very wide range of porosities (Dunnavan et al., 2019). Determining their partic- ulate densities and bulk snow-to-liquid ratios (SLR) once fallen on the ground is important to a wide range of ﬁelds including hydrology (Rango and Martinec, 1995; Sturm et al., 2010), climatology (Dickinson, 1983), remote sensing at wavelengths ranging from the visible to the microwave (Kendra et al., 1994; Kokhanovsky and Zege, 2004; Gergely et al., 2010), and the parameterization of snowﬂake fall speeds in weather and climate models (Rutledge and Hobbs, 1984; Hong et al., 2004; Fovell 20 and Su, 2007; Alcott and Steenburgh, 2010; Finlon et al., 2019). Because hydrometeor porosity is invisible to most imag- ing techniques, obtaining accurate snowﬂake-density estimates has proven to be a signiﬁcant challenge where even the best estimates have required the use of sophisticated ﬁeld programs using multiple instruments (Tiira et al., 2016). parameterization of snowﬂake fall speeds in weather and climate models (Rutledge and Hobbs, 1984; Hong et al., 2004; Fovell 20 and Su, 2007; Alcott and Steenburgh, 2010; Finlon et al., 2019). Because hydrometeor porosity is invisible to most imag- ing techniques, obtaining accurate snowﬂake-density estimates has proven to be a signiﬁcant challenge where even the best estimates have required the use of sophisticated ﬁeld programs using multiple instruments (Tiira et al., 2016). 1 1 https://doi.org/10.5194/amt-2023-148 Preprint. Discussion started: 18 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. Improvements to weather prediction are currently hampered by an inability to assimilate information about ongoing variabil- ity in frozen and semi-frozen precipitation particles (Rasmussen et al., 2011). 2.1 Particle mass measurement (2) (2) Where, Lvv = CwTp,max + Leqv (that is, the sum of the internal energy per unit mass of a liquid droplet and its latent heat of fusion and vaporization). Mass estimates were shown in wind-tunnel calibrations to be nearly independent of environmental conditions, including wind speed, relative humidity, and ambient temperature (Singh et al., 2021). Speciﬁcally, wind-tunnel 70 experiments with the DEID showed less than 4% variability in mass measurements of hydrometeors for a wide range of wind speeds, relative humidities, and air temperatures (Singh et al., 2021). The reason for the low sensitivity to environmental conditions is that the DEID directly measures the energy required to melt and evaporate a droplet, mLeqv. For example, the heat-transfer rate to a droplet is dependent on parameters such as wind speed through the Reynolds number (Kosky et al., 2013) conditions, including wind speed, relative humidity, and ambient temperature (Singh et al., 2021). Speciﬁcally, wind-tunnel 70 experiments with the DEID showed less than 4% variability in mass measurements of hydrometeors for a wide range of wind speeds, relative humidities, and air temperatures (Singh et al., 2021). The reason for the low sensitivity to environmental conditions is that the DEID directly measures the energy required to melt and evaporate a droplet, mLeqv. For example, the heat-transfer rate to a droplet is dependent on parameters such as wind speed through the Reynolds number (Kosky et al., 2013) and the temperature. However, while higher winds may accelerate heat transfer they also diminish the time for completing 75 evaporation. Because it is the product of the heat-transfer rate and evaporation time that determines particle mass, winds play a minor role in the calculation of mass. 2.1 Particle mass measurement The DEID methodology for obtaining the mass of a hydrometeor particle has been described previously by Singh et al. (2021), Rees et al. (2021), Rees and Garrett (2021), and Morrison et al. (2023). Here, we present a concise summary including recent Rees et al. (2021), Rees and Garrett (2021), and Morrison et al. (2023). Here, we present a concise summary including recent modiﬁcations to the measurement methodology. Brieﬂy, the mass of individual hydrometeors is obtained by considering the 50 area of each hydrometeor on a heated plate and the temperature difference between the plate and the surface of the melted liquid particle, which is integrated over time from the point of ﬁrst impact of the particle onto the plate surface up to the point of its complete evaporation. 50 modiﬁcations to the measurement methodology. Brieﬂy, the mass of individual hydrometeors is obtained by considering the 50 area of each hydrometeor on a heated plate and the temperature difference between the plate and the surface of the melted liquid particle, which is integrated over time from the point of ﬁrst impact of the particle onto the plate surface up to the point of its complete evaporation. Speciﬁcally, the mass m of an individual snowﬂake is obtained by applying energy conservation to a control volume sur- rounding each hydrometeor after it has melted. The heat gained by a snowﬂake from the heated plate is assumed to be equivalent 55 2 2 to the heat required to increase the snowﬂake’s internal energy plus the heat lost during melting and evaporation as described by the following equation κ ∆tevap Z (T (t) T (t))A(t) dt (1) https://doi.org/10.5194/amt-2023-148 Preprint. Discussion started: 18 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/amt-2023-148 Preprint. Discussion started: 18 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/amt-2023-148 Preprint. Discussion started: 18 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. to the heat required to increase the snowﬂake’s internal energy plus the heat lost during melting and evaporation as described by the following equation to the heat required to increase the snowﬂake’s internal energy plus the heat lost during melting and evaporation as described by the following equation m = κ CwTp,max + Leqv ∆tevap Z 0 (Tp(t) −Tw(t))A(t) dt. m = κ CwTp,max + Leqv ∆tevap Z 0 (Tp(t) −Tw(t))A(t) dt. (1) m = κ CwTp,max + Leqv ∆tevap Z 0 (Tp(t) −Tw(t))A(t) dt. 2.1 Particle mass measurement (1) ap (Tp(t) −Tw(t))A(t) dt. (1) (1) Here, Leqv = Lv is applied to liquid hydrometeors, and Leqv = Lv + Lf is applied to frozen hydrometeors, where Lf is latent Here, Leqv = Lv is applied to liquid hydrometeors, and Leqv = Lv + Lf is applied to frozen hy Here, Leqv = Lv is applied to liquid hydrometeors, and Leqv = Lv + Lf is applied to frozen hydrometeors, where Lf is latent Here, Leqv Lv is applied to liquid hydrometeors, and Leqv Lv + Lf is applied to frozen hydrometeors, where Lf is latent heat of fusion for water and Lv is the latent heat of vaporization of water, Cw is the speciﬁc heat of water, A(t) is the area of 60 each snowﬂake at time t, Tp(t) is the surface temperature of the hotplate at time t and it is approximately constant with time, Tp,max is the maximum surface temperature of the hotplate, and Tw(t) is the temperature of the water droplet at time t. ∆tevap is the time required to evaporate the water droplet, and κ is an empirical calibration coefﬁcient determined to be 7.01±0.01×103 W m−2 K−1 (equivalent to (k/d)eﬀin Singh et al. (2021)). Rees et al. (2021) demonstrated that mass can be calculated from ∆T R ∆tevap A( )d i l f R ∆tevap(T ( ) T ( ))A( ) d l i h i i ∆T T ( ) T ( ) Th heat of fusion for water and Lv is the latent heat of vaporization of water, Cw is the speciﬁc heat of water, A(t) is the area of 60 each snowﬂake at time t, Tp(t) is the surface temperature of the hotplate at time t and it is approximately constant with time, Tp,max is the maximum surface temperature of the hotplate, and Tw(t) is the temperature of the water droplet at time t. ∆tevap is the time required to evaporate the water droplet, and κ is an empirical calibration coefﬁcient determined to be 7.01±0.01×103 W m−2 K−1 (equivalent to (k/d)eﬀin Singh et al. (2021)). Rees et al. (2021) demonstrated that mass can be calculated from 60 ∆Tevap R ∆tevap 0 A(t)dt in place of R ∆tevap 0 (Tp(t)−Tw(t))A(t) dt employing the approximation ∆Tevap = Tp(t) −Tw(t). Then, 65 Eq. (1) may be expressed as Eq. (1) may be expressed as m = κ Lvv ∆Tevap ∆tevap Z 0 A(t) dt. 2.2 Particle density Obtaining frozen hydrometeor density from hydrometeor mass requires an estimate of the particle volume while it is in its frozen state. While the DEID can provide an accurate estimate of snowﬂake mass m and initial snowﬂake projected area after 80 it impacts the hotplate Ap, it cannot provide a direct measure of a particle’s effective thickness in the direction normal to the hotplate h as illustrated in Fig. 1. In its place, we have developed a method for estimating h based on a ‘melting speed’ vmelt such that h = vmelt∆tmelt, where ∆tmelt is the time required to melt an individual snowﬂake. Using these substitutions, the density of a frozen hydrometeor can be written as Obtaining frozen hydrometeor density from hydrometeor mass requires an estimate of the p ρMS = m Vs = m Apvmelt∆tmelt , (3) 85 ρMS = m Vs = m Apvmelt∆tmelt , 85 (3) ρMS = Vs = Apvmelt∆tmelt , 85 3 3 Control volume h !"($) !&($) m – mass of snow particle Heat input (conduction) ⇒ κ ) * ∆,-./0 1 $ (!" $ −!& $ 3$ = 567 89:;< = = ∆,-./0 = >(∆!) Aluminum plate 1" at all environmental conditions 1 $ = 0 = 1" Figure 1. Schematic of heat transfer from an aluminum hotplate to a solid hydrometeor during melting. h is the thickness and Ap is the base https://doi.org/10.5194/amt-2023-148 Preprint. Discussion started: 18 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/amt-2023-148 Preprint. Discussion started: 18 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. Control volume h !"($) !&($) m – mass of snow particle Heat input (conduction) ⇒ κ ) * ∆,-./0 1 $ (!" $ −!& $ 3$ = 567 89:;< = = ∆,-./0 = >(∆!) Aluminum plate 1" at all environmental conditions 1 $ = 0 = 1" Control volume h !"($) !&($) m – mass of snow particle Heat input (conduction) ⇒ κ ) * ∆,-./0 1 $ (!" $ −!& $ 3$ = 567 89:;< = = ∆,-./0 = >(∆!) Aluminum plate 1" at all environmental conditions 1 $ = 0 = 1" Control volume m – mass of snow particle Heat input (conduction) ⇒ ) ∆,-./0 Figure 1. Schematic of heat transfer from an aluminum hotplate to a solid hydrometeor during melting. h is the thickness and Ap is the base area of hydrometeor on the hotplate. 2.2 Particle density For the methodology presented here, a control volume is deﬁned to wrap around the hydrometeor. where ρMS indicates the density computed using the ‘melting speed’ method and Vs is the volume of a snowﬂake estimated as Apv∆tmelt. Apv∆tmelt. We propose a method to measure vmelt as a function of the temperature difference across a hydrometeor (∆Tmelt) – and hence the heat-transfer rate – as illustrated in Fig. 1. During the time it takes to melt a snowﬂake, ∆tmelt, a hydrometeor receives a quantity of energy equal to mLﬀfrom the hotplate, and we may write the average conductive heat-transfer rate ( ˙q) from the hotplate to a hydrometeor during melting as We propose a method to measure vmelt as a function of the temperature difference across a hydrometeor (∆Tmelt) – and hence the heat-transfer rate – as illustrated in Fig. 1. During the time it takes to melt a snowﬂake, ∆tmelt, a hydrometeor receives a quantity of energy equal to mLﬀfrom the hotplate, and we may write the average conductive heat-transfer rate ( ˙q) from the hotplate to a hydrometeor during melting as 90 90 ˙q = mLﬀ ∆tmelt = κ∆Tmelt ∆tmelt ∆tmelt Z 0 A(t) dt = κ∆Tmelt Z dA, ˙q = mLﬀ ∆tmelt = κ∆Tmelt ∆tmelt ∆tmelt Z 0 A(t) dt = κ∆Tmelt Z dA, (4) t A(t) dt = κ∆Tmelt Z dA, (4) (4) where ∆Tmelt = Tp(t) −Ts(t) during the melting process and Ts(t) is the surface temperature of the frozen portion of the particle during melting. This average conductive heat-transfer rate can be interpreted as the ‘effective mechanical work rate’ or mechanical power ( ˙w) required to melt a snowﬂake, that is 95 where ∆Tmelt = Tp(t) −Ts(t) during the melting process and Ts(t) is the surface temperature of the frozen portion of the particle during melting. This average conductive heat-transfer rate can be interpreted as the ‘effective mechanical work rate’ or mechanical power ( ˙w) required to melt a snowﬂake, that is 95 95 ˙w = 1 ∆tmelt ∆tmelt Z 0 P0 dVs = P0 ∆tmelt ∆tmelt Z 0 d(Ah) = P0h ∆tmelt Z dA, (5) (5) where h is the effective thickness of the hydrometeor and P0 (N m−2) is the constant ‘melting pressure’ determined experi- mentally (see section 4.1). Combining Eq. (4) and Eq. (5), we can write the vmelt as vmelt = κ∆Tmelt P0 . vmelt = κ∆Tmelt P0 . 2.2 Particle density (6) Note that the melting speed, vmelt, at all environmental conditions depends on ∆Tmelt only and is independent of hydrometeors 100 density. Note that the melting speed, vmelt, at all environmental conditions depends on ∆Tmelt only and is independent of hydrometeors 100 density. Now, if vmelt from Eq. (6) is substituted into Eq. (3), the MS density equation can now be written as Now, if vmelt from Eq. (6) is substituted into Eq. (3), the MS density equation can now be written as ρMS = P0m κAp∆Tmelt∆tmelt . ρMS = P0m κAp∆Tmelt∆tmelt . (7) (7) 4 Figure 2. Observed melting and evaporation of a laboratory-made ice particle on the DEID hotplate. (a) Time series of the area of the ice particle (dashed black line) and the melted portion of the ice particle (solid black line). (b) The minimum surface temperature of the ice particle (dashed black line), and the temperature of the melted portion of the ice particle (solid black line) immediately after being placed on the hotplate. The horizontal dotted line represents a temperature partition in the melting and evaporation process. The shaded region denotes the period of melting. Details on the manufacture of the laboratory-made ice particles and experiments are provided in Section 3.1. Figure 2. Observed melting and evaporation of a laboratory-made ice particle on the DEID hotplate. (a) Time series of the area of the ice particle (dashed black line) and the melted portion of the ice particle (solid black line). (b) The minimum surface temperature of the ice particle (dashed black line), and the temperature of the melted portion of the ice particle (solid black line) immediately after being placed on the hotplate. The horizontal dotted line represents a temperature partition in the melting and evaporation process. The shaded region denotes the period of melting. Details on the manufacture of the laboratory-made ice particles and experiments are provided in Section 3.1. Figure 2. Observed melting and evaporation of a laboratory-made ice particle on the DEID hotplate. (a) Ti Figure 2. Observed melting and evaporation of a laboratory-made ice particle on the DEID hotplate. (a) Time series of the area of the ice particle (dashed black line) and the melted portion of the ice particle (solid black line). 2.2 Particle density Figure 2 shows how the area of the ice particle prior to melting is similar to the maximum area of the liquid 120 droplet showing that the area of solid hydrometeors is preserved after melting. 2.2 Particle density (b) The minimum surface temperature of the ice particle (dashed black line), and the temperature of the melted portion of the ice particle (solid black line) immediately after being placed on the hotplate. The horizontal dotted line represents a temperature partition in the melting and evaporation process. The shaded region denotes the period of melting. Details on the manufacture of the laboratory-made ice particles and experiments are provided in Section 3.1. The melting parameter ∆tmelt is quite short for low-density snowﬂakes, and hence requires high-frequency recording of ther- mal images resulting in the generation of a tremendous amount of data, which is not convenient for ﬁeld experiments. In 105 ﬁeld experiments, the DEID measures ∆tevap, which is much longer than ∆tmelt. Fortunately, a relation between ∆tmelt and ∆tevap can be easily derived (see Appendix A). By estimating the average conductive heat-transfer rate during the melting and evaporation processes, we may substitute ∆Tmelt∆tmelt ≈(Lf/Lv)(∆Tevap∆tevap) into Eq. (7), which yields 105 ρMS = c m Ap∆Tevap∆tevap . (8) Here, the constant c is given by 110 Here, the constant c is given by 110 (9) c = (LfP0)/(Lvκ), (9) 5 which is derived from a combination of thermodynamic and laboratory calibration constants that must be determined experi- mentally (see section 4.1). Note that in Eq. (8), ∆tevap and ∆Tevap are impacted by variability in environmental conditions. A sample time series of temperature and hydrometeor area during melting and evaporation is shown in Fig. 2. During the melting process, the area of 115 the particle that is in its frozen state decreases to zero from a maximum immediately after having fallen on the plate. At the same time, the observed liquid component of the hydrometeor increases to a maximum before abruptly disappearing. The sum of these two areas is nearly constant, at least accounting for inevitable uncertainties in the binary thresholding associated with discriminating the hydrometeor from its background. Notably, the sum is also equal to the initial area of the frozen particle prior to its melting. Figure 2 shows how the area of the ice particle prior to melting is similar to the maximum area of the liquid 120 droplet showing that the area of solid hydrometeors is preserved after melting. 115 prior to its melting. 3 Experimental Methods Two laboratory experiments and one ﬁeld experiment were designed to calibrate and validate the MS method for determining snowﬂake density. The ﬁrst lab experiment was used to estimate vmelt of ice particles for a given set of environmental conditions and validate the density measurements of ice particles. The second lab experiment investigated the impact of environmental factors on vmelt. A ﬁeld experiment was conducted at Alta Ski Area’s mid-Collins snow-study plot to provide an opportunity to 145 validate the MS method against manual measurements, ultrasonic snow-depth sensors, and a weighing gauge using an industry standard method. factors on vmelt. A ﬁeld experiment was conducted at Alta Ski Area’s mid-Collins snow-study plot to provide an opportunity to 145 validate the MS method against manual measurements, ultrasonic snow-depth sensors, and a weighing gauge using an industry standard method. 145 2.3 Use of the DEID to determine bulk snowpack-derived quantities Finally, the total accumulated snow over H (mm) a given time interval ∆tres (h) is given by Finally, the total accumulated snow over H (mm) a given time interval ∆tres (h) is given by H = ∆tres Z 0 ˙Hdt. (14) H = ∆tres Z 0 ˙Hdt. 140 (14) 2.3 Use of the DEID to determine bulk snowpack-derived quantities In additional to individual hydrometeor mass and density measurements, the DEID can be used to provide useful bulk snowpack quantities. Precipitation intensity or snow water equivalent rate of precipitation, ˙ SWE (in mm hr−1), can be estimated from the cumulative particle mass measured by the DEID over a given time period (∆tres) as 125 In additional to individual hydrometeor mass and density measurements, the DEID can be used to provide useful bulk snowpack quantities. Precipitation intensity or snow water equivalent rate of precipitation, ˙ SWE (in mm hr−1), can be estimated from the cumulative particle mass measured by the DEID over a given time period (∆tres) as 125 the cumulative particle mass measured by the DEID over a given time period (∆tres) as 125 ˙ SWE = k ∆m ρwAhp∆tres , (10) where k is a conversion factor from m s−1 to mm h−1 (3.6 × 106 mm h−1 m−1 s), ∆tres is sampling time (h), ∆m (kg) is the total hydrometeor mass that falls on the hotplate in given time, where individual mass of hydrometeor is estimated using Eq. (1), ρw (kg m−3) is the bulk density of water and Ahp (m2) is a rectangular sampling area on the hotplate that captures all hydrometeors. The accumulated SWE (mm) can be calculated over a given time interval ∆tres (h) as 130 SWE = ∆tres Z 0 ˙ SWE dt. SWE = ∆tres Z 0 ˙ SWE dt. (11) (11) The average density (ρMS) of a freshly fallen snowpack layer can be estimated using the DEID from the ratio of the cumu- lative measured mass and the total volume of all snowﬂakes sampled in a given time interval (∆tres), ρMS = P N i=1 mi P N i=1 mi/ρMS,i , (12) ρMS = P N i=1 mi P N i=1 mi/ρMS,i , (12) where mi (kg) is the mass of ith snowﬂake, ρMS,i (kg m−3) the density of the ith snowﬂake, and N is the total number of 135 snowﬂakes collected on the plate during the given time interval (∆tres). From the average density of the snowﬂakes, the new snow accumulation rate ˙H (mm h−1) is then, 135 ˙H = k ∆m ρMSAhp∆tres . (13) ˙H = k ∆m ρMSAhp∆tres . (13) 6 https://doi.org/10.5194/amt-2023-148 Preprint. Discussion started: 18 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. 3.1 Laboratory experiments method and validation Laboratory experiments were conducted using: a DEID disdrometer, a 3D sonic anemometer (Campbell Scientiﬁc, Inc. CSAT3, sampling rate 20 Hz and accuracy ± 0.05 ms−1), a temperature/relative-humidity sensor (Vaisala HMP 155, sample rate 1 Hz 150 and accuracy ± 5% relative humidity, ± 1◦C in temperature), a high-precision gravity scale (Sartorius model ENTRIS64-1S with a readability of 0.1 mg and a standard deviation of 0.1 mg), a micropipette (accuracy of 1.00/1.20 %/µL), a silicon mold, and a freezer with a minimum temperature -37 ◦C. The precise setup of the DEID is modiﬁable, but for this study, the thermal camera measured surface temperatures of a hotplate with dimension ≈9 cm × 6 cm at 15 fps with 531 pixels × 362 pixels which yields a spatial resolution on the plate of about 0.2 mm/pixel. Continuous thermal-camera imagery provided all relevant 155 parameters for calculating hydrometeor mass, except for the effective thermal-conduction coefﬁcient between the hotplate and hydrometeor (κ), which was determined through laboratory calibration. We used an Infratec thermal camera that writes out infrared binary (IRB) ﬁles that store each pixel’s absolute temperature. In post-processing, a gray-scale thermal image ranging in intensity from 0 to 255 is created from the IRB ﬁles based on a preset infrared temperature range. For determining ∆tmelt and ∆Tmelt, the temperature range used was [– 40, 0] ◦C, and for the m, Ap, ∆tevap, and ∆Tevap measurements, the temperature 160 range used was [0, 85] ◦C. In a table-top experiment, the DEID was operated at 85 ◦C as determined using the thermal camera. To measure vmelt using h and ∆tmelt, the DEID was placed in a 0.25 m per side open-topped cubic enclosure with in an environment with a near-zero wind-tunnel wind speed of 0.02 m s−1, a constant ambient temperature of 18 ◦C, and a constant relative humidity of 38%. Eighty hemispherically-shaped ice particles with a range of known masses and volumes were made in a laboratory freezer using a micropipettor by applying a distilled water droplet to a ﬂat silicon mold. Droplet volumes of 5, 165 10, 20, 30, 40, 50, 60, and 70 µL, with ten samples per volume, were used to manufacture the ice particles. The height of the ice particles h was measured in the freezer as illustrated in Fig. 3d. A U-shaped rigid metallic frame was mounted on a translational stage. 3.1 Laboratory experiments method and validation A millimeter ruler was attached to one arm of the U-frame, and a laser pointer was afﬁxed perpendicularly to the second arm facing towards the ruler on the opposite arm in such a manner that when turned on, the in a laboratory freezer using a micropipettor by applying a distilled water droplet to a ﬂat silicon mold. Droplet volumes of 5, 165 10, 20, 30, 40, 50, 60, and 70 µL, with ten samples per volume, were used to manufacture the ice particles. The height of the ice particles h was measured in the freezer as illustrated in Fig. 3d. A U-shaped rigid metallic frame was mounted on a translational stage. A millimeter ruler was attached to one arm of the U-frame, and a laser pointer was afﬁxed perpendicularly to the second arm facing towards the ruler on the opposite arm in such a manner that when turned on, the in a laboratory freezer using a micropipettor by applying a distilled water droplet to a ﬂat silicon mold. Droplet volumes of 5, 165 10, 20, 30, 40, 50, 60, and 70 µL, with ten samples per volume, were used to manufacture the ice particles. The height of the ice particles h was measured in the freezer as illustrated in Fig. 3d. A U-shaped rigid metallic frame was mounted on a translational stage. A millimeter ruler was attached to one arm of the U-frame, and a laser pointer was afﬁxed perpendicularly to the second arm facing towards the ruler on the opposite arm in such a manner that when turned on, the µ p p p The height of the ice particles h was measured in the freezer as illustrated in Fig. 3d. A U-shaped rigid metallic frame was mounted on a translational stage. A millimeter ruler was attached to one arm of the U-frame, and a laser pointer was afﬁxed perpendicularly to the second arm facing towards the ruler on the opposite arm in such a manner that when turned on, the 7 7 (a) (b) ! ~ 0.1 ! ~ 0.96 ! ~ 0.95 Kapton tape Water droplet AL plate )++ h 10 mm 2 mm , Translational stage Laser Millimeter ruler (c) (d) SOLIDWORKS Educational Product. For Instructional Use Only. Thermal camera Background T [−40, 0]3C Hotplate T [0, 85]3C Singh et al. 2021 Low e plate Frozen ice particle Melted ice particle Figure 3. 3.1 Laboratory experiments method and validation Details of the DEID MS measurement technique.(a) A hemispherical ice particle applied to the hotplate seen as a bright circular region after melting alongside a rectangular piece of Kapton tape (ϵ ≈0.95) used to measure hotplate surface temperature (Tp). (b) Side-view of a surface temperature contour plot of an ice particle obtained using the thermal camera. (c) Schematic of the DEID showing the imaging of melting and evaporating particles, respectively. The black and white contrast of the ice and water particles is optimized by adjusting the camera’s temperature range. (d) Schematic of the ice-particle height measurement apparatus. (a) (b) ! ~ 0.1 ! ~ 0.96 ! ~ 0.95 Kapton tape Water droplet AL plate )++ h 10 mm 2 mm , Translational stage Laser Millimeter ruler (c) (d) Thermal camera Background T [−40, 0]3C Hotplate T [0, 85]3C Singh et al. 2021 Low e plate Frozen ice particle Melted ice particle (b) )*++ h 2 mm (a) Translational stage Laser Millimeter ruler (d) (d) (c) Millimeter ruler SOLIDWORKS Educational Product. For Instructional Use Only. Figure 3. Details of the DEID MS measurement technique.(a) A hemispherical ice particle applied to the hotplate seen as a bright circular region after melting alongside a rectangular piece of Kapton tape (ϵ ≈0.95) used to measure hotplate surface temperature (Tp). (b) Side-view of a surface temperature contour plot of an ice particle obtained using the thermal camera. (c) Schematic of the DEID showing the imaging of melting and evaporating particles, respectively. The black and white contrast of the ice and water particles is optimized by adjusting the camera’s temperature range. (d) Schematic of the ice-particle height measurement apparatus. Details of the DEID MS measurement technique.(a) A hemispherical ice particle applied to the hotplate seen SOLIDWORKS Educational Product. For Instructional Use Only. Figure 3. Details of the DEID MS measurement technique.(a) A hemispherical ice particle applied to the hotplate seen as a bright circular region after melting alongside a rectangular piece of Kapton tape (ϵ ≈0.95) used to measure hotplate surface temperature (Tp). (b) Side-view of a surface temperature contour plot of an ice particle obtained using the thermal camera. (c) Schematic of the DEID showing the imaging of melting and evaporating particles, respectively. The black and white contrast of the ice and water particles is optimized by adjusting the camera’s temperature range. (d) Schematic of the ice-particle height measurement apparatus. 3.2 Environmental impacts on vmelt measurement: wind-tunnel experiments For any given ice-particle mass m, independent of the rate of melting or evaporation, the ≈m(Lf +Lv) constant total quantity 180 of energy is required both to melt and to evaporate a particle from the hotplate. However, the conductive heat rate from the hotplate to the ice particle is a function of environmental conditions through the temperature difference ∆T. To determine how environmental variability affects measurement of the melting velocity vmelt, a portable wind tunnel was set on one side of the DEID’s hotplate, allowing different wind velocities to pass over the hotplate and ice particles. A pitot-static probe and an automated weather station measured air speed, ambient temperature and relative humidity. 60-µL (0.06 g) ice particles 185 with thickness h = 1.95 mm and area A = 5.02 × 10−5 m2 were placed on the DEID’s hotplate. Three experiments were performed. First, the hotplate temperature and the ambient relative humidity were maintained constant at 85◦C and 38%, respectively, while the wind tunnel was adjusted for air speeds of 0.0, 1.5, 3.0, 4.7, 5.9, and 8.3 m s−1. Second, ice particle experiments were performed for surface plate temperatures of 65 ◦C, 85 ◦C, and 95 ◦C for near-zero wind speed and 38% relative humidity. Finally, the relative humidity was varied to cover 38%, 68%, and 91% with near-zero wind speed and 190 constant hotplate temperature 85◦C. an automated weather station measured air speed, ambient temperature and relative humidity. 60-µL (0.06 g) ice particles 185 with thickness h = 1.95 mm and area A = 5.02 × 10−5 m2 were placed on the DEID’s hotplate. Three experiments were performed. First, the hotplate temperature and the ambient relative humidity were maintained constant at 85◦C and 38%, respectively, while the wind tunnel was adjusted for air speeds of 0.0, 1.5, 3.0, 4.7, 5.9, and 8.3 m s−1. Second, ice particle experiments were performed for surface plate temperatures of 65 ◦C, 85 ◦C, and 95 ◦C for near-zero wind speed and 38% relative humidity. Finally, the relative humidity was varied to cover 38%, 68%, and 91% with near-zero wind speed and 190 constant hotplate temperature 85◦C. relative humidity. Finally, the relative humidity was varied to cover 38%, 68%, and 91% with near-zero wind speed and 190 constant hotplate temperature 85◦C. 3.1 Laboratory experiments method and validation pointer’s laser beam would strike a spot on the ruler bar. The pointer was horizontally aligned independently using a precision 170 bubble level. The ice particle was thus positioned between the two arms of the U-frame. The ruler scale’s purpose was to obtain the vertical height of the ice particles. The U-frame was vertically displaced when the ice particle occluded the laser beam and did not reach the ruler scale. Upon emerging at the top of the ice particle, when the beam spot hit the ruler scale, the reading was taken as illustrated in Fig. 3d. Furthermore, ice particle mass was also measured with a gravimetric scale prior to pointer’s laser beam would strike a spot on the ruler bar. The pointer was horizontally aligned independently using a precision 170 bubble level. The ice particle was thus positioned between the two arms of the U-frame. The ruler scale’s purpose was to obtain the vertical height of the ice particles. The U-frame was vertically displaced when the ice particle occluded the laser beam and did not reach the ruler scale. Upon emerging at the top of the ice particle, when the beam spot hit the ruler scale, the reading was taken as illustrated in Fig. 3d. Furthermore, ice particle mass was also measured with a gravimetric scale prior to i li i h h l its application on the hotplate. 175 Individual frozen droplets were placed on the hotplate, and the cross-sectional area Ap, m, ∆Tmelt, ∆Tevap, ∆tmelt and ∆tevap were measured using the DEID from Eq. (2). vmelt was then calculated using two different formulas vmelt = h/∆tmelt and using Eq. (6), where h determined from the laser pointer system. 8 https://doi.org/10.5194/amt-2023-148 Preprint. Discussion started: 18 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. 3.3 Density of individual frozen salt-water particles To test the MS method on a wide range of particle densities, a method was required to produce particles of varying densities. This was done by creating frozen droplets composed of 5%, 10%, 20%, 30%, and 40% of sodium chloride solution by weight (i.e., distilled water dilutions). The densities of these particles were then measured using the DEID and the same methods 195 described above for the pure frozen-water tests. To test the MS method on a wide range of particle densities, a method was required to produce particles of varying densities. This was done by creating frozen droplets composed of 5%, 10%, 20%, 30%, and 40% of sodium chloride solution by weight To test the MS method on a wide range of particle densities, a method was required to produc This was done by creating frozen droplets composed of 5%, 10%, 20%, 30%, and 40% of sodi This was done by creating frozen droplets composed of 5%, 10%, 20%, 30%, and 40% of sodium chloride solution by weight (i.e., distilled water dilutions). The densities of these particles were then measured using the DEID and the same methods 195 described above for the pure frozen-water tests. (i.e., distilled water dilutions). The densities of these particles were then measured using the DEID and the same methods 195 described above for the pure frozen-water tests. (i.e., distilled water dilutions). The densities of these particles were then measured using the DEID and the same methods 195 described above for the pure frozen-water tests. At approximately the same height, a Campbell Scientiﬁc, Inc. CSAT3 3D sonic anemometer was deployed (sampling rate 20 Hz). Images from the SLR camera were combined with mass measurements from the DEID to compute snowﬂake density in the ﬁeld and validate the MS method. With this SLR-DEID method, the geometrical volume of each free falling snowﬂake was estimated using images from the SLR camera. The mass of each hydrometeor was determined by following individual snowﬂakes through the laser sheet until they hit the DEID hotplate. This method was applied to approximately 1000 snowﬂakes. Selected thermal images of aggregate snowﬂakes and graupel on the hotplate are illustrated in Appendix B. 215 215 3.4 Field validation experiments https://doi.org/10.5194/amt-2023-148 Preprint. Discussion started: 18 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/amt-2023-148 Preprint. Discussion started: 18 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. 4 2 3 5 6 1 7 8 9 cm 6 cm DEID Laser sheet 7 cm Hotplate (a) (b) x y Figure 4. (a) Field experimental set-up for measurements of microphysical properties of snowﬂakes and snowﬂakes visualization. The experimental set-up consists of (1) 20 – m tower (2) a thermal camera (3) Hotplate (4) Three 10-W lasers and optical lens (5) D850 Nikon SLR camera (6) 3-D sonic anemometer (7) data logger/computer and (8) relative humidity and temperature sensor. (b) Top-view schematic showing the co-location of the laser system and DEID hotplate. 9 cm 6 cm DEID Laser sheet 7 cm Hotplate (b) x y 4 2 3 5 6 1 7 8 (a) (b) (a) Figure 4. (a) Field experimental set-up for measurements of microphysical properties of snowﬂakes and snowﬂakes visualization. The experimental set-up consists of (1) 20 – m tower (2) a thermal camera (3) Hotplate (4) Three 10-W lasers and optical lens (5) D850 Nikon SLR camera (6) 3-D sonic anemometer (7) data logger/computer and (8) relative humidity and temperature sensor. (b) Top-view schematic showing the co-location of the laser system and DEID hotplate. At approximately the same height, a Campbell Scientiﬁc, Inc. CSAT3 3D sonic anemometer was deployed (sampling rate 20 Hz). 3.4 Field validation experiments Data were obtained from ﬁeld experiments conducted during the winter between October 2020 and April 2021 comprising seventeen snowfall events in Upper Little Cottonwood Canyon, Utah, USA at the Alta Ski Area’s mid-Collins snow-study Plot (Alcott and Steenburgh, 2010) (40.5763 ◦N, 111.6383 ◦W, 2920 m above sea level). A 10-m crank-up measurement tower 200 at the site included a DEID for measurement of microphysical properties of snowﬂakes. In addition to the DEID, a particle imaging system was simultaneously deployed that consisted of a laser sheet with a sampling volume of 10 cm × 18 cm × 7 cm that was oriented normal to the viewing angle of a Nikon D850 single-lens reﬂex (SLR) camera as shown in Fig. 4a. The SLR camera recorded 1920 pixel × 1080 pixel images at a spatial resolution ≈160 µm/pixel at 120 fps within a vertical laser sheet created using three 10-W, 520-nm diode lasers and a collimator lens. The laser beam spread angle of ≈6.8◦allowed for 205 a light sheet with near constant thickness of ≈7 cm throughout the region of interest. A single focal length Nikon AF-S VR Micro-Nikkor 105 mm f/2.8 G IF-ED lens permitted a depth-of-ﬁeld greater than the thickness of the laser light sheet. The DEID was deployed 2 cm below the lower end of laser sheet, which permits measurement of the microphysical properties of snowﬂakes that pass the laser sheet and fall on the hotplate. A Vaisala HMP 155 temperature and relative humidity sensor (1 Hz sampling rate) was also located on the tower and maintained at a height of approximately 1.5 m above the new snow level. 210 Hz sampling rate) was also located on the tower and maintained at a height of approximately 1.5 m above the new snow level. 210 9 4 2 3 5 6 1 7 8 9 cm 6 cm DEID Laser sheet 7 cm Hotplate (a) (b) x y e 4. (a) Field experimental set-up for measurements of microphysical properties of snowﬂakes and snowﬂakes visualization. The mental set-up consists of (1) 20 – m tower (2) a thermal camera (3) Hotplate (4) Three 10-W lasers and optical lens (5) D850 Nikon camera (6) 3-D sonic anemometer (7) data logger/computer and (8) relative humidity and temperature sensor. (b) Top-view schematic ng the co-location of the laser system and DEID hotplate. //doi.org/10.5194/amt-2023-148 int. Discussion started: 18 August 2023 uthor(s) 2023. CC BY 4.0 License. 3.5 Hydrometeor density calculation exploiting concurrent imagery during their fall The volume of planar crystals was approximated as a disc, hence, VSLR = AmaxDmin. Here, Amax is the maximum 230 area of a planar crystal in a 2-D plane within all images that are visible by the camera and Dmin is the minimum dimension in the 2-D plane as indicated in Fig. 5b. The volume of graupel was estimated as a sphere VSLR = π/6D3 eﬀ, where DReﬀ= q 4 πA. A is the mean area of all images as illustrated in Fig. 5b. The volume of aggregates was estimated by ﬁtting an ellipsoid such that VSLR = π 6 DmaxDminDv, where Dmax, Dmin and Dv are illustrated in Fig. 5b. The volume of columnar crystals was estimated from VSLR = π 4 D 2 minDmax, where Dmax and Dmin are the average of the maximum and minimum dimensions of 235 single camera with multiple images was found to represent a three-dimensional picture of a snowﬂake and provide geometrical volume more accurately than using a single image (Li et al., 2022). Five sequential selected images of each crystal type are illustrated in Fig. 5a. A schematic showing how snowﬂake volume is computed is illustrated in Fig. 5b and formulated in illustrated in Fig. 5a. A schematic showing how snowﬂake volume is computed is illustrated in Fig. 5b and formulated in Table 1 . The volume of planar crystals was approximated as a disc, hence, VSLR = AmaxDmin. Here, Amax is the maximum 230 area of a planar crystal in a 2-D plane within all images that are visible by the camera and Dmin is the minimum dimension in the 2-D plane as indicated in Fig. 5b. The volume of graupel was estimated as a sphere VSLR = π/6D3 eﬀ, where DReﬀ= q 4 πA. A is the mean area of all images as illustrated in Fig. 5b. The volume of aggregates was estimated by ﬁtting an ellipsoid such that VSLR = π 6 DmaxDminDv, where Dmax, Dmin and Dv are illustrated in Fig. 5b. The volume of columnar crystals was estimated from VSLR = π 4 D 2 minDmax, where Dmax and Dmin are the average of the maximum and minimum dimensions of 235 Table 1 . The volume of planar crystals was approximated as a disc, hence, VSLR = AmaxDmin. 3.5 Hydrometeor density calculation exploiting concurrent imagery during their fall The geometrical volume VSLR of a snowﬂake can be estimated independently from the DEID by imaging falling snowﬂakes using the particle tracking system discussed above. In this case, the density is determined from 220 ρSLR-DEID = m VSLR , (15) ρSLR-DEID = m VSLR , (15) where snowﬂake mass m is determined with the DEID from Eq. (2). where snowﬂake mass m is determined with the DEID from Eq. (2). We categorized ﬁve snowﬂake habits based on the international classiﬁcation for seasonal snow on the ground (Fierz et al., 2009; Praz et al., 2017), planar crystal (combining stellar and plates), graupel (combining hail and graupel), columnar crystal, aggregate (combining irregular crystal), and small particles. Graupel and small particle crystals were classiﬁed based on size. 225 Each snowﬂake category contained approximately 200 samples. Taking advantage of how snowﬂakes rotate while falling, a We categorized ﬁve snowﬂake habits based on the international classiﬁcation for seasonal snow on the ground (Fierz et al., 2009; Praz et al., 2017), planar crystal (combining stellar and plates), graupel (combining hail and graupel), columnar crystal, aggregate (combining irregular crystal), and small particles. Graupel and small particle crystals were classiﬁed based on size. 225 Each snowﬂake category contained approximately 200 samples. Taking advantage of how snowﬂakes rotate while falling, a 10 https://doi.org/10.5194/amt-2023-148 Preprint. Discussion started: 18 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/amt-2023-148 Preprint. Discussion started: 18 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/amt-2023-148 Preprint. Discussion started: 18 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. Aggregate Planar crystal Graupel Columnar crystal Small particle !"#$ %"&' A %"&' %"#$ %( %"#$ %"&' %"#$ (a) (b) Figure 5. (a) For ﬁve snowﬂake types, ﬁve images of each snowﬂake separated due to rotation just prior to settling on the DEID’s hotplate. (b) Volume measurement method (b) Figure 5. (a) For ﬁve snowﬂake types, ﬁve images of each snowﬂake separated due to rotation just prior to settling on the DEID’s hotplate. (b) Volume measurement method single camera with multiple images was found to represent a three-dimensional picture of a snowﬂake and provide geometrical volume more accurately than using a single image (Li et al., 2022). Five sequential selected images of each crystal type are illustrated in Fig. 5a. A schematic showing how snowﬂake volume is computed is illustrated in Fig. 5b and formulated in Table 1 . 3.5 Hydrometeor density calculation exploiting concurrent imagery during their fall Here, Amax is the maximum 230 area of a planar crystal in a 2-D plane within all images that are visible by the camera and Dmin is the minimum dimension in the 2-D plane as indicated in Fig. 5b. The volume of graupel was estimated as a sphere VSLR = π/6D3 eﬀ, where DReﬀ= q 4 πA. A is the mean area of all images as illustrated in Fig. 5b. The volume of aggregates was estimated by ﬁtting an ellipsoid such that VSLR = π 6 DmaxDminDv, where Dmax, Dmin and Dv are illustrated in Fig. 5b. The volume of columnar crystals was estimated from VSLR = π 4 D 2 minDmax, where Dmax and Dmin are the average of the maximum and minimum dimensions of 235 Table 1 . The volume of planar crystals was approximated as a disc, hence, VSLR = AmaxDmin. Here, Amax is the maximum 230 area of a planar crystal in a 2-D plane within all images that are visible by the camera and Dmin is the minimum dimension in the 2-D plane as indicated in Fig. 5b. The volume of graupel was estimated as a sphere VSLR = π/6D3 eﬀ, where DReﬀ= q 4 πA. A is the mean area of all images as illustrated in Fig. 5b. The volume of aggregates was estimated by ﬁtting an ellipsoid such that VSLR = π 6 DmaxDminDv, where Dmax, Dmin and Dv are illustrated in Fig. 5b. The volume of columnar crystals was estimated from VSLR = π 4 D 2 minDmax, where Dmax and Dmin are the average of the maximum and minimum dimensions of 235 230 11 https://doi.org/10.5194/amt-2023-148 Preprint. Discussion started: 18 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/amt-2023-148 Preprint. Discussion started: 18 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/amt-2023-148 Preprint. Discussion started: 18 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. ﬁve images as illustrated in Fig. 5b. The volume of small particles were estimated using a spherical volume VSLR = π 6 D 3 max, where Dmax is the average of the maximum dimension of ﬁve small particle images ( Fig. 5b). Table 1. Classiﬁcation of snowﬂakes and corresponding volume estimation based on crystal geometry. Table 1. Classiﬁcation of snowﬂakes and corresponding volume estimation based on crystal geometry. 3.5 Hydrometeor density calculation exploiting concurrent imagery during their fall Snow crystal type Geometrical shape Volume (VSLR) Planar Disc AmaxDmin Columnar Solid cylinder (π/4)D 2 minDmax Graupel Spherical (π/6)D3 eff Small particle Spherical (π/6)D 3 max Aggregate Ellipsoid (π/6)DmaxDminDv 4.1 MS density method laboratory calibration (8), the average measured density of 80 ice particles with different shapes and sizes, with the above listed environmental conditions and plate temperatures, was 928 ± 56 kg m−3, which shows the MS method works for a wide range of environmental conditions to within experimental uncertainty. 285 3.6 Manual measurements of the snowpack: Bulk-density snowpack calculations The mean bulk density of a fresh snowpack( ρm s ) can also be determined using manual measurements of the ratio of the snow water equivalent depth (SWE) to the new snow depth (H). This can be done with the DEID by recalling that SWE = 240 k∆m/ρwAhp and H = k∆m/ρm s Ahp, 240 240 ρm s = ρw SWE H . (16) where ρw is the density of water of 1000 kg m−3. At the Alta-Collins snow-study plot these measurements were obtained every 12 hours. Note that changes within the snowpack due to such processes as densiﬁcation, heat transfer, wind shear, etc., are not considered here as analyses are limited to consideration of freshly fallen snow. 245 where ρw is the density of water of 1000 kg m−3. At the Alta-Collins snow-study plot these measurements were obtained every 12 hours. Note that changes within the snowpack due to such processes as densiﬁcation, heat transfer, wind shear, etc., are not considered here as analyses are limited to consideration of freshly fallen snow. 245 For further comparison, the average snowpack density can also be estimated at hourly intervals based on measurements obtained from an ETI Instrument Systems Noah-II precipitation weighing gauge (SWEETI) and the snow depth from a Camp- bell Scientiﬁc, Inc. SR50 ultrasonic snow-depth sensors. The ETI and SR50 sensors were deployed 4 m from the DEID at the Alta-Collins site. A windshield was deployed around the ETI bucket to increase catchment efﬁciency. The ETI reported SWE measurements once every hour with a resolution, threshold, and accuracy of 0.25 mm, 0.25 mm, and ± 0.25 mm respectively. 250 The SR50 sensor recorded snow depth every hour to provide running totals of snow depth. The measurement range of the ultra-sonic sensor was 0.5 m to 10 m with an accuracy of 0.4% and a resolution of 0.1 mm. Raw DEID data sampled at a rate of 15 Hz were integrated to produce hourly measurements for comparison with ETI data. ETI and SR50 data were collected throughout the winter of 2020, but data from 0700 UTC 12 Dec to 1900 UTC 12 Dec 2020 were used to compare SWE and snow accumulation with the DEID obtained using the MS density measurement method. 255 12 12 https://doi.org/10.5194/amt-2023-148 Preprint. Discussion started: 18 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. 4.1 MS density method laboratory calibration In order to use the MS method for determining density, the melting pressure constant P0 and the calibration constant c in Eqs. (6) and (8), respectively, must ﬁrst be determined empirically. To do this, eighty ice particles with different sizes and masses were applied to the DEID hotplate. Furthermore, the experiments were conducted in an environmentally controlled chamber 260 with the air temperature ﬁxed at 18 ◦C, near-zero wind velocity (0.05 m s−1), a hotplate temperature of 85◦C, and 38% relative humidity. Results from experiments conducted at these standard conditions are identiﬁed with a subscript 0. The variables ∆tmelt, ∆tevap, ∆Tmelt, and ∆Tevap for each particle were determined using the thermal camera, while h was measured directly using the laser-pointer system. The measured values of h and ∆tmelt are plotted in Fig. 6a. The slope of the h-∆tmelt curve were applied to the DEID hotplate. Furthermore, the experiments were conducted in an environmentally controlled chamber 260 with the air temperature ﬁxed at 18 ◦C, near-zero wind velocity (0.05 m s−1), a hotplate temperature of 85◦C, and 38% relative humidity. Results from experiments conducted at these standard conditions are identiﬁed with a subscript 0. The variables ∆tmelt, ∆tevap, ∆Tmelt, and ∆Tevap for each particle were determined using the thermal camera, while h was measured directly using the laser-pointer system. The measured values of h and ∆tmelt are plotted in Fig. 6a. The slope of the h-∆tmelt curve is vmelt, which is approximately constant (v0,melt = 2.11 ± 0.10 × 10−4 m s−1) because the experiments were performed with 265 ice-particles in an environmentally controlled chamber where the average measured value of ∆T0,melt was also found to be approximately constant (101.43 ± 0.82 K). The measured vmelt and ∆Tmelt for each particle can be substituted into Eq. (6) to solve for the melting pressure constant P0, and then the constant c can easily be determined from Eq. (9). This was done and the results were averaged over all 80 samples, yielding P0 = 3.41±0.21×109 N m−2 and c = 7.14±0.33×104. With a derived value of c and particle mass measured with 270 the DEID, the particle density can now be inferred from Eq. (8). This yields an average ice-particle density of ρice MS = 919±65 kg m−3. This is very close to the expected value of ice density at temperatures near 0◦C (i.e., 917 kg m−3). 4.1 MS density method laboratory calibration A summary of the following measured parameters for laboratory-created ice particles are presented in Table 2: vmelt, ρice MS, mDEID, mgravity and Vpipette. Here, Vpipette is volume of the ice particle created by pipetting a known volume of water. Since these experiments were conducted in an enclosure where environmental variability was negligible and the effect of convective 275 cooling on the measurement of m, Ap, ∆tmelt ∆tevap, and ∆Tevap did not play a role. However, in nature, winds can affect ∆tmelt ∆tevap, and ∆Tevap as well as vmelt. To determine how environmental variability affects vmelt and to generalize the validation of Eq. (6), 60-µL (0.06 g) ice particles with thickness h = 1.95 mm and area Ap = 5.02 × 10−5 m2 were placed on the DEID’s hotplate and the wind speed particles with thickness h = 1.95 mm and area Ap = 5.02 × 10 m were placed on the DEID s hotplate and the wind speed was varied from 0.0 to 8.3 m s−1, relative humidity varied from 38% to 91%, and plate temperature varied from 65◦C to 280 95◦C. vmelt was computed using direct measurements and computed as v = h/∆tmelt. It was also computed using Eq. (6, i.e., vmelt = κ∆Tmelt/P0. Results are shown in Fig. 6b. The coefﬁcient of determination R2 between vmelt computed using the two methods is 0.99, and the RMS error is 3.46 × 10−6 m s−1. Using Eq. (8), the average measured density of 80 ice particles with different shapes and sizes, with the above listed environmental conditions and plate temperatures, was 928 ± 56 kg m−3, which shows the MS method works for a wide range of environmental conditions to within experimental uncertainty. 285 was varied from 0.0 to 8.3 m s−1, relative humidity varied from 38% to 91%, and plate temperature varied from 65◦C to 280 95◦C. vmelt was computed using direct measurements and computed as v = h/∆tmelt. It was also computed using Eq. (6, i.e., vmelt = κ∆Tmelt/P0. Results are shown in Fig. 6b. The coefﬁcient of determination R2 between vmelt computed using the two methods is 0.99, and the RMS error is 3.46 × 10−6 m s−1. Using Eq. 4.2 Density of individual frozen salt-water particles To test the MS method on a wider range of particle densities, frozen salt-water particles with different salt (NaCl) concen- trations were applied to the DEID hotplate. The estimated density of the frozen salt-water particles calculated using the MS 13 Figure 6. (a) Ice-particle height as a function of ∆tmelt, the slope of this line determines v0,melt, the melting speed under ﬁxed standard conditions. That is, ice particles of different maximum thickness [0.22, 0.56] mm and their melting time at a plate temperature of 85◦C, near-zero wind velocity (0.05 m s−1), and 38% relative humidity. (b) Plot of vmelt versus κ∆Tmelt/P0 illustrating the validity of Eq. (6). vmelt is determined directly from measurements of particle maximum thickness, h and melting time, ∆tmelt and then compared to κ∆Tmelt/P0 for a range of environmental conditions. Figure 6. (a) Ice-particle height as a function of ∆tmelt, the slope of this line determines v0,melt, the melting speed under ﬁxed standard conditions. That is, ice particles of different maximum thickness [0.22, 0.56] mm and their melting time at a plate temperature of 85◦C, near-zero wind velocity (0.05 m s−1), and 38% relative humidity. (b) Plot of vmelt versus κ∆Tmelt/P0 illustrating the validity of Eq. (6). vmelt is determined directly from measurements of particle maximum thickness, h and melting time, ∆tmelt and then compared to κ∆Tmelt/P0 for a range of environmental conditions. Table 2. Mean and standard deviation of the mass of eighty experimentally manufactured ice particles the water droplet volume applied by pipette (Vpipette) prior to freezing, determined using a gravimetric scale (mgravity) and the DEID (mDEID). The density of ice particles estimated using the DEID MS method (ρice MS), and the melting velocity v0,melt (ms−1) under the standard conditions described in the text. Table 2. Mean and standard deviation of the mass of eighty experimentally manufactured ice particles the water droplet volume applied by pipette (Vpipette) prior to freezing, determined using a gravimetric scale (mgravity) and the DEID (mDEID). The density of ice particles estimated using the DEID MS method (ρice MS), and the melting velocity v0,melt (ms−1) under the standard conditions described in the text. method agrees with the density expected from the percentage of salt in the solution (ρtheoretical) to within 3%. The results are summarized in Table 3. 290 Table 3. The density of frozen salt-water particles measured using the MS method and determined theoretically based on a salt-water ratio. Table 3. The density of frozen salt-water particles measured using the MS method and determined theoretically based on a salt-water ratio. The density of frozen salt-water particles measured using the MS method and determined theoretically based o Percentage of salt ρMS (kg m−3) ρtheoretical (kg m−3) 5 969 ± 37 1008 10 988 ± 26 1015 20 1002 ± 38 1028 30 1018 ± 24 1040 40 1028 ± 31 1050 Percentage of salt ρMS (kg m−3) ρtheoretical (kg m−3) 5 969 ± 37 1008 10 988 ± 26 1015 20 1002 ± 38 1028 30 1018 ± 24 1040 40 1028 ± 31 1050 Figure 7. Measured density of a range of snowﬂake types using the particle imaging system compared with values obtained using the DEID MS method with associated coefﬁcients of determination, slopes and RMS errors. Figure 7. Measured density of a range of snowﬂake types using the particle imaging system compared with values obtained using the DEID MS method with associated coefﬁcients of determination, slopes and RMS errors. 4.2 Density of individual frozen salt-water particles Vpipette ( µL) mgravity (mg) mDEID (mg) ρice MS (kg m−3) v0,melt × 10−4 ( m s−1) 05 5.6 ± 0.4 5.1 ± 0.2 926±32 2.15±0.14 10 12.3 ± 0.7 14.0 ± 0.8 916±58 2.09±0.12 20 21.6 ± 0.8 22.1 ± 1.0 942±61 2.11±0.12 30 30.8 ± 2.7 29.8 ± 2.0 938±84 2.16±0.16 40 42.0 ± 3.2 43.1 ± 3.4 906±58 2.04±0.09 50 53.1 ± 4.1 52.1 ± 2.4 901±67 2.08±0.11 60 61.8 ± 3.8 63.1 ± 3.8 928±76 2.12±0.07 70 74.1 ± 4.1 76.2 ± 4.2 899±86 2.14±0.04 919±65 2.11±0.10 method agrees with the density expected from the percentage of salt in the solution (ρtheoretical) to within 3%. The results are summarized in Table 3. 290 14 https://doi.org/10.5194/amt-2023-148 Preprint. Discussion started: 18 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. 4.4 Frequency distribution of individual hydrometeor densities and the temperature dependence of bulk density The spherical assumption underestimates snowﬂake density by a factor ≈1.5 compared to the MS method. Table 4. Camparision between two density methods, MS and SLR-DEID of ﬁve types of snow crystal. Range Table 4. Camparision between two density methods, MS and SLR-DEID of ﬁve types of snow crystal. Range of Deff for each type of crystal. Snow crystal type Deﬀ(mm) ρMS (kg m−3) ρSLR-DEID (kg m−3) Uncertainty (%) R2 Planar 2.4 - 4.3 89 ± 40 98 ± 46 9.6 0.96 Columnar 1.4 - 3.4 157 ± 82 140 ± 92 11.4 0.95 Graupel 1.4 - 4.6 120 ± 87 130 ± 89 3.7 0.97 Small particle 0.8 - 1.2 141 ± 109 138 ± 110 2.1 0.98 Aggregate 3.1 - 10.2 188 ± 72 170 ± 64 10.1 0.91 density methods, MS and SLR-DEID of ﬁve types of snow crystal. Range of Deff for each type of crystal. parision between two density methods, MS and SLR-DEID of ﬁve types of snow crystal. Range of Deff for eac 4.4 Frequency distribution of individual hydrometeor densities and the temperature dependence of bulk density Figure 8a shows a probability distribution function (PDF) for the densities of individual snowﬂakes using the MS method applied to data acquired at the Alta-Collins snow-study plot for seven snow storms selected to encompass a broad range of 305 environmental conditions: mean ambient temperatures [-13.45, -4.82] ◦C, mean wind speeds [0.30, 0.89] ms−1, and mean relative humidities [72, 91]% as listed in Table 5. The kurtosis (Kr) and skewness (Sk) of the normalized density distribution functions vary from 2.02 to 2.42 and 0.01 to 0.10, respectively. We found that the PDFs of snow density are symmetric about the mean (Sk = 0.01) and near Gaussian (Kr = 2.41) when the ambient temperature is lowest, while Sk = 0.10 and Kr = 2.02 when the ambient temperature is highest, which is shown in Table 5. Figure 8b includes results from snowﬂake 310 densities computed assuming a spherical particle volume but also using the DEID, as done in (Rees et al., 2021). The spherical assumption underestimates snowﬂake density by a factor ≈1.5 compared to the MS method. Figure 8a shows a probability distribution function (PDF) for the densities of individual snowﬂakes using the MS method applied to data acquired at the Alta-Collins snow-study plot for seven snow storms selected to encompass a broad range of 305 environmental conditions: mean ambient temperatures [-13.45, -4.82] ◦C, mean wind speeds [0.30, 0.89] ms−1, and mean relative humidities [72, 91]% as listed in Table 5. The kurtosis (Kr) and skewness (Sk) of the normalized density distribution functions vary from 2.02 to 2.42 and 0.01 to 0.10, respectively. We found that the PDFs of snow density are symmetric about the mean (Sk = 0.01) and near Gaussian (Kr = 2.41) when the ambient temperature is lowest, while Sk = 0.10 and Kr = 2.02 when the ambient temperature is highest, which is shown in Table 5. Figure 8b includes results from snowﬂake 310 densities computed assuming a spherical particle volume but also using the DEID, as done in (Rees et al., 2021). The spherical assumption underestimates snowﬂake density by a factor ≈1.5 compared to the MS method. Kr = 2.02 when the ambient temperature is highest, which is shown in Table 5. Figure 8b includes results from snowﬂake 310 densities computed assuming a spherical particle volume but also using the DEID, as done in (Rees et al., 2021). 4.3 Field evaluation of the MS method for snow particles of different types We collected data at Alta Ski Area’s mid-Collins snow-study plot from 07 November 2020 to 27 April 2021. During this time, a snow accumulation of 12.35 m and a SWE accumulation of 1.38 m were observed with the DEID. The ambient air temperature varied from –21◦C to 2◦C, relative humidity varied from 64 to 97%, and wind speed varied from 0.2 to 8 m s−1. Generally, the observed densities of freshly fallen individual snowﬂakes varied from 9 to 495 kg m−3. The average densities of each storm 295 15 https://doi.org/10.5194/amt-2023-148 Preprint. Discussion started: 18 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. varied from 35 to 115 kg m−3. Figure 7 shows estimated snowﬂake densities using both the SLR-DEID and MS methods for ﬁve types of snowﬂakes. A comparison between the MS and SLR-DEID density methods for ﬁve crystal types is summarized in Table 4. The coefﬁcient of determination between the two methods is highest for small particles and graupel and least for aggregates. The measured size (Deﬀ) of each type of crystal is summarized in Table 4. The uncertainty that arises between the two methods for aggregate snowﬂakes may be due to the SLR-DEID’s method used to estimate the geometrical volume because aggregate have have a more irregular shape than the other types. For all snowﬂakes, the mean estimated density is 131 ± 83 using the SLR-DEID method and 142 ± 87 using the MS method, yielding an uncertainty of 3.9% and an R2 of 0.95. 4.5 Validation of SWE measurements SWE determined with the DEID can be compared to manual measurements collected SWE determined with the DEID can be compared to manual measurements collected at the Alta-Collins study plot. Since manual measurements are made infrequently at intervals of 12 hours, the comparisons are made on a storm-by-storm basis as 315 shown in Fig. 9. The relationship between DEID observations and the bulk standard manual measurement techniques is shown in Fig. 9a. A best ﬁt relationship between the two methods yields an R2 of 0.994 with a slope of 0.94 ± 0.04. p y p manual measurements are made infrequently at intervals of 12 hours, the comparisons are made on a storm-by-storm basis as 315 shown in Fig. 9. The relationship between DEID observations and the bulk standard manual measurement techniques is shown in Fig. 9a. A best ﬁt relationship between the two methods yields an R2 of 0.994 with a slope of 0.94 ± 0.04. 16 Figure 8. (a) Probability distribution functions of density of individual snowﬂakes measured using the MS method for seven storms. (b) Comparison between density determined with two methods employing DEID measurements alone, the MS method and the spherical par- ticle method. The solid line, dash line, and dot line are the mean density of a storm using a spherical method, MS method and manual measurements, respectively. https://doi.org/10.5194/amt-2023-148 Preprint. Discussion started: 18 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/amt-2023-148 Preprint. Discussion started: 18 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/amt-2023-148 Preprint. Discussion started: 18 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. Figure 8. (a) Probability distribution functions of density of individual snowﬂakes measured using the MS method for seven storms. (b) Comparison between density determined with two methods employing DEID measurements alone, the MS method and the spherical par- ticle method. The solid line, dash line, and dot line are the mean density of a storm using a spherical method, MS method and manual measurements, respectively. Figure 8. (a) Probability distribution functions of density of individual snowﬂakes measured using the MS method for seven storms. (b) Comparison between density determined with two methods employing DEID measurements alone, the MS method and the spherical par- ticle method. The solid line, dash line, and dot line are the mean density of a storm using a spherical method, MS method and manual measurements, respectively. 4.5 Validation of SWE measurements The accumulated SWE integrated over one-minute intervals is compared to ETI data in Fig. 9b. DEID SWE accumulation observations match those from the ETI gauge to within ± 6% over the 12-hour measurement period (storm duration). SWE accumulation measured by the DEID is slightly higher than that obtained by the ETI because the minimum resolution of the ETI is 0.254 mm, whereas the minimum DEID resolution is 0.001 mm (Singh et al., 2021). Furthermore, the ETI gauge has been shown to undercatch snowﬂakes compared to the DEID under high-wind conditions (Singh et al., 2021). 4.6 Validation of Snow depth measurements Snow accumulation (H) was computed with the DEID using Eq. (13) and Eq. (14), where Snow accumulation (H) was computed with the DEID using Eq. (13) and Eq. (14), where 1 min average MS density was used. The total snow accumulated in each storm measured using the DEID MS density method compares well with manual 325 measurements obtained every 12 hours at the Alta-Collins snow-study plot with an R2 value of 0.983 and a slope of 1.12 ± 0.07 as shown in Fig. 10a. A 12-hour period with snowfall between 0700 UTC and 1900 UTC 12 Dec 2020 is shown in Fig. 10b. There is also good agreement to within ± 5% between snow accumulation measurements obtained from the DEID using the MS-density method and those obtained using an ultrasonic snow-depth sensor obtained once per hour. The bulk density of a used. The total snow accumulated in each storm measured using the DEID MS density method compares well with manual 325 measurements obtained every 12 hours at the Alta-Collins snow-study plot with an R2 value of 0.983 and a slope of 1.12 ± 0.07 as shown in Fig. 10a. A 12-hour period with snowfall between 0700 UTC and 1900 UTC 12 Dec 2020 is shown in Fig. 10b. There is also good agreement to within ± 5% between snow accumulation measurements obtained from the DEID using the MS-density method and those obtained using an ultrasonic snow-depth sensor obtained once per hour. The bulk density of a fresh snowpack can differ from the average density of individual snowﬂakes and the 1-min average because snowﬂake settling 330 and compaction on the ground depends on such considerations as their settling characteristics, fall angle, wind speed, the 17 https://doi.org/10.5194/amt-2023-148 Preprint. Discussion started: 18 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/amt-2023-148 Preprint. Discussion started: 18 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. (b) (a) (a) SWE accumulation from DEID and manual measurements for seventeen storms. Each data point represents an individual anual measurements were made every 12 hours (1100 UTC and 2300 UTC), and the DEID sampled at 15 Hz. (b) Time series of umulation and SWE rate measured using the DEID and ETI gauge. Each DEID data point represents a 1-min average and each e data point a 1-hour average. (a) (a) (b) Figure 9. (a) SWE accumulation from DEID and manual measurements for seventeen storms. Each data point represents an individual storm. Manual measurements were made every 12 hours (1100 UTC and 2300 UTC), and the DEID sampled at 15 Hz. (b) Time series of SWE accumulation and SWE rate measured using the DEID and ETI gauge. Each DEID data point represents a 1-min average and each ETI-gauge data point a 1-hour average. 18 https://doi.org/10.5194/amt-2023-148 Preprint. Discussion started: 18 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. Nonetheless, The bulk density of the snowpack measured during seventeen storms using the DEID MS method, can also be compared with manual gravimetric snow-density measurements of SWE depth (SWEm) and snow depth (H) (Eq. 16). The R2 value relating the DEID and manual bulk-density measurements is 335 0.88 with a slope 0.90 ± 0.15, as shown in Fig. 11. The implication of these two comparisons, somewhat surprisingly, is that the DEID reproduces measurements made with more traditional techniques of freshly fallen snowpack density and accumulation without considering the quite complex physics of how individual snowﬂakes pack and stack. https://doi.org/10.5194/amt-2023-148 Preprint. Discussion started: 18 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/amt-2023-148 Preprint. Discussion started: 18 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/amt-2023-148 Preprint. Discussion started: 18 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. (b) (a) For seventeen storms, comparison between snow accumulation from the DEID obtained using the MS density method and measurements made every 12 hours (1100 UTC and 2300 UTC). Each data point represents a storm. (b) Time series of lation and snowfall rate measured using 1-min averaged DEID results and 1-hour averaged ultrasonic snow-depth sensor during a storm on 12 December 2020. (a) (a) (b) Figure 10. (a) For seventeen storms, comparison between snow accumulation from the DEID obtained using the MS density method and from manual measurements made every 12 hours (1100 UTC and 2300 UTC). Each data point represents a storm. (b) Time series of snow accumulation and snowfall rate measured using 1-min averaged DEID results and 1-hour averaged ultrasonic snow-depth sensor measurements during a storm on 12 December 2020. 19 https://doi.org/10.5194/amt-2023-148 Preprint. Discussion started: 18 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. Storm Day/duration (Hours) N Deﬀ (mm) ρMS (kg m−3) T amb (◦C) U (m s−1) RH (%) total Snow (mm) total SWE (mm) Kr Sk Dec 12 2020/20 242,643 1.60 ±0.74 41.86 -13.45 ± 1.37 0.58 85.24 292 10.66 2.42 0.01 Dec 17 2020/25 482,152 1.50 ±0.71 56.25 -6.75 ± 2.30 0.30 90.86 413 27.00 2.18 -0.07 Dec 22 2020/24 251,532 1.50 ±0.71 49.19 -12.08 ± 4.72 0.76 81.15 314 15.38 2.39 0.03 Jan 22 2021/45 570,590 1.50 ±0.68 90.54 -6.82 ± 2.29 0.65 90.28 457 34.37 2.14 0.05 Feb 03 2021/62 653,976 1.40 ±0.65 65.30 -9.98 ± 2.54 0.76 89.55 488 31.75 2.27 0.02 Feb 11 2021/67 1,275,102 1.50 ±0.59 56.27 -7.29 ± 3.17 0.56 86.56 862 51.81 2.16 0.03 March 20 2021/24 629,870 1.60 ±0.78 88.76 -4.82 ± 3.07 0.89 71.71 425 35.35 2.02 0.10 Table 5. Summary of DEID-derived snowﬂake parameters for a series of storms in Alta, Utah. Mean diameter Deﬀ, mean density ρMS, a total number of snowﬂakes N captured during seven storms with a mean ambient temperature T amb, mean wind speed U, mean relati humidity RH, total snow and total accumulated snow water equivalent for seven storms using the DEID. Kr is the Kurtosis and Sk t Skewness of the density distribution. Table 5. Summary of DEID-derived snowﬂake parameters for a series of storms in Alta, Utah. https://doi.org/10.5194/amt-2023-148 Preprint. Discussion started: 18 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. Mean diameter Deﬀ, mean density ρMS, and total number of snowﬂakes N captured during seven storms with a mean ambient temperature T amb, mean wind speed U, mean relative humidity RH, total snow and total accumulated snow water equivalent for seven storms using the DEID. Kr is the Kurtosis and Sk the Skewness of the density distribution. Table 5. Summary of DEID-derived snowﬂake parameters for a series of storms in Alta, Utah. Mean diameter Deﬀ, mean density ρMS, and total number of snowﬂakes N captured during seven storms with a mean ambient temperature T amb, mean wind speed U, mean relative humidity RH, total snow and total accumulated snow water equivalent for seven storms using the DEID. Kr is the Kurtosis and Sk the Skewness of the density distribution. structure of snowﬂakes, and the ambient temperature. We do not account for these processes in the calculation of the volume of freshly fallen snow layers as the impacts are largely unknown. Nonetheless, The bulk density of the snowpack measured during seventeen storms using the DEID MS method, can also be compared with manual gravimetric snow-density measurements of SWE depth (SWEm) and snow depth (H) (Eq. 16). The R2 value relating the DEID and manual bulk-density measurements is 335 0.88 with a slope 0.90 ± 0.15, as shown in Fig. 11. The implication of these two comparisons, somewhat surprisingly, is that the DEID reproduces measurements made with more traditional techniques of freshly fallen snowpack density and accumulation without considering the quite complex physics of how individual snowﬂakes pack and stack. structure of snowﬂakes, and the ambient temperature. We do not account for these processes in the calculation of the volume of freshly fallen snow layers as the impacts are largely unknown. Nonetheless, The bulk density of the snowpack measured during seventeen storms using the DEID MS method, can also be compared with manual gravimetric snow-density measurements of SWE depth (SWEm) and snow depth (H) (Eq. 16). The R2 value relating the DEID and manual bulk-density measurements is 335 0 88 with a slope 0 90 ± 0 15 as shown in Fig 11 The implication of these two comparisons somewhat surprisingly is that the structure of snowﬂakes, and the ambient temperature. We do not account for these processes in the calculation of the volume of freshly fallen snow layers as the impacts are largely unknown. 5 Conclusions Automated determination of the density of individual snowﬂakes has been a long-standing challenge. In this study, we present 340 a novel method for accomplishing this goal that exploits a new hotplate instrument, the Differential Emissivity Imaging Dis- drometer or DEID, which has previously been shown capable of obtaining highly accurate direct measurements of particle mass. Particle-by-particle density estimates are obtained from measurements of particle mass, projected are onto the plate, and Automated determination of the density of individual snowﬂakes has been a long-standing challenge. In this study, we present 340 a novel method for accomplishing this goal that exploits a new hotplate instrument, the Differential Emissivity Imaging Dis- drometer or DEID, which has previously been shown capable of obtaining highly accurate direct measurements of particle mass. Particle-by-particle density estimates are obtained from measurements of particle mass, projected are onto the plate, and 20 https://doi.org/10.5194/amt-2023-148 Preprint. Discussion started: 18 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. ρ"#$%#&(kg m+,) ̅/01 (kg m+,) ̅/01 = 0.90 ± 0.15 ρ"#$%#& Correlation between ̅/01 & ρ"#$%#&: 0.88 RMS: 8.8 (kg m+,) Figure 11. For seventeen storms, comparison between bulk density of snowpack from the DEID obtained using the MS density method and from manual gravimetric snow-density measurements made every 12 hours (1100 UTC and 2300 UTC). Each data point represents a storm. ρ"#$%#&(kg m+,) ̅/01 (kg m+,) ̅/01 = 0.90 ± 0.15 ρ"#$%#& Correlation between ̅/01 & ρ"#$%#&: 0.88 RMS: 8.8 (kg m+,) Figure 11. For seventeen storms, comparison between bulk density of snowpack from the DEID obtained using the MS density method and from manual gravimetric snow-density measurements made every 12 hours (1100 UTC and 2300 UTC). Each data point represents a storm. an estimate of the speed with which the particle thickness on the plate diminishes as it melts. A particle’s thickness normal to an estimate of the speed with which the particle thickness on the plate diminishes as it melts. A particle’s thickness normal to the hotplate is a product of the melting speed and melting time from which, in combination with a particle’s area, an estimate 345 can be obtained of particle volume and hence its density. For individual hydrometeors, this ‘melting-speed method’ was val- idated using laboratory ice particles of known density showing a maximum uncertainty of 6.3%, as well as with videos from ﬁeld measurements of a range of natural falling snowﬂakes with an uncertainty of 3.7%. Appendix A: Relation between the melting and evaporation statistics The average conductive heat-transfer rate during melting of hydrometeors (mLf/∆tmelt) can be written as 365 mLf/∆tmelt ≈κAp∆Tmelt. (A1) mLf/∆tmelt ≈κAp∆Tmelt. The average conductive heat-transfer rate during evaporation of hydrometeors (mLv/∆tevap) can be written as The average conductive heat-transfer rate during evaporation of hydrometeors (mLv/∆tevap) can be written as (A2) mLv/∆tevap ≈κAp∆Tevap. By using Eq. (A1) and Eq. (A2) we may write By using Eq. (A1) and Eq. (A2) we may write ∆Tmelt∆tmelt Lf ≈∆Tevap∆tevap Lv . (A3) 370 Experimentally determined values for ∆tevap and ∆tmelt for different ice-particle masses and sizes for a large range of environmental conditions are shown in Fig. A1. As expected, the slope between the two-time scales agrees to be within 5% of the ratio of the latent heat of vaporization to the latent heat of fusion. With the DEID, ∆tmelt and ∆tevap are directly measured using a thermal camera and counting the number of frames between the ﬁrst frame when an ice particle hits the hotplate and (A3) Experimentally determined values for ∆tevap and ∆tmelt for different ice-particle masses and sizes for a large range of environmental conditions are shown in Fig. A1. As expected, the slope between the two-time scales agrees to be within 5% of the ratio of the latent heat of vaporization to the latent heat of fusion. With the DEID, ∆tmelt and ∆tevap are directly measured using a thermal camera and counting the number of frames between the ﬁrst frame when an ice particle hits the hotplate and last fame when the particle has completely melted or evaporated. 375 last fame when the particle has completely melted or evaporated. 375 5 Conclusions The codes used for pro- 360 cessing follow the methodologies and equations described herein. Data availability. Data from the ETI Noah II precipitation gauge and ultrasonic snow-depth are openly accessible from https://mesowest.utah.edu (University of Utah, 2021), Station ID: CLN. All other datasets are available upon request. Data availability. Data from the ETI Noah II precipitation gauge and ultrasonic snow-depth are openly accessible from https://mesowest.utah.edu (University of Utah, 2021), Station ID: CLN. All other datasets are available upon request. 5 Conclusions DEID observations at a high elevation mountain site of snow water equivalent (SWE) accumulation, snow depth accumulation (H), and bulk snow density (ρMS) the hotplate is a product of the melting speed and melting time from which, in combination with a particle’s area, an estimate 345 can be obtained of particle volume and hence its density. For individual hydrometeors, this ‘melting-speed method’ was val- idated using laboratory ice particles of known density showing a maximum uncertainty of 6.3%, as well as with videos from ﬁeld measurements of a range of natural falling snowﬂakes with an uncertainty of 3.7%. DEID observations at a high elevation mountain site of snow water equivalent (SWE) accumulation, snow depth accumulation (H), and bulk snow density (ρMS) from seventeen storms taken at the Alta-Collins snow-study plot at Alta Ski Area during winter 2020-2021 agreed well with 350 traditional manual techniques measurements with R2 values of 0.994, 0.983 and 0.88, with slope 0.94 ± 0.04, 1.12 ± 0.07 and 0.90 ± 0.15, respectively independent of environmental conditions, including wind speed, ambient temperature, relative humidity, and hotplate temperature. Using the melting-speed method, the utility of the DEID design can be extended from hydrometeor mass measurement to Using the melting-speed method, the utility of the DEID design can be extended from hydrometeor mass measurement to measurement of the density of irregularly shaped hydrometeors, in real-time with high accuracy. Such information, whether 355 taken on a particle-by-particle basis or assessed as a cumulative bulk quantity has applications for high-resolution measurement of vertical density variability in the snowpack (e.g., Morrison et al. (2023))– critical for assessment of its stability – as well as measurement of the density of irregularly shaped hydrometeors, in real-time with high accuracy. Such information, whether 355 taken on a particle-by-particle basis or assessed as a cumulative bulk quantity has applications for high-resolution measurement of vertical density variability in the snowpack (e.g., Morrison et al. (2023))– critical for assessment of its stability – as well as 21 https://doi.org/10.5194/amt-2023-148 Preprint. Discussion started: 18 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. for studies of snow metamorphism, assessment of transitions between rain and snow, determinations of rates of hydrometeor settling and its response to turbulence, and the scattering by snow particles of visible light and radar pulses. Code availability. The data processing codes are protected through a patent and are not available for distribution. Appendix B: DEID Thermal Imagery In this section, we present example thermal images of different types of snowﬂakes after melting on the DEID hotplate. Figures B1 and B2 show aggregate and graupel snow particles, respectively. These data were taken at the Alta-Collins snow-study plot 22 December 2020. 22 Figure A1. Experimentally measured melting and evaporation times for laboratory ice particles. Lv and Lf are the latent heat of evaporation and fusion, respectively. The slope of the line is quite close to Lv/Lf. https://doi.org/10.5194/amt-2023-148 Preprint. Discussion started: 18 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/amt-2023-148 Preprint. Discussion started: 18 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. Figure A1. Experimentally measured melting and evaporation times for laboratory ice particles. Lv and Lf are the latent heat of evaporation and fusion, respectively. The slope of the line is quite close to Lv/Lf. 2 mm Figure B1. Illustrative examples showing thermal image of aggregate snowﬂakes on the DEID hotplate. Figure B1. Illustrative examples showing thermal image of aggregate snowﬂakes on the DEID hotplate. 2 mm Figure B2. Illustrative examples showing thermal images of graupel snow particles on the DEID hotplate. Figure B2. Illustrative examples showing thermal images of graupel snow particles on the DEID hotplate. 23 Author contributions. D.K.S., E.R.P. and T.J.G. designed the experiments and D.K.S performed the experiments in consultation with E.R.P 0 and T.J.G.; All authors analyzed the data and contributed to writing and editing the paper. https://doi.org/10.5194/amt-2023-148 Preprint. Discussion started: 18 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/amt-2023-148 Preprint. Discussion started: 18 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. Author contributions. D.K.S., E.R.P. and T.J.G. designed the experiments and D.K.S performed the experiments in consultation with E.R.P 380 and T.J.G.; All authors analyzed the data and contributed to writing and editing the paper. Author contributions. D.K.S., E.R.P. and T.J.G. designed the experiments and D.K.S performed the experiments in consultation with E.R.P 380 and T.J.G.; All authors analyzed the data and contributed to writing and editing the paper. Author contributions. D.K.S., E.R.P. and T.J.G. designed the experiments and D.K.S performed the experiments in consultation with E.R.P 380 and T.J.G.; All authors analyzed the data and contributed to writing and editing the paper. Competing interests. Conﬂict of interest: The DEID technology is protected through patent US20210172855A1 co-authored with D.K.S., E.R.P., and T.J.G. and is commercially available through Particle Flux Analytics, Inc. T.J.G. is a co-owner of Particle Flux Analytics, Inc. Acknowledgements. We thank Allan Reaburn and his colleagues at Particle Flux Analytics, Inc. for their contributions to the development 385 of the DEID, Dave Richards and the Alta Ski Patrol for ﬁeld support, and Spencer Donovan for contributions to the experimental setup. This work was supported by the U.S. National Science Foundation through the following grants: PDM-1841870 and PDM-2210179) References Alcott, T. I. and Steenburgh, W. 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P.: Scattering optics of snow, Applied optics, 43, 1589–1602, 2004. Kosky, P., Balmer, R., Keat, W., and Wise, G.: Exploring engineering 3rd edition, Book, pp. 451–462, 2013. Appendix B: DEID Thermal Imagery which has a license from the University of Utah to commercialize the DEID. Competing interests. Conﬂict of interest: The DEID technology is protected through patent US20210172855A1 co-authored with D.K.S., E.R.P., and T.J.G. and is commercially available through Particle Flux Analytics, Inc. T.J.G. is a co-owner of Particle Flux Analytics, Inc. which has a license from the University of Utah to commercialize the DEID. Acknowledgements. We thank Allan Reaburn and his colleagues at Particle Flux Analytics, Inc. for their contributions to the development 385 of the DEID, Dave Richards and the Alta Ski Patrol for ﬁeld support, and Spencer Donovan for contributions to the experimental setup. This work was supported by the U.S. National Science Foundation through the following grants: PDM-1841870 and PDM-2210179) Acknowledgements. We thank Allan Reaburn and his colleagues at Particle Flux Analytics, Inc. for their contributions to the development 385 of the DEID, Dave Richards and the Alta Ski Patrol for ﬁeld support, and Spencer Donovan for contributions to the experimental setup. This work was supported by the U.S. National Science Foundation through the following grants: PDM-1841870 and PDM-2210179) 24 24 24 https://doi.org/10.5194/amt-2023-148 Preprint. Discussion started: 18 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/amt-2023-148 Preprint. Discussion started: 18 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. References Li, J., Guala, M., and Hong, J.: Snow particle analyzer for simultaneous measurements of snow density and morphology, arXiv preprint arXiv:2209.11129, 2022. Kokhanovsky, A. A. and Zege, E. 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https://openalex.org/W4390669635	https://www.nature.com/articles/s41398-023-02712-y.pdf	English	null	Normalized affective responsiveness following deep brain stimulation of the medial forebrain bundle in depression	Translational psychiatry	2,024	cc-by	8,069	INTRODUCTION symptom improvement are rarely studied [21, 22] and the mechanisms of slMFB DBS improving poor social functioning in depression are unknown, so far [20]. Thus, the current study systematically investigates DBS treatment effects on social skills, the basis of successful social functioning [23]. Speciﬁcally, three higher-order social skills termed affective empathy, compassion, and theory of mind (ToM) are being investigated. Affective empathy, compassion, and ToM were assessed behaviorally before and three months after the onset of active slMFB DBS in patients with TRD and compared to a sample of age- and gender-matched healthy controls. INTRODUCTION Approximately 30% of patients with depression do not respon conventional treatment methods such as psychotherapy pharmacotherapy [1]. Resistance to antidepressant treatment associated with a reduced quality of life for the patients [2 Currently, deep brain stimulation (DBS) is under investigation new emerging treatment method in psychiatry [4–7]. DBS is invasive, non-lesional and highly focal treatment method involves the bilateral implantation of electrodes into a selec brain area as well as the constant application of electrical impu to this brain target. Electrical current is delivered from a p generator implanted subcutaneously in the region of clavicular [8]. The supero-lateral medial forebrain bundle (slM represents one of the brain targets for DBS electrode placem currently investigated in treatment-resistant depression (T [9, 10]. DBS of the slMFB has shown promising results in term rapid and sustained antidepressant effects [11–15]. Beyond t patients subjectively reported social functioning improveme [13, 16]. Considering that normal social functioning is crucial f good quality of life [17, 18], reduces the mortality risk [19] further plays an important role in the long-term stabilization a chronic diseases [20], it can be considered an impor therapeutic outcome [9]. However, slMFB DBS effects bey Received: 12 March 2023 Revised: 6 December 2023 Accepted: 1Division of Interventional Biological Psychiatry, Department of Psychiatry and Germany. 2Department of Psychology, Laboratory for Biological Psychology, Cli ✉email: Hannah.kilian@szvt.rhap.de Approximately 30% of patients with depression do not respond to conventional treatment methods such as psychotherapy and pharmacotherapy [1]. Resistance to antidepressant treatments is associated with a reduced quality of life for the patients [2, 3]. Currently, deep brain stimulation (DBS) is under investigation as new emerging treatment method in psychiatry [4–7]. Translational Psychiatry Translational Psychiatry www.nature.com/tp 1Division of Interventional Biological Psychiatry, Department of Psychiatry and Psychotherapy Medical Center - University of Freiburg, Faculty of Medicine, DE-79104 Freiburg, Germany. 2Department of Psychology, Laboratory for Biological Psychology, Clinical Psychology and Psychotherapy, University of Freiburg, DE-79104 Freiburg, Germany. ✉email: Hannah kilian@szvt rhap de ARTICLE OPEN Normalized affective responsiveness following deep brain stimulation of the medial forebrain bundle in depression Hannah Marlene Kilian 1✉, Bastian Schiller 2, Dora Margarete Meyer-Doll 1, Markus Heinrichs2 and Thomas Eduard Schläpfer © The Author(s) 2024 © The Author(s) 2024 Deep brain stimulation (DBS) of the supero-lateral medial forebrain bundle (slMFB) is associated with rapid and sustained antidepressant effects in treatment-resistant depression (TRD). Beyond that, improvements in social functioning have been reported. However, it is unclear whether social skills, the basis of successful social functioning, are systematically altered following slMFB DBS. Therefore, the current study investigated speciﬁc social skills (affective empathy, compassion, and theory of mind) in patients with TRD undergoing slMFB DBS in comparison to healthy subjects. 12 patients with TRD and 12 age- and gender-matched healthy subjects (5 females) performed the EmpaToM, a video-based naturalistic paradigm differentiating between affective empathy, compassion, and theory of mind. Patients were assessed before and three months after DBS onset and compared to an age- and gender-matched sample of healthy controls. All data were analyzed using non-parametric Mann-Whitney U tests. DBS treatment signiﬁcantly affected patients’ affective responsiveness towards emotional versus neutral situations (i.e. affective empathy): While their affective responsiveness was reduced compared to healthy subjects at baseline, they showed normalized affective responsiveness three months after slMFB DBS onset. No effects occurred in other domains with persisting deﬁcits in compassion and intact socio-cognitive skills. Active slMFB DBS resulted in a normalized affective responsiveness in patients with TRD. This speciﬁc effect might represent one factor supporting the resumption of social activities after recovery from chronic depression. Considering the small size of this unique sample as well as the explorative nature of this study, future studies are needed to investigate the robustness of these effects. Translational Psychiatry (2024) 14:6 ; https://doi.org/10.1038/s41398-023-02712-y 1Division of Interventional Biological Psychiatry, Department of Psychiatry and Psychotherapy Medical Center - University of Freiburg, Faculty of Medicine, DE-79104 Freiburg, Germany. 2Department of Psychology, Laboratory for Biological Psychology, Clinical Psychology and Psychotherapy, University of Freiburg, DE-79104 Freiburg, Germany. ✉email: Hannah.kilian@szvt.rhap.de Received: 12 March 2023 Revised: 6 December 2023 Accepted: 12 December 2023 INTRODUCTION Pharmacotherapy as well as speciﬁcally developed psychotherapy (e.g. cognitive behavioral system of psychotherapy (CBASP) [37]; interpersonal therapy (IPT) [38]) have an impact on social skill deﬁcits in depression, but effects are only small to moderate [20, 39, 40]. DBS of the slMFB, however, might directly inﬂuence social skills. This idea is based on neuroimaging data demonstrating that the slMFB as a connecting structure of brain regions of the mesolimbic pathway not only induces brain metabolism changes in the stimulated area but also distal to the stimulated target [41–43]. Importantly, neuronal regions asso- ciated with affective empathy, compassion and ToM partly overlap with these regions, e.g. the medial prefrontal cortex, the ventral tegmental area and the ventral striatum [24, 44–52]. It thus seems plausible to assume that slMFB DBS modulates social skill deﬁcits associated with depression. Fig. 1 Study Procedure. Socio-affective and socio-cognitive skills were assessed in a sample of 12 patients with TRD (5 females) before and after three months of slMFB DBS (gray boxes) and compared to social skills of 12 age- and gender-matched healthy control subjects (HC). descripition of inclusion and exclusion criteria see clinicaltrials.gov (NCT03653858) or previous publications, e.g. [11]). Data of 12 patients with TRD were analyzed and compared to 12 age- and gender-matched HC. The baseline data before DBS surgery (n = 21) have previously been published [26] and the patients reported in this study represent a subsample (n = 15) of participants who underwent surgery. Of this subsample, three patients did not take part in the follow-up measurement, resulting in a ﬁnal sample of 12 patients. Healthy control subjects completed an online questionnaire and were eligible if they had no history of neurological or psychiatric disorders, no previous or current psychiatric or psychotherapeutic treatment and no current alcohol or drug abuse, as well as no current depressive symptoms (BDI < 10). All participants were ﬂuent in German. In this study, we aim to illuminate social functioning changes following slMFB DBS by systematically investigating behaviorally assessed affective empathy, compassion, and ToM before and after the onset of stimulation in a unique sample of patients with TRD. In order to do so, we used a naturalistic test paradigm based on video stimuli, the EmpaToM [24]. INTRODUCTION DBS is an invasive, non-lesional and highly focal treatment method that involves the bilateral implantation of electrodes into a selected brain area as well as the constant application of electrical impulses to this brain target. Electrical current is delivered from a pulse generator implanted subcutaneously in the region of the clavicular [8]. The supero-lateral medial forebrain bundle (slMFB) represents one of the brain targets for DBS electrode placement currently investigated in treatment-resistant depression (TRD) [9, 10]. DBS of the slMFB has shown promising results in terms of rapid and sustained antidepressant effects [11–15]. Beyond that, patients subjectively reported social functioning improvements [13, 16]. Considering that normal social functioning is crucial for a good quality of life [17, 18], reduces the mortality risk [19] and further plays an important role in the long-term stabilization after chronic diseases [20], it can be considered an important therapeutic outcome [9]. However, slMFB DBS effects beyond y Affective empathy is deﬁned as the ability to share positive and negative feelings of a counterpart [24]. Feelings of affective empathy might further induce positive feelings of warmth and care including the motivation to help another person and reduce their suffering (compassion/ concern). Study evidence shows that patients with depressive symptoms feel less affective empathy and compassion [20, 25, 26]. On the other side, feelings of affective empathy might also cause aversive feelings of stress [27], which seem to be prominent in patients with depression [25, 28–30]. Theory of Mind (ToM), also known as perspective- taking or mentalizing, describes the ability to understand and infer mental states of another person [31]. Data from studies Received: 12 March 2023 Revised: 6 December 2023 Accepted: 12 December 2023 H.M. Kilian et al. 2 Fig. 1 Study Procedure. Socio-affective and socio-cognitive skills were assessed in a sample of 12 patients with TRD (5 females) before and after three months of slMFB DBS (gray boxes) and compared to social skills of 12 age- and gender-matched healthy control subjects (HC). investigating ToM in depression have been inconsistent [20, 28]. Meta-analyses have yielded impaired ToM skills [32, 33], while recent single studies revealed intact ToM skills either from self- report questionnaires or assessed with new naturalistic paradigms [25, 26, 30]. Considering the role social skill deﬁcits play in the development, maintenance and re-occurrence of depressive symptoms [34–36], improving these deﬁcits represents an important outcome in the antidepressant treatment. Procedure P i Patients with TRD were tested two to four weeks before stereotactic surgery was performed (see Fig. 1). Stereotactic surgery contains the bilateral implantation of DBS electrodes in the selected brain target (slMFB) under local anesthesia as well as the implantation of the pulse generator in the region of the clavicular under general anesthesia (for a detailed description of the surgery procedure see [59]). The slMFB has been proposed as DBS target in depression considering its central location, interconnections with other DBS targets in depression (e.g. ventral striatum), and its association with reward and motivation seeking behavior [9, 10]. Follow-up data of patients with TRD were analyzed three months after the stimulation onset of slMFB DBS (active DBS). Patients were asked not to change pharmacotherapy or psy- chotherapy during the study trial. The assessment in the healthy subject sample was repeated three to four months after the ﬁrst measurement (without intervention). This study was registered at the ‘Deutsches Register Klinischer Studien (DRKS)‘ (identiﬁer DRKS00019092). Patients were recruited from the ongoing FORESEE III trial (clinicaltrials.gov with identiﬁer: NCT03653858). The authors assert that all procedures contributing to this work comply with the ethical standards of the relevant national and institutional committees on human experimentation (afﬁrmative vote of the University of Freiburgs’s Ethics Committee on 12/21/ 2017) and with the Helsinki Declaration of 1975, as revised in 2008. Written informed consents were signed by all participants before study participation. INTRODUCTION The EmpaToM has previously been validated using an established empathy task for behavioural outcomes as well as on a neuronal level by comparison of activation clusters with previous ﬁndings of meta-analyses [24]. Furthermore, this paradigm has been shown to signiﬁcantly differentiate between affective empathy, compassion and ToM in studies with different (patient) samples [53–55]. Patients with TRD (n = 12) performed the EmpaToM both before the neurosurgical procedure with implantation of the DBS system and three months after the onset of active slMFB DBS. These data were then compared to an age- and gender-matched sample of healthy control subjects (HC) (n = 12). Based on reports of subjectively improved social functioning after slMFB DBS [26] and the neuronal overlap of regions stimulated by slMFB DBS and associated with social skills, we hypothesized that DBS normalizes impaired social skills in patients with TRD. MATERIALS, SUBJECTS AND METHODS Sample description and recruitment p p Patients were recruited through the outpatient clinic of the Division of Interventional Biological Psychiatry, Department of Psychiatry and Psychotherapy, Medical Center, University of Freiburg. Inclusion criteria were a primary diagnosis of major depressive disorder, a current chronic episode ( > two years) or at least four previous episodes of depression, a minimum score of 21 of the Hamilton Depression Rating Scale (HDRS) [56] and a score of less than 45 in the Global Assessment of Functioning (GAF) [57]. All patients included were diagnosed with unipolar depression. Treatment-resistant depression (TRD) was deﬁned as a lacking or inadequate response to all these treatments: (1) three different classes of antidepressants, (2) augmentation/combination therapy of primary antidepressants with other agents, (3) electroconvulsive therapy ( > 6 session) and (4) individual psychotherapy ( > 20 h). Adequacy of previous treatments was assessed with the Antidepressive Treatment History Form (ATHF) [58]. Furthermore, patients with a diagnosis of non-affective psychotic disorder, neurological disorder or medical illness affecting brain function, current or unstably remitted substance abuse, severe personality disorder and acute suicidal ideation were excluded (for a detailed Measures In terms of social skills, patients with TRD experienced reduced affective responsiveness (U = 26, z = −2.66, p = 0.01, r = 0.59) and generally reduced feelings of compassion (U = 37, z = −2.02, p = 0.04, r = 0.45) but intact ToM (U = 70.5, z = −0.09, p = 0.93, r = 0.02) at baseline compared to HC (see Supplementary Table S2). For more information about EmpaToM test data at baseline, see additional analyses in the supplement (Supplement 1). Social skills. The EmpaToM [24] is a video-based naturalistic test paradigm. In total, 48 short videos ( ~ 15 s) are presented that display people talking about a situation with either neutral or emotional (negative) valence (24 videos per condition) (for exemplary video stories see [24]). While neutral videos represent the control condition, videos with emotionally negative content represent the experimental condition. To measure affective empathy, participants are asked to rate their own current feelings (‚How do you feel?‘) on a dimensional scale ranging from ‚negative‘ (-2) to ‚positive‘ (2) after each video. The affective responsiveness is calculated as a difference score of empathic responses to emotional and neutral videos describing the ability to affectively resonate with others in response to different situations. For compassion, another question (‚How much compassion do you feel?‘) has to be answered on a dimensional scale ranging from ‚none‘ (0) to ‚very much‘ (6). Theory of mind is assessed by a multiple choice question referring to the thoughts of the person in the video (e.g. ‚Anna thinks that…‘). Participants have to select one out of three options and the accuracy (correct answers/total number of videos; min = 0, max = 1) is calculated. To control for attention and concentration abilities, half of the multiple choice questions demand factual reasoning skills (‚It is correct that…‘). The test thus comprises four conditions (12 trials per condition) with two video categories (neutral and emotional) and two task categories (ToM and non-ToM) (1: neutral, non-ToM; 2: emotional, non-ToM; 3: neutral, ToM; 4: emotional, ToM) (for a detailed description see [24]). For the main analyses, we combined the four categories so that only neutral and emotional or ToM and non-ToM were compared. The videos are presented in a different order for each participant and parallelized test versions presenting new videos were utilized for the follow-up assessment. Changes of social skills following DBS onset g g To analyze the effects following three months of active slMFB DBS regarding social skills, the difference scores from baseline to follow-up assessment were compared between TRD patients (n = 12) and HC (n = 12) using non-parametric Mann-Whitney U tests (Table 2). Signiﬁcant effects of medium effect size occurred in the affective empathy domain, i.e. with regard to the affective responsiveness to emotional compared to neutral stimuli (TRD: M = 0.10, SD = 0.80; HC: M = −0.44, SD = 0.56; TRD vs. HC: U = 38, z = −1.96, p = 0.05, r = 0.44) (see Fig. 2A). Single comparisons revealed that the affective responsiveness signiﬁcantly differed between HC and patients with TRD at baseline (U = 26, z = −2.66, p =0.01, r = 0.59) but not at the follow-up assessement (U = 54, z = −1.04, p = 0.30, r = 0.23) indicating a normalized affective responsiveness (see Fig. 2B). This effect was mainly driven by changes from baseline to follow-up in the neutral condition indicating a reduction of the depression-associated negativity bias in patients with TRD (TRD: M = 0.21, SD = 0.40; HC: M = −0.18, SD = 0.32; TRD vs. HC: U = 28, z = −2.54, p = 0.01, r = 0.57) (Supplementary Fig. S1). To determine any equivalence in affective responsiveness at follow-up between the two groups, we conducted equivalence testing. As those results failed to reach Statistical analysis. Demographic and clinical characteristics are only displayed descriptively as the main study (FORESEE III) is still ongoing. Difference scores from baseline to follow-up were calculated for affective empathy, compassion, and ToM separately for each group (patients with TRD and HC). Difference scores (baseline-follow- up) were then compared between groups via non-parametric Mann- Whitney U tests for two independent samples. Additionally, effect sizes “r” were calculated with r < 0.3 representing small, r < 0.5 medium and r > 0.5 strong effects [63]. We conducted non- parametric tests as the assumptions for parametric tests were not given for all variables of interest (tested using Kolmogorov-Smirnov tests for normal distribution and Levene’s test for homogeneity of variances) and considering our sample’s small size. We also ran equivalence tests [64, 65] to examine the practical similarity of affective responsiveness at follow-up between TRD patients and HC. Changes of social skills following DBS onset We set the smallest effect size of interest to a large effect, with bounds of d = −0.80 (lower) and d = 0.80 (upper), and conducted a one-sided test procedure via Welch’s tests for two independent samples [66]. To analyze reliability scores, non-parametric correlation analyses of test and re-test data were calculated exclusively in the HC sample (see Supplementary Table S1). Data were analyzed using MATLAB and IBM SPSS Statistics 20. For all statistical comparisons, p- values ≤0.05 were considered signiﬁcant (two-tailed). Table 1. Demographic and clinical characteristics. Baseline Follow-Up TRD N (male/female) 12 (5/7) Age (mean in years) (SD) 44.08 (8.08) Verbal Intelligence* (SD) 112.33 (14.64) 114.33 (14.62) HDRS (sum) (SD) 28.00 (4.26) 19.08 (8.89) MADRS (sum) (SD) 33.08 (5.96) 22.50 (10.54) BDI-II (sum) (SD) 38.25 (6.54) 26.83 (12.32) HC N (male/female) 12 (5/7) Age (mean in years) (SD) 45.33 (9.26) Verbal Intelligence* (SD) 114.67 (12.77) 123.33 (10.25) HDRS (sum) (SD) 0.83 (0.84) 0.42 (0.67) MADRS (sum) (SD) 0.67 (0.78) 0.42 (0.52) BDI-II (sum) (SD) 0.75 (1.36) 1.17 (2.37) TRD Treatment-resistant depression, HC Healthy control subjects, SD Standard deviation, HDRS Hamilton Depression Rating Scale, MADRS Montgomery-Åsberg Depression Rating Scale, BDI-II Beck Depression Inventory II. assessed by the Multiple Choice Vocabulary Test [67]. Table 1. Demographic and clinical characteristics. Measures In the current study, time to respond is generally extended by two seconds compared to the original task because a small pilot trial with ﬁve psychiatric patients revealed increased response times in comparison to healthy samples. Measures Clinical symptoms. Severity of symptoms of depression was assessed using self-report (Beck Depression Inventory (BDI), Hautzinger et al. [60]) as well as expert-rating instruments (Montgomery-Åsberg Depression Rating Scale (MADRS), Montgom- ery & Åsberg [61]; Hamilton Depression Rating Scale (HDRS), Translational Psychiatry (2024) 14:6 H.M. Kilian et al. 3 Hamilton [56]). These scales have reached the status of a gold standard for the evaluation of symptoms of depression according to the diagnostic criteria [62]. MHC = 45.33, SDHC = 9.26; TRD vs. HC: U = 62, z = −0.58, p = 0.56, r = 0.13) (Table 1). Furthermore, both groups were comparable with regard to a measure linked to verbal intelligence, namely the multiple choice vocabulary test (MCVT) [67] (TRD: M = 112.33, SD = 14.64; HC: M = 114.67, SD = 12.77; TRD vs. HC: U = 62.5, z = −0.55, p = 0.58, r = 0.12). Descriptively, severity of depression assessed with MADRS, HDRS and BDI decreased in the TRD sample after the onset of DBS (MADRS: M = −10.58, SD = 9.92; HDRS: M = −8.92, SD = 8.79; BDI: M = −11.42, SD = 12.06) (Table 1). In terms of social skills, patients with TRD experienced reduced affective responsiveness (U = 26, z = −2.66, p = 0.01, r = 0.59) and generally reduced feelings of compassion (U = 37, z = −2.02, p = 0.04, r = 0.45) but intact ToM (U = 70.5, z = −0.09, p = 0.93, r = 0.02) at baseline compared to HC (see Supplementary Table S2). For more information about EmpaToM test data at baseline, see additional analyses in the supplement (Supplement 1). MHC = 45.33, SDHC = 9.26; TRD vs. HC: U = 62, z = −0.58, p = 0.56, r = 0.13) (Table 1). Furthermore, both groups were comparable with regard to a measure linked to verbal intelligence, namely the multiple choice vocabulary test (MCVT) [67] (TRD: M = 112.33, SD = 14.64; HC: M = 114.67, SD = 12.77; TRD vs. HC: U = 62.5, z = −0.55, p = 0.58, r = 0.12). Descriptively, severity of depression assessed with MADRS, HDRS and BDI decreased in the TRD sample after the onset of DBS (MADRS: M = −10.58, SD = 9.92; HDRS: M = −8.92, SD = 8.79; BDI: M = −11.42, SD = 12.06) (Table 1). RESULTS TRD Treatment-resistant depression, HC Healthy control subjects, SD Standard deviation, HDRS Hamilton Depression Rating Scale, MADRS Montgomery-Åsberg Depression Rating Scale, BDI-II Beck Depression Inventory II. assessed by the Multiple Choice Vocabulary Test [67]. Sample description Sample description As both groups were matched, gender distribution (male = 7, female = 5) and age were comparable (MTRD = 44.08, SDTRD = 8.08; Translational Psychiatry (2024) 14:6 H.M. Kilian et al. H.M. Kilian et al. 4 Table 2. Group differences of changes from baseline to follow-up (difference scores follow-up - baseline). TRD (n = 12) HC (n = 12) Mann-Whitney U Test Mean (SD) Mean (SD) U, z, p, r Affective Empathy Affective responsiveness 0.10 (0.80) −0.44 (0.56) U = 38, z = −1.96, p = 0.05, r = 0.44 Neutral 0.21 (0.40) −0.18 (0.32) U = 28, z = −2.54, p = 0.01, r = 0.57 Emotional −0.11 (0.52) −0.25 (0.31) U = 49.5, z = −1.30, p = 0.19, r = 0.29 Compassion Both conditions −0.26 (0.74) −0.01 (0.65) U = 63, z = −0.52, p = 0.60, r = 0.12 Neutral 0.01 (0.64) −0.02 (0.96) U = 67.5, z = −0.26, p = 0.80, r = 0.06 Emotional −0.53 (1.13) 0.01 (0.49) U = 53, z = −1.10, p = 0.27, r = 0.25 Theory of mind Theory of Mind 0.04 (0.05) 0.02 (0.13) U = 64, z = −0.47, p = 0.64, r = 0.11 Factual Reasoning −0.02 (0.12) −0.05 (0.15) U = 67.5, z = −0.26, p = 0.79, r = 0.06 TRD Treatment-resistant depression, HC Healthy control subjects, SD Standard deviation. Table 2. Group differences of changes from baseline to follow-up (difference scores follow-up - baseline). Fig. 2 Effects on affective empathy following DBS. Shown are mean scores of differences in affect rating between negative and neutral situations (i.e. affective responsiveness) in patients with treatment-resistant depression (TRD, n = 12; in grey) and healthy control subjects (HC, n = 12; in white). Error bars represent 95% conﬁdence intervals. Asteriks indicate a statistically signiﬁcant difference (p ≤0.05, two-sided). Small dots represent individual data points. A Change of affective responsiveness from baseline to follow-up (difference follow-up – baseline) differed signiﬁcantly between TRD patients in comparison to HC (p = 0.05). B Affective responsiveness scores separately displayed for baseline (left side) and follow-up (right side). At baseline, patients with TRD experienced signiﬁcantly reduced affective responsiveness compared to HC. At follow-up no difference between the groups was found indicating a normalized affective responsiveness in patients with TRD after three months of active slMFB DBS. n.s. not signiﬁcant. Fig. 2 Effects on affective empathy following DBS. Sample description Shown are mean scores of differences in affect rating between negative and neutral situations (i.e. affective responsiveness) in patients with treatment-resistant depression (TRD, n = 12; in grey) and healthy control subjects (HC, n = 12; in white). Error bars represent 95% conﬁdence intervals. Asteriks indicate a statistically signiﬁcant difference (p ≤0.05, two-sided). Small dots represent individual data points. A Change of affective responsiveness from baseline to follow-up (difference follow-up – baseline) differed signiﬁcantly between TRD patients in comparison to HC (p = 0.05). B Affective responsiveness scores separately displayed for baseline (left side) and follow-up (right side). At baseline, patients with TRD experienced signiﬁcantly reduced affective responsiveness compared to HC. At follow-up no difference between the groups was found indicating a normalized affective responsiveness in patients with TRD after three months of active slMFB DBS. n.s. not signiﬁcant. statistical signiﬁcance (T (15,17) = −1.04, p = 0.32), our data provide insufﬁcient evidence to assume similar affective respon- siveness at follow-up. Taken together, these ﬁndings add to the evidence of speciﬁc effects following slMFB DBS in the domain of affective empathy in terms of normalized affective responsiveness. No effects regarding other social skills (compassion, theory of mind) were revealed in the course of slMFB DBS (all p ≥0.27; for details see Table 2 and Supplementary Table S3). slMFB DBS. Deﬁcits in compassion remained unchanged and socio-cognitive skills remained intact in the TRD sample. g p By behaviorally assessing social skills in the course of slMFB DBS treatment using a naturalistic paradigm, this study contributes to a better understanding of DBS’s effects on social functioning. Three months following the onset of slMFB DBS (follow-up), preopera- tively reduced affective responsiveness (baseline) was normalized in patients with TRD. Normalized affective responsiveness follow- ing slMFB DBS onset could represent one factor facilitating the social re-integration of these chronically ill patients [17, 20]. The increased negative affect towards neutral stimuli at baseline (e.g. depression-associated negativity bias) was signiﬁcantly weaker (strong effect size) following slMFB DBS in patients with TRD compared to HC [23, 68]. This ﬁnding is in line with a previous study demonstrating a reduced negativity bias six months after the onset of DBS of the subcallosal cingulate gyrus (SCG) in nine patients with TRD [69]. Considering that the SCG and slMFB are part of the same reward-network and that the SCG is anatomically and functionally coupled with regions connected with the medial Translational Psychiatry (2024) 14:6 REFERENCES 1. Rush AJ, Trivedi MH, Wisniewski SR, Nierenberg AA, Stewart JW, Warden D, et al. Acute and longer-term outcomes in depressed outpatients requiring one or several treatment steps: a STAR* D report. Am J Psychiatry. 2006;163:1905–17. 1. Rush AJ, Trivedi MH, Wisniewski SR, Nierenberg AA, Stewart JW, Warden D, et al. Acute and longer-term outcomes in depressed outpatients requiring one or several treatment steps: a STAR* D report. Am J Psychiatry. 2006;163:1905–17. 2. Rathod S, Denee T, Eva J, Kerr C, Jacobsen N, Desai M, et al. 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A longitudinal study on deep brain stimulation of the medial forebrain bundle for treatment-resistant depression. Transl Psychiatry. 2018;8:1–11. Altogether, the key strengths of the current study are the recruitment of a unique patient sample, and the differentiated assessment of social skills following slMFB DBS by means of a naturalistic paradigm. Alongside these strengths, our study has also limitations. The patient sample of the current study is small due to the experimental status of slMFB DBS for patients with TRD in Germany. The current study thus does not allow to differentiate between DBS treatment responders and non-responders as well as 16. Kilian HM, Bewernick BH, Klein M, Meyer DM, Spanier S, Reinacher PC, et al. Deep Brain Stimulation for Major Depression and Obsessive-Compulsive Disorder— Discontinuation of Ongoing Stimulation. Psych. 2020;2:174–85. 17. Hirschfeld R, Montgomery SA, Keller MB, Kasper S, Schatzberg AF, Möller H-J, et al. Social functioning in depression: a review. J Clin Psychiatry. 2000;61:268–75. 18. Schiller B, Tönsing D, Kleinert T, Böhm R, Heinrichs M. Effects of the COVID-19 pandemic nationwide lockdown on mental health, environmental concern, and prejudice against other social groups. DATA AVAILABILITY Data are available on reasonable request. Data are available on reasonable request In contrast to affective empathy, we observed no effects following DBS on (preoperatively impaired) compassion in the TRD sample. This ﬁnding is unexpected taking into account that the slMFB is directly interconnected with the ventral striatum [41, 42], a region that has been linked to compassion [24, 73]. Although slMFB DBS is known to have rapid antidepressant effects [13, 14], the follow-up period of three months might have been too short to demonstrate effects of DBS altering compassion. Considering that affective empathy represents the basis for compassion [27, 74], feelings of compassion might only improve in the longer-term outcome subsequent to normalized affective responsiveness. Considering the crucial role of compassion in social functioning [75], it could turn out to accelerate these effects by augmenting DBS’s effects on compassion-related brain regions with speciﬁc compassion training. To date, there is no study investigating the effects of a social skills training on the antidepressant efﬁcacy of DBS in depression. However, the value of combining DBS treatment with psychother- apy has already been established regarding other mental disorders (e.g. obsessive-compuslive disorder [76]). Therefore, it could prove worthwile to combine DBS therapy in TRD with speciﬁc trainings targeting social skill deﬁcits. Supporting the potential of such an approach, compassion training successfully increased feelings of compassion in healthy participants accompanied by increased brain activations in the medial prefrontal cortex [77] and the ventral striatum [73]. DISCUSSION Nevertheless, considering that our equivalence tests revealed a non-signiﬁcant result of affective responsiveness at follow-up, we cannot conclude that patients with TRD under- going DBS perform as well as the HC group regarding social skills. to compare the effects of active and sham stimulation. Future studies are necessary to test the long-term stability of the demonstrated effects with the stimulation turned on and turned off. This is highly relevant given the fact that a discontinuation of stimulation is associated with a relapse of symptoms [16] as well as a reduction of quality of life [84]. y In sum, our research demonstrated speciﬁc effects following slMFB DBS onset in depression in terms of a normalized affective responsiveness. This effect might facilitate the resumption of social activities after recovery from chronic depression thereby contributing to a stable long-term antidepressant response to DBS. Nevertheless, deﬁcits in compassion persisted. Thus, our data support the idea to combine DBS with speciﬁc psychotherapeutic interventions for full recovery. Translational Psychiatry (2024) 14:6 DISCUSSION This study systematically investigated speciﬁc social skills (affec- tive empathy, compassion, and theory of mind (ToM)) in patients with treatment-resistant depression (TRD) before and three months after the onset of deep brain stimulation (DBS) of the supero-lateral medial forebrain bundle (slMFB). Active DBS of the slMFB resulted in a normalized affective responsiveness towards emotionally negative versus neutral stimuli in patients with TRD. None of the other social skills was signiﬁcantly altered following Translational Psychiatry (2024) 14:6 H.M. Kilian et al. 5 forebrain bundle [9], our data together with this previous study imply a network-speciﬁc effect of DBS in depression. Given the importance to reverse the depression-associated negativity bias for a successful antidepressant treatment [20, 70, 71], this effect might play a signiﬁcant role in DBS’s antidepressant effects. Furthermore, our results appear to be promising with regard to the social skill deﬁcits hypothesis [72], according to which persisting social skill deﬁcits in patients with depression contribute decisively to both relapsing into depression and to chronic depressive symptoms due to the loss of positive reinforcement during social interactions. Thus, the ﬁnding of normalized affective responsiveness in the course of DBS treatment might reduce the probability of a relapse into depression and thereby contribute to a stable, long-term antidepressant effect by enabling positive social interactions. Nevertheless, considering that our equivalence tests revealed a non-signiﬁcant result of affective responsiveness at follow-up, we cannot conclude that patients with TRD under- going DBS perform as well as the HC group regarding social skills. I ff i h b d ff f ll i forebrain bundle [9], our data together with this previous study imply a network-speciﬁc effect of DBS in depression. 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https://openalex.org/W3217646951	https://www.frontiersin.org/articles/10.3389/fcosc.2021.766086/pdf	English	null	A Need for Context-Based Conservation: Incorporating Local Knowledge to Mitigate Livestock Predation by Large Carnivores	Frontiers in conservation science	2,021	cc-by	8,414	COMMUNITY CASE STUDY published: 25 November 2021 doi: 10.3389/fcosc.2021.766086 A Need for Context-Based Conservation: Incorporating Local Knowledge to Mitigate Livestock Predation by Large Carnivores Ajay Bijoor 1,2, Munib Khanyari 1,3,4, Rigzen Dorjay 1, Sherab Lobzang 1 and Kulbhushansingh Suryawanshi 1,2,5 1 Nature Conservation Foundation, Mysore, India, 2 Snow Leopard Trust, Seattle, WA, United States, 3 Department of Biological Sciences, University of Bristol, Bristol, United Kingdom, 4 Department of Zoology, Interdisciplinary Centre for Conservation Sciences, Oxford, United Kingdom, 5 Wissenschaftskolleg zu Berlin, Institude for Advanced Studies, Berlin, Germany Mitigating livestock predation by carnivores is crucial to ensure carnivore conservation and facilitate human-carnivore coexistence. Mitigation measures proposed by conservation agencies, however, are often technocratic and perceived as being an external imposition on the local community. Herders affected by the depredation may have the knowledge to design locally relevant solutions, but they might lack ﬁnancial and technical support to implement these effectively. Their inability to act can result in the communities being viewed as antagonistic rather than a part of the solution. We present a case study on co-development of a conservation intervention by a traditional pastoral community together with a conservation NGO, to mitigate livestock depredation inside night-time corrals in Ladakh, India. Between January and June 2020, livestock corrals in Sumdoo TR village were attacked 10 times by carnivores such as snow leopards and wolves, killing over 100 sheep, goat, yak, and horses and causing loses of over 10,400 USD. Local people were agitated, and there were strong demands for capture or removal of the carnivores from the area. We operationalized the PARTNERS (Presence, Aptness, Respect, Transparency, Negotiation, Empathy, Responsiveness, and Strategic Support) principles framework for community-based conservation to help the village effectively implement an intervention based on a novel predator-proof corral design conceptualized by the community. We demonstrate that empowering the community to design and implement a conservation intervention helped them take ownership of the effort, improve trust with conservation agencies, and hence likely to be a long-term solution to conservation conﬂicts in the region. Our approach of using the PARTNERS principles has relevance for conservation agencies who are trying to implement interventions, particularly those geared toward reducing livestock depredation by carnivores. Our approach further helps communities to view themselves as part of the solution and not the problem. Keywords: community-based conservation, coexistence, co-development, interventions, herders, mitigation, carnivores Edited by: Sahil Nijhawan, University College London, United Kingdom Reviewed by: Sathyakumar Sambandam, Wildlife Institute of India, India Phuntsho Thinley, University of New England, Australia Correspondence: Munib Khanyari munib@ncf-india.org orcid.org/0000-0003-4624-5073 Reviewed by: Sathyakumar Sambandam, Wildlife Institute of India, India Phuntsho Thinley, University of New England, Australia *Correspondence: Munib Khanyari munib@ncf-india.org orcid.org/0000-0003-4624-5073 Specialty section: This article was submitted to Human-Wildlife Dynamics, a section of the journal Frontiers in Conservation Science Received: 28 August 2021 Accepted: 08 November 2021 Published: 25 November 2021 INTRODUCTION are co-developed by aﬀected communities as equal partners with conservation agencies (e.g., Govt., NGOs). This is particularly important and relevant in areas of the world where challenges of poverty, weak institutions, and poor governance exist simultaneously, further limiting the opportunity of local communities to participate in biodiversity conservation. Livestock depredation by carnivores is a conservation concern globally. It can cause severe economic and emotional trauma for livestock owners, and retaliatory killing of carnivores (Woodroﬀe et al., 2005; Barua et al., 2013). Mitigating livestock losses is crucial for conserving large carnivores and facilitating coexistence with people (Treves and Karanth, 2003). The costs of coexistence, however, are often borne locally by the communities co-inhabiting areas with wildlife. Exclusionary conservation approaches like protected area-based approaches have often come with signiﬁcant social cost and conﬂict. This has caused a further alienation of local communities turning potential conservation allies into adversaries (Lele et al., 2010). Although numerous measures have been developed to prevent livestock predation, the decision on which measure to adopt is often taken by the government or conservation agencies with little or no consultation with the aﬀected community. Inputs on technical feasibility and local relevance of such measures are seldom sought from local stakeholders, and their eﬃcacy is rarely measured. Livestock herders may be able to design locally relevant solutions, but often lack ﬁnancial and technical support to implement them eﬀectively. This relegates aﬀected pastoral communities to appear as part of the problem and not the solution. p p y We aim to highlight the approach to implementing a conservation intervention to mitigate livestock depredation inside night-time corrals of a traditional pastoral community in a remote area of Ladakh, India. Here, we present a case study on how a predator-proof corral was co-developed and co-designed with local communities. We present the case study in light of the PARTNERS (Presence, Aptness, Respect, Transparency, Negotiation, Empathy, Responsiveness, and Strategic Support) principles for community-based conservation developed by Mishra (2016) through 20 years of experience across the snow leopard Panthera uncia habitats of Central and South Asia. The eight PARTNERS principles for community-based conservation build on the ideas that have been developed in diverse ﬁelds such as applied ecology, natural resource management, health, social psychology, rural development, negotiation theory, and ethics. Please see Mishra et al. (2017) for more details of the PARTNERS principles which are summarized in Table 1. INTRODUCTION For ease of understanding the principles in a nutshell, they are as follows. Presence alludes to the immersion of conservation practitioners to better understand the social-ecological context of a community. Aptness encourages practitioners to identify locally relevant interventions. Respect urges establishment of equal partnerships with local community. Transparency highlights the importance of establishing an honest decision- making partnership with the community. Negotiation cautions against taking extreme positional or either-or stances in conﬂict mitigation. Empathy reminds practitioners that conservation and conﬂict mitigation is often one of many realities of the community. Responsiveness emphasizes that timely responses to events are crucial. Lastly, strategic support illustrates the importance of formalizing conservation interventions by working using a multi-sectoral approach, including with relevant government agencies. Several non-lethal techniques are used by herders and government agencies to minimize livestock depredation by carnivores in livestock grazing pastures (Breitenmoser et al., 2005). These include use of guard dogs and avoidance of grazing in conﬂict hotspots (Breitenmoser et al., 2005). Visual repellents (e.g., ﬂadry, fox lights, strobe lights) and acoustic repellents (sirens) are also deployed (e.g., Shivik et al., 2003). However, the issue of livestock predation inside night-time pens is more serious because attacks in these pens often lead to surplus killing of livestock i.e., when carnivores kill several tens of livestock even when they cannot eat them all (Kruuk, 1972). Such instances have a much bigger negative impact on herders than predation of a few livestock in the pastures. Interventions to protect livestock inside night-time corrals include fencing, ranging from building basic stone wall fences to those reinforced with electric fencing or netted fences (e.g., Samelius et al., 2020). While fencing is an eﬀective intervention, there are challenges and limitations including the high cost, labor, and technical knowledge that is necessary for installation and maintenance (Kioko et al., 2008). Maintenance of such installations proves challenging without sustained technical support or training imparted to those using the fences. Additionally, the eﬀectiveness of such interventions is rarely tested (Samelius et al., 2020). Collectively, local acceptance of fencing as a means to reduce depredation by carnivores can prove challenging if the aﬀected herders cannot eﬀectively manage the setup post installation. This can quickly lead to a perception that the intervention is ineﬀective. Our case-study demonstrates how these principles can be used on the ground to co-develop conservation interventions together with local communities to minimize livestock predation by large carnivores. Citation: Bijoor A, Khanyari M, Dorjay R, Lobzang S and Suryawanshi K (2021) A Need for Context-Based Conservation: Incorporating Local Knowledge to Mitigate Livestock Predation by Large Carnivores. Front. Conserv. Sci. 2:766086. doi: 10.3389/fcosc.2021.766086 November 2021 \| Volume 2 \| Article 766086 Frontiers in Conservation Science \| www.frontiersin.org Mitigating Livestock Predation by Carnivores Bijoor et al. Frontiers in Conservation Science \| www.frontiersin.org INTRODUCTION This approach has relevance for conservation agencies across the world who are trying to work together with local communities to implement interventions to reduce livestock predation by carnivores. A Case Study From the Western Indian Trans-Himalaya The practical challenges of achieving eﬀective community engagement are considerable (Waylen et al., 2010) and are fraught with diﬃculties and ethical considerations (Chan et al., 2007). Often the opportunities and challenges of implementing eﬀective community engagement are seldom discussed. There is a need for more case studies where conservation interventions Across the mountain ranges of Central and South Asia, livestock depredation by snow leopards P. uncia, wolves Canis lupus and Lynx Lynx lynx is a concern for the local pastoralists (Jackson and Wangchuk, 2004; Mishra et al., 2017; Samelius et al., 2020). Spread over c. 17,000 km2, the Changthang region November 2021 \| Volume 2 \| Article 766086 2 Mitigating Livestock Predation by Carnivores Bijoor et al. TABLE 1 \| The eight PARTNERS principles for effective implementation of community-based programs as deﬁned by Mishra et al. (2017). Principle Description Presence This principle highlights the importance of immersion by practitioners to gain a nuanced understanding of the community and their way of life in order to build a resilient relationship. Aptness Aptness centers on ensuring that community-based conservation programs are relevant and sensitive to the local context. This principle urges practitioners to be mindful when scaling up and encourages practitioners to understand the local threats to the species or ecosystem of interest, the ecology of the area, socio-cultural acceptance, the scientiﬁc basis of the proposed conservation interventions, the social-economic situation of the community, and its culture and value orientations. Respect It is easy for conservation practitioners to fall into the trap of viewing local communities as recipients of aid and themselves as providers. This principle guards practitioners against such a pitfall while fostering partnerships with the local communities. Transparency This principle implies disclosure of one’s goals and purpose. It is the conservationists’ responsibility to clearly outline the shared conservation objectives, norms and interventions, the roles/responsibilities of all involved, and, the rationale behind choices and their potential effects—including any uncertainties. Negotiations Every community-based engagement requires negotiations to arrive at a joint agreement. The principle of negotiation encourages conservation practitioners to embrace an integrative approach to negotiation that is grounded in shared information and interests, use of objective standards, incentive building, and tangible stakes in the conservation interventions. Empathy This principle encourages practitioners to understand conservation from the perspective of local people. What may seem critical to practitioners may seem trivial to the local communities. Thus, empathizing is to understand the local context. Study Area h ll f of Eastern Ladakh is a high altitude rangeland that is inhabited by the Changpa people who practice nomadic pastoralism. This sparsely populated region is home to 22 pastoral villages. Between January and June 2020, 24 instances of small and large livestock depredation by predators (snow leopard, wolves and lynx) were recorded from this region, and 14 of these were incidents of surplus killing inside night-time corrals i.e., when carnivores kill a great many more animals than can possibly be consumed at the time. A single herding community in Sumdoo Tibetan Refugees (TR), consisting of 68 herders (Figure 1), reported 12 instances of livestock predation, and 10 of these were instances of surplus killing inside night-time corrals (Supplementary Material 1). A total of 102 small-bodied livestock (sheep and goat), 11 yak and two horses were killed in these incidents amounting to a ﬁnancial loss of c. INR 0.7 million (USD 10,400). Unsurprisingly, the villagers of Sumdoo TR demanded the capture and removal of these carnivores by the Department of Wildlife Protection. A common preventive intervention to this problem is the reinforcement of vulnerable night-time corrals to make them predator-proof. While this intervention has been occurring in Ladakh for over a decade, facilitated by the Department of Wildlife Protection, Sheep Husbandry Department, and various conservation organizations (e.g., Jackson and Wangchuk, 2004; Maheshwari and Sathyakumar, 2019, 2020; Bhatia et al., 2021), our primary aim is to highlight how conservation interventions can be done collaboratively with communities; as often local communities are recipient of interventions rather than being equitable partners in developing and implementing them. The village of Sumdoo TR (Figure 1) is located in the Changthang region of Ladakh with 68 predominantly pastoral households. The topography is primarily characterized by undulating terrain interspaced with rugged regions with elevation ranging from 4,000 to 6,000 m. This region is characterized by extreme cold and frigid winters, high aridity, and strong winds. Owing to the relatively low temperatures and low precipitation, the primary productivity is low as well (Rawat and Adhikari, 2005). The growing seasons is restricted to a few months in the summer (June–August) and the vegetation is characterized as dry alpine steppe. The large mammals of the area include Blue sheep Pseudois nayaur, Tibetan Wild Ass Equus kiang, snow leopards, wolves, and lynx. The people of Sumdoo TR are ethnically Tibetan. A Case Study From the Western Indian Trans-Himalaya It encourages that practitioners consider both rational and emotional aspects into decision making. Responsiveness Given the dynamism of social-ecological systems, this principle reiterates the need to be responsive to changing threats to biodiversity, to changes within communities, and to the need for addressing any shortcomings in conservation interventions. It also underscores the importance of setting up mechanisms to monitor and periodically evaluate conservation interventions. Strategic support Governments are often a key stakeholder in conservation decision-making and interventions. This principle highlights the importance of strategic government support to local communities. This can be through policy reforms, management planning and implementation of interventions with proactive involvement of conservation practitioners, and legal support. Where applicable, we have highlighted the use of these principles in our case study in brackets. TABLE 1 \| The eight PARTNERS principles for effective implementation of community-based programs as deﬁned by Mishra et al. (2017). This principle highlights the importance of immersion by practitioners to gain a nuanced understanding of the community and their way of life in order to build a resilient relationship. Aptness centers on ensuring that community-based conservation programs are relevant and sensitive to the local context. This principle urges practitioners to be mindful when scaling up and encourages practitioners to understand the local threats to the species or ecosystem of interest, the ecology of the area, socio-cultural acceptance, the scientiﬁc basis of the proposed conservation interventions, the social-economic situation of the community, and its culture and value orientations. t is easy for conservation practitioners to fall into the trap of viewing local communities as recipients of aid and themselves as providers. This principle guards practitioners against such a pitfall while fostering partnerships with the local communities. This principle implies disclosure of one’s goals and purpose. It is the conservationists’ responsibility to clearly outline the shared conservation objectives, norms and interventions, the roles/responsibilities of all involved, and, the rationale behind choices and their potential effects—including any uncertainties. Every community-based engagement requires negotiations to arrive at a joint agreement. The principle of negotiation encourages conservation practitioners to embrace an integrative approach to negotiation that is grounded in shared information and interests, use of objective standards, incentive building, and tangible stakes in the conservation interventions. This principle encourages practitioners to understand conservation from the perspective of local people. What may seem critical to practitioners may seem trivial to the local communities. A Case Study From the Western Indian Trans-Himalaya Thus, empathizing is to understand the local context. It encourages that practitioners consider both rational and emotional aspects into decision making. Given the dynamism of social-ecological systems, this principle reiterates the need to be responsive to changing threats to biodiversity, to changes within communities, and to the need for addressing any shortcomings in conservation interventions. It also underscores the importance of setting up mechanisms to monitor and periodically evaluate conservation interventions. Governments are often a key stakeholder in conservation decision-making and interventions. This principle highlights the importance of strategic government support to local communities. This can be through policy reforms, management planning and implementation of interventions with proactive involvement of conservation practitioners, and legal support. Where applicable, we have highlighted the use of these principles in our case study in brackets. Study Area h ll f At large, the 68 households are of similar socio-economic background, albeit with some variation which is reﬂected predominantly in the type of livestock owned. Usually households with smaller holdings were socio-economically worse-oﬀrelative to those with larger holdings. While largely pastoralists, most households engage in agriculture which is primarily for livestock fodder during winter.. Unlike other regions in Changthang like Korzok and Hanle, tourism isn’t a mainstay of the people of Sumdoo TR. Livestock herds in Sumdoo TR are managed at the household level and primarily comprise of Changluk sheep and Changra goat. The Changluk sheep, a breed indigenous to Changthang, is predominantly used for meat, while the Changra goat yields the pashmina/cashmere ﬁber that is sold to prospective buyers (Singh et al., 2013). The herders move through the year along with their November 2021 \| Volume 2 \| Article 766086 Frontiers in Conservation Science \| www.frontiersin.org 3 Mitigating Livestock Predation by Carnivores Bijoor et al. FIGURE 1 \| Map displaying the Eastern Ladakh region (primarily consisting of Gya-Miru, Rong valley, and Changthang) and the location of Sumdoo TR. FIGURE 1 \| Map displaying the Eastern Ladakh region (primarily consisting of Gya-Miru, Rong valley, and Changthang) and the location of Sumdoo TR. the Eastern Ladakh region (primarily consisting of Gya-Miru, Rong valley, and Changthang) and the location of Sumdoo TR. ﬂocks following a ﬁxed path, spending between a few weeks to a few months in the pastures that are communally accessed by the entire community. The sheep and goat are herded during the day and bought back to night-time corral. Other livestock such as yaks and horses are also kept. These are predominantly free-ranging and only bought back to the village for plowing in the case of yaks, riding and trekking in case of horses, or during severe winter conditions for both. as snow leopards and wolves, these are overwhelmingly limited compared to negative sentiments because of their tendency to prey on livestock (Bhatia et al., 2021). Consequently, herders often retaliate against carnivores to protect their livestock (Suryawanshi et al., 2013). The Department of Wildlife Protection in Ladakh is the primary government agency responsible for wildlife protection and conservation. They have primarily a legislative and enforcement role. However, administering all of Ladakh (c. 60,000 km2) with a small team, in diﬃcult ﬁeld conditions, and with limited resources, is challenging. Study Area h ll f NCF’s team comprised of conservation practitioners from Ladakh and other parts of India. Both the members (RD and SL) that worked directly with the local community are Ladakhi, while three non-local members (AB, MK, and KS) gave inputs and guided the process over a series of audio calls. Each of the local and non-local members have over 5 years of experience working across the Indian trans- Himalayas. NCF’s overall aim is to partake in socially responsible conservation which in our context includes facilitating positive- human relationships (Mishra et al., 2017). While the Department of Wildlife Protection and diﬀerent conservation agencies have engaged in various conservation interventions including predator-prooﬁng of corrals across Ladakh, from our knowledge no such engagement had previously occurred in Sumdoo TR from conservation agencies. The lack of previous conservation engagement along with prevalent negative interactions with predators was the primary reasons we chose to engage in Sumdoo TR. In June 2020, we (members of the NGO Nature Conservation Foundation—NCF led locally by RD and SL) met oﬃcers of the Department of Wildlife Protection and jointly decided to initiate work with the Sumdoo TR community (Strategic Support, Responsiveness; for emphasis, we have indicated the relevant PARTNERS principle being used). Positionality of Stakeholders Co-designing the Intervention d d ﬁ f Respect, Negotiation). An experienced NCF staﬀfrom Ladakh (RD) led the entire eﬀort thus building trust from the outset (Presence). Based on the estimated costs, in the ﬁfth meeting, we mutually agreed to start the eﬀort with the construction of seven corrals. The community helped decide which seven corrals would be re-built based on their perception of risk of depredation and their size. Led by the village headman and the corral committee, the villagers produced a list of most vulnerable corrals in the village. As corral sizes are linked to livestock number, which in turn reﬂects the socio-economic status of a household in Sumdoo TR, the village headman, and members of NCF negotiated with villagers to factor in corral sizes along with risk to predators in producing the vulnerable corral list (Negotiation). Corrals of diﬀerent sizes that were at high risk were chosen for predator-prooﬁng, so that this could prove a useful test case. In Sumdoo TR, each corral belongs to an individual family. Four of these corrals were built in or near the village in relatively ﬂat terrain, while three were built close to cliﬀs in rugged regions. These seven corrals housed 1,840 sheep/goat, which accounted for nearly 15% of the total livestock in the village. A written agreement was prepared in Ladakhi (the local dialect of Tibetan) between the community and NCF (Figure 2C) outlining timelines, milestones, and the responsibilities that each would, respectively, fulﬁll. NCF was responsible for providing fabricated materials and its transportation, while the herders were responsible for construction of the corrals (Figure 2B). If a selected corral owner did not follow timelines, the corral committee had identiﬁed backup corrals that would be supported through this pilot eﬀort. A copy of the agreement was kept with the community, while another copy was kept with NCF (Transparency). Herders highlighted that they bred Changra goats to produce pashmina (cashmere) wool. They listed speciﬁc design requirements for the corrals to ensure the health of their animals and wool production: (i) the chilly winter winds facilitate wool growth hence it was important for structures to allow breeze to circulate; (ii) since this region receives snowfall, it was critical that the structure prevented snow from collecting within the corral and allow the wind to blow it oﬀ. A logistical challenge was that this region has very few stones—a key resource that is necessary for constructing the walls of the coral. Respect, Negotiation). g In the fourth meeting, we held a round of discussion with the corral committee of the village to identify the optimum design for the corrals. It was evident that the design suggested by the herders was the most suitable while the other options would either prove suboptimal or diﬃcult to implement (Negotiation). Line drawings of this new design were made and vetted by experts of snow leopard and wolf ecology from the Snow Leopard Trust (Figure 2D). This was done as an additional means to triangulate the aptness of the new design. Interestingly, the experts’ knowledge on snow leopard and wolf behavior with respect to depredation corroborated with the knowledge of the herders. To check for the structural soundness, we also took advice from local civil engineers and fabricators in Leh—the regional headquarter (Aptness). These inputs provided critical adjustments to the design and were also crucial in accurately estimating their cost. We (RD) started by meeting the village heads to explain our intentions (Figure 2A). The discussion was subsequently expanded to include the whole village to ensure that everyone could participate (Respect). These two meetings over the span of 2 days formed the basis for the community engagement. We ensured clarity in communication, i.e., we wanted to understand existing conservation conﬂict and subsequently assess how we could jointly identify and implement a preventive solution (Transparency). We also spoke about various interventions that are used to mitigate impacts of livestock depredation in other agro-pastoralist communities in the trans-Himalayas (e.g., reinforcing corrals and community-run livestock insurance programs). These meetings conﬁrmed that snow leopard and wolves had caused depredation in night-time corrals, and reinforcing them would be the most eﬀective solution. Corrals in winter pastures seemed particularly vulnerable to depredation (Aptness). Due to resource and time limitations, we mutually agreed to reinforce/rebuild corrals in the winter pastures that were at a higher risk of being attacked by predators (Transparency, Negotiation). Community members agreed that predator-prooﬁng of corrals could help, but only if the design was locally relevant (Aptness). The people of Sumdoo TR constituted a committee of four people from the village who were to lead the discussions on behalf of the community. This committee was tasked with coordinating the eﬀort with NCF and discussing key points with the entire community so that fair decisions could be taken through community-level consultations (Transparency). Co-designing the Intervention Agro-pastoral communities across the Central and South Asian mountains have economic and emotional linkages to their livestock. Many of them have been living in these regions for several millennia (Mishra et al., 2017). Most of these rangelands are outside formal strictly Protected Area network, therefore, people and wildlife tend to live in close proximity (Mishra, 2016). This is true for the villagers of Sumdoo TR as well. Locally, while there are some positive symbolic associations of predators such Having engaged in predator-prooﬁng of over 100 corrals with several communities in Ladakh and Himachal Pradesh over the past decade, we were aware that pre-determined corral designs can be built quickly and eﬃciently. However, not factoring in the communities’ views in implementation could also lead to the community questioning the eﬀectiveness of the intervention and continuing their demand for removal of the carnivores November 2021 \| Volume 2 \| Article 766086 Frontiers in Conservation Science \| www.frontiersin.org 4 Mitigating Livestock Predation by Carnivores Bijoor et al. from the region. This is problematic, not least because relocated predators are known to cause higher incidence of negative human–wildlife interactions (Athreya et al., 2011). Persistence of this situation could also lead to long-term distrust between the community and conservation agencies. An ineﬀective corral design can aﬀect the health and well-being of the livestock and in some cases fail at preventing depredation (Empathy). Our eﬀorts in Sumdo TR were guided by multiple Focus Group Discussions (FGD) (Nyumba et al., 2018) while ensuring that views of all the community members were sought and taken into consideration at every step along the way (Aptness, Respect, Negotiation). Table 2). During this meeting, herders also provided designs based on their personal experiences and local understanding. The herders developed a design that was inspired by the shape of tent, one that requires less construction, provides better access to sunlight, and allows for wind to pass through the structure (Figures 2D,E). Key components of this design were essentially that sunlight could enter in the day to dry the soil inside and shorter walls so that wind can blow. All the designs were collated and tabled before the corral-building committee in order to start a wider consultation with the community (Transparency). Frontiers in Conservation Science \| www.frontiersin.org Challenges Needs large quantity of stones to build the walls. The structure could reduce or completely prevent wind through the corrals. High walls may reduce access to sunlight inside the corral during winter. Soil inside the corral likely to remain wet due to lack of sunlight, livestock could be prone to disease. U t t d d i th t d d il ti Needs large quantity of stones to build the walls. The structure could reduce or completely prevent wind through the corrals. High walls may reduce access to sunlight inside the corral during winter. Soil inside the corral likely to remain wet due to lack of sunlight, livestock could be prone to disease. Needs large quantity of stones to build the walls. The structure could reduce or completely prevent wind through the corrals. High walls may reduce access to sunlight inside the corral during winter. Soil inside the corral likely to remain wet due to lack of sunlight, livestock could be prone to disease. Untested design that needed piloting. Needed to be constructed under skilled supervision since design elements required attention to detail and structural strength Tested design that has been used successfully in other parts of Ladakh Simplicity of design Design proposed by the herders and NGO partner checked its potential efﬁcacy through ecological experts. The proposed design was also validated by engineers for its structural strength. Fulﬁlls all requirement. Untested design that needed piloting. Needed to be constructed under skilled supervision since design elements required attention to detail and structural strength. Simple design, tested successfully in another region. No additional construction of structures required in areas of undulating terrain, hence requirement for additional stones is canceled. The design is not suitable for areas around cliff and high rocks. Many of the vulnerable corrals were near such structures. Would require the herders to shift their original corral and reconstruct them in open areas, away from cliffs and rugged areas. Expensive with need for electric fence, solar panels and batteries. Maintenance requires technical knowhow. Challenges FIGURE 2 \| (A) A discussion between the community members and NCF staff, (B) Corral building in progress, (C) the signed agreement between the community members and NCF staff written in Ladakhi (a local dialect of Tibetan), (D) Line drawing of the corral design inspired by local herders—the walls are around the perimeter indicated by the sold lines, whilst the net (wire-mesh) is on the top indicated by the light shaded line, (E) The ﬁnished predator-proof corral, and (F) Sheep and goat within the new predator-proof corrals. Verbal consent was obtainedfrom people in the photographs before they were taken. FIGURE 2 \| (A) A discussion between the community members and NCF staff, (B) Corral building in progress, (C) the signed agreement between the community members and NCF staff written in Ladakhi (a local dialect of Tibetan), (D) Line drawing of the corral design inspired by local herders—the walls are around the perimeter indicated by the sold lines, whilst the net (wire-mesh) is on the top indicated by the light shaded line, (E) The ﬁnished predator-proof corral, and (F) Sheep and goat within the new predator-proof corrals. Verbal consent was obtainedfrom people in the photographs before they were taken. Implementing the Solution instance when fabricated material was incorrectly designed, the herders remained patient with the NCF team and worked jointly with them and the manufacturers to have these corrected, despite this leading to some delays. All seven corrals were constructed and put to use by November 2020, before the onset of winter. It was important to complete the construction before the coming winter and given the novelty of the design, NCF’s ﬁeld team and the corral committee were closely involved in monitoring the construction along with the corral owner. This process was not without unanticipated challenges. For example, in one instance some of the corral owners delayed the start of construction from the agreed timelines. In this case the corral-building committee stepped in to understand the cause of the delay and when it was veriﬁed that these delays were for genuine reasons, they agreed to a marginal relaxation in timelines (Empathy). In another Respect, Negotiation). Beneﬁts Challenges TABLE 2 \| Reports the beneﬁts, challenges, and arrangements required for each designs, in particular in the Changthang region of Ladakh. Respect, Negotiation). The design had to be such that requirement of stones was minimum. These requirements were speciﬁc but needed careful consideration (Aptness). In the third meeting with the community, we shared designs from our experience and from conservation science literature (e.g., Samelius et al., 2020), and discussed the beneﬁts and shortcomings of each (see Supplementary Material 2 and November 2021 \| Volume 2 \| Article 766086 Frontiers in Conservation Science \| www.frontiersin.org 5 Mitigating Livestock Predation by Carnivores Bijoor et al. TABLE 2 \| Reports the beneﬁts, challenges, and arrangements required for each designs, in particular in the Changthang region of Ladakh. Design Beneﬁts Challenges Traditional Tested design that has been used successfully in other parts of Ladakh Simplicity of design Needs large quantity of stones to The structure could reduce or co through the corrals. High walls may reduce access to corral during winter. Soil inside the corral likely to rema sunlight, livestock could be prone Tent Design proposed by the herders and NGO partner checked its potential efﬁcacy through ecological experts. The proposed design was also validated by engineers for its structural strength. Fulﬁlls all requirement. Untested design that needed pilo Needed to be constructed under since design elements required a structural strength. Fenced Samelius et al. (2020) Simple design, tested successfully in another region. No additional construction of structures required in areas of undulating terrain, hence requirement for additional stones is canceled. The design is not suitable for area rocks. Many of the vulnerable cor structures. Would require the herders to shift reconstruct them in open areas, a rugged areas TABLE 2 \| Reports the beneﬁts, challenges, and arrangements required for each designs, in particular in the Changthang region of Ladakh. Design Beneﬁts Challenges Traditional Tested design that has been used successfully in other parts of Ladakh Simplicity of design Needs large quantity of stone The structure could reduce or through the corrals. Hi h ll d TABLE 2 \| Reports the beneﬁts, challenges, and arrangements required for each designs, in particular in the Changthang region of Ladakh. Design Beneﬁts Challenges Traditional Tested design that has been used successfully in other parts of Ladakh Simplicity of design Needs large quantity of stone The structure could reduce o through the corrals. 2 \| Reports the beneﬁts, challenges, and arrangements required for each designs, in particular in the Changthang region of Ladakh. Fenced Samelius et al. (2020) POST-INTERVENTION SCENARIO Empowering the herders of Sumdoo TR to implement their preferred solution to a conservation problem also ensured that all this work could be done even during Covid-19 restrictions, which were particularly stringent with respect to outsider entry into Ladakh. After the success of the ﬁrst seven corrals, herders of Sumdoo TR and NCF are now in discussions to re-enforce the remaining existing corrals in the village. In the September 2021 visit, seven new corrals were identiﬁed by the community to be reinforced. This is inspired by the PARTNERS approach which emphasizes the importance of long-term partnerships with local communities (Young et al., 2021). to eﬀectively implement conservation interventions together with local communities. NCF provided most, if not all, design inputs in corrals built or reinforced in communities before the engagement in Sumdoo TR. Employing the PARTNERS approach with the Sumdoo TR community highlighted that empowering the community to design and implement a conservation intervention resulted in them taking ownership of the eﬀort, building trust with conservation agencies, and hence is potentially a long-term solution to conservation conﬂicts in the region. At the outset, a good working relation with local government agencies and an experienced ﬁeld team facilitated a timely response to a situation of acute conﬂict. Building presence took time, but was vital in establishing mutual trust and this was boosted by having local Ladakhi team members. A community although a collective, is often a heterogenous mix of individual aspirations, thought processes, and opinions (Mishra, 2016). Engaging with the wider community helped us understand the challenges being faced and a possible solution relevant to the local context. For instance, often during the various meeting in Sumdoo TR, there was diversity of perspective on topics concerning which corrals were most vulnerable and what the most appropriate design would be. The various members of the community and the NGO were provided the same platform—the community meetings—to bring their expertise while remaining open to explore possibilities. Rather than imposing thoughts or actions onto the community members, we tried to facilitate a consultative process, which was led by the village headman. Creation of unrealistic expectations by local communities from conservation organization can be an unintended output of community engagements (e.g., Dahlberg and Burlando, 2009). To safeguard against this, transparent and clear communication allowed for a time-bound agreement with ﬁxed roles, responsibilities, and expected outcomes. POST-INTERVENTION SCENARIO Despite challenges and occasional delays, the work was completed on time and to the prescribed design. As part of this eﬀort, the herders were not merely recipients of support; they were part of setting up the solution to prevent livestock depredation by carnivores. The co-developed solution relied on local knowledge of the herders thus ensuring that it was locally relevant. While there is growing recognition for conservation to be evidence-based (Sutherland et al., 2004), we believe there is an equal need for it to be context-based and inclusive of local traditional knowledge. This is an important step in de-colonizing conservation, dismantling discrimination, and achieving inclusion and representation (Chaudhury and Colla, 2020; Trisos et al., 2021). Prioritizing external ideas— often more technically sophisticated—over local traditional knowledge can create a deep-seeded sense of discrimination leading to long-term conservation conﬂicts. The knowledge and experience of the local community, who are the primary stakeholder in a conservation situation, needs to be considered on a par with conservation evidence coming from other parts of the world. Without this sensitivity conservation interventions risk becoming technocratic solutions. We believe POST-INTERVENTION SCENARIO The cost of building each of the corrals in Sumdoo TR was higher than most of the previous corrals built by NCF in November 2021 \| Volume 2 \| Article 766086 Frontiers in Conservation Science \| www.frontiersin.org 6 Mitigating Livestock Predation by Carnivores Bijoor et al. Ladakh and Himachal Pradesh. This was because most of the previous corrals were much smaller and housed fewer livestock. Nonetheless, the Sumdoo TR corrals were more cost eﬀective per livestock. In most communities where NCF has worked before, livestock is mostly corralled indoors, hence predator-prooﬁng essentially entailed reinforcing the doors and windows of pre- existing structure. However, in Sumdoo TR entire structures were needed to be made. Our ﬁeld team returned to Sumdoo TR in March 2021 to check how the corrals had fared over the ﬁrst winter. We conducted a FGD with the village headman and all the seven herders whose corrals were reinforced (Nyumba et al., 2018). All seven herders had used the corral through the winter and housed all the 1,840 sheep/goats. Herders did not report any instance of depredation in any of the re-built corrals. Herders believed the new structures did not negatively impact wool growth of their Changra goats and were eﬀective in preventing snow from accumulating inside. The herders are conﬁdent that periodic repairs or maintenance that these structures may require can be handled by them with minimal support using locally available resources. The seven herders reiterated that not having the new corral design would have likely meant accumulation of snow during the winter which often leads to livestock deaths due to hypothermia (Yatoo et al., 2014). While this indicates the success of the structures, how they persist in the long-run needs careful monitoring (Aptness). In depth details regarding visitation of predators to these structure also needs to be monitored. The village headman suggested during the FGD that demands for removal of carnivores have reduced as a result of this intervention, and the trust that has been built between the herders and conservation agencies. In another follow up visit in September 2021, we visited each of the seven herders individually as they were placed in dispersed summer camps, away from the village. They conﬁrmed the structures were in good condition and the same seven herders are planning to use them in the upcoming winter. We visited the structures as well and conﬁrmed their condition. Frontiers in Conservation Science \| www.frontiersin.org ETHICS STATEMENT Ajay Bijoor works with local communities and government agencies to plan and implement conservation action in parts of Ladakh and Himachal Pradesh in India. In over 10 years of experience, Ajay has worked with community partners to implement interventions including reinforcing nearly 50 livestock corrals and coordinating several community-based livestock insurance programs to facilitate human–wildlife coexistence. Alongside, he works with government agencies to implement various projects such as the Project Snow Leopard within India. He also supports research activities and is Assistant Program Head of the High Altitude Programme in NCF. Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements. Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements. CONCLUSION While there are multiple frameworks to engage communities in conservation (e.g., Berkes, 2007; Mishra et al., 2017), operationalizing these is challenging. We demonstrate how the PARTNERS principles framework for community based conservation (Mishra et al., 2017) could be used on the ground November 2021 \| Volume 2 \| Article 766086 Frontiers in Conservation Science \| www.frontiersin.org 7 Bijoor et al. Mitigating Livestock Predation by Carnivores ACKNOWLEDGMENTS p g Sherab Lobzang has been working for over 5 years toward nurturing positive human-nature relationships primarily through education modules and other on-ground conservation eﬀorts across Ladakh. These include predator-prooﬁng of corrals, wildlife surveys, and livestock insurance programs. She hails from an agro-pastoralost family in Kumdok village, Ladakh. She has had ﬁrst-hand experiences of the various interactions pastoralist experience with wild carnivores in the landscape. Sherab Lobzang has been working for over 5 years toward nurturing positive human-nature relationships primarily through education modules and other on-ground conservation eﬀorts across Ladakh. These include predator-prooﬁng of corrals, wildlife surveys, and livestock insurance programs. She hails from an agro-pastoralost family in Kumdok village, Ladakh. She has had ﬁrst-hand experiences of the various interactions pastoralist experience with wild carnivores in the landscape. We would like to thank the Department of Wildlife Protection in Ladakh, and especially the ranger of Changthang, for their support and encouragement to work with the community in Sumdoo TR. Karma Sonam played an important role during the community engagement and corral building by providing invaluable suggestions. We would like to thank Charudutt Mishra, Justine Alexander and Juliette Young for helping conceptualize the PARTNERS principles. Juliette Young also provided key inputs on an earlier draft. Lastly but most importantly, we would like to thank the community members of Sumdoo TR for their patience and enthusiasm in leading this eﬀort on the ground. Kulbhushansingh Suryawanshi splits his time between conservation practice and conservation science. Over the past 15 year he has worked across the mountains of Central and South Asia. He has been helping set-up and guide on ground conservation eﬀorts to facilitate co-existence between local pastoral communities and large carnivores. He has helped set-up and run conservation programs such as SHEN—an initiative to help local people, especially women, to earn a livelihood and engage with conservation, Snow Leopard Friendly Pashmina to help herders make their practices more wildlife friendly, livestock insurance, predator-prooﬁng corral, and village reserves. DATA AVAILABILITY STATEMENT that equitable partnerships between local communities and conservation agencies can help local communities be (and see themselves as) a part of conservation solutions rather than conservation problems. The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author. FUNDING Rigzen Dorjay has been leading on-ground conservation eﬀorts such as building predator-proof corrals, creating village reserves for wild herbivores, instituting, and helping run livestock insurance programs, and conducting wildlife surveys in remote corners of his homeland, Ladakh, for over 7 year. He hails from Saspoche village in Ladakh. This work was made possible by the generous support of the NatWest India Foundation (erstwhile RBS Foundation India), Caroline Ten Have and the Rainbow Foundation, and the Snow Leopard Trust. AUTHOR CONTRIBUTIONS RD led and coordinated the work on ground and assisted in this by SL. AB and RD conceived the project with critical inputs from KS. MK provided inputs throughout the life of the project. AB led the writing of the manuscript, with help from MK. KS critically revised the manuscript. All authors gave ﬁnal approval for publication. Munib Khanyari is interested in studying various factors that aﬀect human-wildlife coexistence. While Munib’s Ph.D. topic is to study disease transmission between wild and domestic ungulates in Trans-Himalayan India, he aims to support his colleagues who work with local communities to mitigate negative human-wildlife interactions. REFERENCES Samelius, G., Suryawanshi, K., Frank, J., Agvaantseren, B., Baasandamba, E., Mijiddorj, T., et al. (2020). Keeping predators out: testing fences to reduce livestock depredation at night-time corrals. Oryx 55, 466–472. doi: 10.1017/S0030605319000565 Athreya, V., Odden, M., Linnell, J. D., and Karanth, K. U. (2011). Translocation as a tool for mitigating conﬂict with leopards in human-dominated landscapes of India. Conser. Biol. 25, 133–141. Shivik, J. A., Treves, A., and Callahan, P. (2003). 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https://openalex.org/W2963737810	http://www.mitpressjournals.org/doi/pdf/10.1162/tacl_a_00113	English	null	The Galactic Dependencies Treebanks: Getting More Data by Synthesizing New Languages	Transactions of the Association for Computational Linguistics	2,016	cc-by	12,874	Abstract Unfortunately, we usually have only from 1 to 40 languages to work with. In contrast, machine learn- ing methods thrive on data, and recent AI successes have mainly been on tasks where one can train richly parameterized predictors on a huge set of IID (input, output) examples. Even 7,000 training examples— one for each language or dialect on Earth—would be a small dataset by contemporary standards. We release Galactic Dependencies 1.0—a large set of synthetic languages not found on Earth, but annotated in Universal Dependen- cies format. This new resource aims to pro- vide training and development data for NLP methods that aim to adapt to unfamiliar lan- guages. Each synthetic treebank is produced from a real treebank by stochastically permut- ing the dependents of nouns and/or verbs to match the word order of other real languages. We discuss the usefulness, realism, parsabil- ity, perplexity, and diversity of the synthetic languages. As a simple demonstration of the use of Galactic Dependencies, we consider single-source transfer, which attempts to parse a real target language using a parser trained on a “nearby” source language. We ﬁnd that including synthetic source languages some- what increases the diversity of the source pool, which signiﬁcantly improves results for most target languages. As a result, it is challenging to develop systems that will discover structure in new languages in the same way that an image segmentation method, for example, will discover structure in new images. The limited resources even make it challenging to de- velop methods that handle new languages by un- supervised, semi-supervised, or transfer learning. Some such projects evaluate their methods on new sentences of the same languages that were used to develop the methods in the ﬁrst place—which leaves one worried that the methods may be inadvertently tuned to the development languages and may not be able to discover correct structure in other languages. Other projects take care to hold out languages for evaluation (Spitkovsky, 2013; Cotterell et al., 2015), but then are left with only a few development lan- guages on which to experiment with different unsu- pervised methods and their hyperparameters. Transactions of the Association for Computational Linguistics, vol. 4, pp. 491–505, 2016. Action Editor: Joakim Nivre. Submission batch: 5/2016; Revision batch: 7/2016; Published 9/2016. c⃝2016 Association for Computational Linguistics. Distributed under a CC-BY 4.0 license. ://www.mitpressjournals.org/doi/pdf/10.1162/tacl_a_00113 by guest on 24 October 2024 Downloaded from http://www.mitpressjournals.org/doi/pdf/10.1162/tacl_a_00113 by guest on 24 October 2024 1 Motivation Some potential NLP tasks have very sparse data by machine learning standards, as each of the IID train- ing examples is an entire language. For instance: If we had many languages, then we could develop better unsupervised language learners. Even better, we could treat linguistic structure discovery as a su- pervised learning problem. That is, we could train a system to extract features from the surface of a language that are predictive of its deeper structure. Principles & Parameters theory (Chomsky, 1981) conjectures that such features exist and that the ju- venile human brain is adapted to extract them. • typological classiﬁcation of a language on var- ious dimensions; • adaptation of any existing NLP system to new, low-resource languages; • induction of a syntactic grammar from text; • discovery of a morphological lexicon from text; • other types of unsupervised discovery of lin- guistic structure. Our goal in this paper is to release a set of about 50,000 high-resource languages that could be used to train supervised learners, or to evaluate less- supervised learners during development. These “un- earthly” languages are intended to be at least sim- Given a corpus or other data about a language, we might aim to predict whether it is an SVO lan- guage, or to learn to pick out its noun phrases. For such problems, a single training or test example cor- responds to an entire human language. 491 ilar to possible human languages. As such, they provide useful additional training and development data that is slightly out of domain (reducing the variance of a system’s learned parameters at the cost of introducing some bias). The initial release as described in this paper (version 1.0) is avail- able at https://github.com/gdtreebank/ gdtreebank. We plan to augment this dataset in future work (§8). (2015) obtain data to train semantic parsers in a new domain by ﬁrst generating synthetic (utterance, log- ical form) pairs and then asking human annotators to paraphrase the synthetic utterances into more natural human language. In speech recognition, morphology-based “vocab- ulary expansion” creates synthetic word forms (Ra- sooli et al., 2014; Varjokallio and Klakow, 2016). 2 Related Work Synthetic data generation is a well-known trick for effectively training a large model on a small dataset. Abu-Mostafa (1995) reviews early work that provided “hints” to a learning system in the form of virtual training examples. While datasets have grown in recent years, so have models: e.g., neural networks have many parameters to train. Thus, it is still common to create synthetic train- ing examples—often by adding noise to real inputs or otherwise transforming them in ways that are expected to preserve their labels. Domains where it is easy to exploit these invariances include im- age recognition (Simard et al., 2003; Krizhevsky et al., 2012), speech recognition (Jaitly and Hinton, 2013; Cui et al., 2015), information retrieval (Vilares et al., 2011), and grammatical error correction (Ro- zovskaya and Roth, 2010). 3 Synthetic Language Generation A treebank is a corpus of parsed sentences of some language. We propose to derive each synthetic tree- bank from some real treebank. By manipulating the existing parse trees, we obtain a useful cor- pus for our synthetic language—a corpus that is already tagged, parsed, and partitioned into train- ing/development/test sets. Additional data in the synthetic language can be obtained, if desired, by automatically parsing additional real-language sen- tences and manipulating these trees in the same way. 1 Motivation Machine translation researchers have often tried to automatically preprocess parse trees of a source language to more closely resemble those of the tar- get language, using either hand-crafted or automati- cally extracted rules (Dorr et al., 2002; Collins et al., 2005, etc.; see review by Howlett and Dras, 2011). In addition to releasing thousands of treebanks, we provide scripts that can be used to “translate” other annotated resources into these synthetic lan- guages. E.g., given a corpus of English sentences la- beled with sentiment, a researcher could reorder the words in each English sentence according to one of our English-based synthetic languages, thereby ob- taining labeled sentences in the synthetic language. 2In practice, this means applying a single permutation model to permute the dependents of every word tagged as NOUN (com- mon noun), PROPN (proper noun), or PRON (pronoun). 1http://universaldependencies.org 2 Downloaded from http://www.mitpressjournals.org/doi/pdf/10.1162/tacl_a_00113 by guest on 24 October 2024 3.1 Method (Moved constituents are underlined.) Each language has a different distribution over surface part-of-speech sequences. about 93% of S’s nodes (Table 2), as UD treats ad- positions and conjunctions as childless dependents. portant properties would not be captured by a sim- ple context-free model of dependency trees, which is why we modify real sentences rather than gen- erating new sentences from such a model. In ad- dition, our method obviously preserves the basic context-free properties, such as the fact that verbs typically subcategorize for one or two nominal ar- guments (Naseem et al., 2010). For example, English[French/N, Hindi/V] is a syn- thetic language based on an English substrate, but which adopts subject-object-verb (SOV) word order from the Hindi superstrate and noun-adjective word order from the French superstrate (Figure 1). Note that it still uses English lexical items. Our terms “substrate” and “superstrate” are bor- rowed from the terminology of creoles, although our synthetic languages are unlike naturally oc- curring creoles. Our substitution notation S′ = S[RN/N, RV/V] is borrowed from the logic and pro- gramming languages communities. Second, by drawing on real superstrate languages, we ensure that our synthetic languages use plausible word orders. For example, if RV is a V2 language that favors SVO word order but also allows OVS, then S′ will match these proportions. Similarly, S′ will place adverbs in reasonable positions with re- spect to the verb. Downloaded from http://www.mitpressjournals.org/doi/pdf/10.1162/tacl_a_00113 by guest on 24 October 2024 3.1 Method We begin with the Universal Dependencies collec- tion version 1.2 (Nivre et al., 2015, 2016),1 or UD. This provides manually edge-labeled dependency treebanks in 37 real languages, in a consistent style and format—the Universal Dependencies format. An example appears in Figure 1. In this paper, we select a substrate language S represented in the UD treebanks, and systematically reorder the dependents of some nodes in the S trees, to obtain trees of a synthetic language S′. Synthetic datasets have also arisen recently for se- mantic tasks in natural language processing. bAbI is a dataset of facts, questions, and answers, gen- erated by random simulation, for training machines to do simple logic (Weston et al., 2016). Hermann et al. (2015) generate reading comprehension ques- tions and their answers, based on a large set of news- summarization pairs, for training machine readers. Serban et al. (2016) used RNNs to generate 30 mil- lion factoid questions about Freebase, with answers, for training question-answering systems. Wang et al. Speciﬁcally, we choose a superstrate language RV, and write S′ = S[RV/V] to denote a (projective) synthetic language obtained from S by permuting the dependents of verbs (V) to match the ordering statistics of the RV treebanks. We can similarly per- mute the dependents of nouns (N).2 This permutes 492 ∗ DET NOUN PROPN VERB VERB DET ADJ NOUN ADV PUNCT ROOT Every move Google makes brings this particular future closer . det nsubj nsubj acl:rel root det amod dobj advmod punct Language Sentence English Every move Google makes brings this particular future closer. English[French/N] Every move Google makes brings this future particular closer. English[Hindi/V] Every move Google makes this particular future closer brings. English[French/N, Hindi/V] Every move Google makes this future particular closer brings. Figure 1: The original UD tree for a short English sentence, and its “translations” into three synthetic languages, which are obtained by manipulating the tree. (Moved constituents are underlined.) Each language has a different distribution over surface part-of-speech sequences. Figure 1: The original UD tree for a short English sentence, and its “translations” into three synthetic languages, which are obtained by manipulating the tree. (Moved constituents are underlined.) Each language has a different distribution over surface part-of-speech sequences. Figure 1: The original UD tree for a short English sentence, and its “translations” into three synthetic languages, which are obtained by manipulating the tree. 3.2 Discussion We note, however, that our synthetic languages might violate some typological universals or typo- logical tendencies. For example, RV might pre- scribe head-initial verb orderings while RN pre- scribes head-ﬁnal noun orderings, yielding an un- usual language. Worse, we could synthesize a lan- guage that uses free word order (from RV) even though nouns (from S) are not marked for case. Such languages are rare, presumably for the func- tionalist reason that sentences would be too ambigu- ous. One could automatically ﬁlter out such an im- plausible language S′, or downweight it, upon dis- covering that a parser for S′ was much less accurate on held-out data than a comparable parser for S. There may be more adventurous ways to manufac- ture synthetic languages (see §8 for some options). However, we emphasize that our current method is designed to produce fairly realistic languages. First, we retain the immediate dominance struc- ture and lexical items of the substrate trees, alter- ing only their linear precedence relations. Thus each sentence remains topically coherent; nouns continue to be distinguished by case according to their role in the clause structure; wh-words continue to c- command gaps; different verbs (e.g., transitive vs. intransitive) continue to be associated with differ- ent subcategorization frames; and so on. These im- 493 abilities and their sum Z(x). abilities and their sum Z(x). We also note that our reordering method (§4) does ignore some linguistic structure. For example, we do not currently condition the order of the depen- dent subtrees on their heaviness or on the length of resulting dependencies, and thus we will not faith- fully model phenomena like heavy-shift (Hawkins, 1994; Eisner and Smith, 2010). Nor will we model the relative order of adjectives. We also treat all verbs interchangeably, and thus use the same word orders—drawn from RV—for both main clauses and embedded clauses. This means that we will never produce a language like German (which uses V2 order in main clauses and SOV order in embedded clauses), even if RV = German. All of these prob- lems could be addressed by enriching the features that are described in the next section. Fortunately, we can compute each unnormalized probability in just O(1) amortized time, if we enu- merate the n! orderings π using the Steinhaus- Johnson-Trotter algorithm (Sedgewick, 1977). 4 Modeling Dependent Order Our feature functions (§4.4) are ﬁxed over all lan- guages. They refer to the 17 node labels (POS tags) and 40 edge labels (dependency relations) that are used consistently throughout the UD treebanks. Let X be a part-of-speech tag, such as Verb. To produce a dependency tree in language S′ = S[RX/X], we start with a projective dependency tree in language S.3 For each node x in the tree that is tagged with X, we stochastically select a new or- dering for its dependent nodes, including a position in this ordering for the head x itself. Thus, if node x has n −1 dependents, then we must sample from a probability distribution over n! orderings. For each UD language L and each POS tag X, we ﬁnd parameters θL X that globally maximize the unregularized log-likelihood: θL X = argmax θ X x log pθ(πx \| x) (2) (2) Here x ranges over all nodes tagged with X in the projective training trees of the L treebank, omitting nodes with n ≥7 for speed. Our job in this section is to deﬁne this probabil- ity distribution. Using π = (π1, . . . , πn) to denote an ordering of these n nodes, we deﬁne a log-linear model over the possible values of π: The expensive part of this computation is the gra- dient of log Z(x), which is an expected feature vec- tor. To compute this expectation efﬁciently, we again take care to loop over the permutations in Steinhaus-Johnson-Trotter order. pθ(π \| x) = 1 Z(x) exp X 1≤i<j≤n θ · f(π, i, j) (1) Here Z(x) is the normalizing constant for node x. θ is the parameter vector of the model. f extracts a sparse feature vector that describes the ordered pair of nodes πi, πj, where the ordering π would place πi to the left of πj. Here Z(x) is the normalizing constant for node x. θ is the parameter vector of the model. f extracts a sparse feature vector that describes the ordered pair of nodes πi, πj, where the ordering π would place πi to the left of πj. A given language L may not use all of the tags and relations. Universal features that mention un- used tags or relations do not affect (2), and their weights remain at 0 during training. 3.2 Discussion This enumeration sequence has the property that any two consecutive permutations π, π′ differ by only a sin- gle swap of some pair of adjacent nodes. Thus their probabilities are closely related: the sum in equa- tion (1) can be updated in O(1) time by subtracting θ·f(π, i, i+1) and adding θ·f(π′, i, i+1) for some i. The other O(n2) summands are unchanged. In addition, if n ≥8, we avoid this computation by omitting the entire tree from our treebank; so we have at most 7! = 5040 summands. 3Our method can only produce projective trees. This is be- cause it recursively generates a node’s dependent subtrees, one at a time, in some chosen order. Thus, to be safe, we only apply our method to trees that were originally projective. See §8. Downloaded from http://www.mitpressjournals.org/doi/pdf/10.1162/tacl_a_00113 by guest on 24 October 2024 4We could alternatively have used MCMC sampling. 5Recall that for each head POS X of language L, we learn a separate ordering model with parameter vector θL X. 4.1 Efﬁcient sampling As a result, the burden of explaining the ordering is dis- tributed over more features, and we hope some of these features will transfer to S. For example, sup- pose RX lacks adverbs and yet we wish to use θRX X to permute a sequence of S that contains adverbs. Thus, a bigram feature such as A.DET.EOS would ﬁre on DET when it falls at the end of the sequence. Even though the resulting order must disrupt some familiar non-adverb bigrams by inserting adverbs, other features—which consider non-adjacent tags— will still favor an RX-like order for the non-adverbs. • H.ti.ri.ti+1.ri+1.....tj.rj, provided that i+2 ≤j ≤i+4. Among features of this form, we keep only the 10% that ﬁre most frequently in the training data. These “higher-order k- gram” features memorize sequences of lengths 3 to 5 that are common in the language. Second, we actually sample the reordering from a distribution pθ with an interpolated parameter vector θ = θS′ X = (1 −λ)θRX X + λθS X, Notice that for each non-H feature that mentions both tags t and relations r, we also deﬁned two backoff features, omitting the t ﬁelds or r ﬁelds re- spectively. where λ = 0.05. This gives a weighted product of experts, in which ties are weakly broken in favor of the substrate ordering. (Ties arise when RX is unfa- miliar with some tags that appear in S, e.g., adverb.) Using the example from Figure 1, for subtree g p g DET ADJ NOUN this particular future det amod the features that ﬁre are Template Features L.ti.ri L.DET.det, L.ADJ.amod L.ti.ri.tj.rj L.DET.det.ADJ.amod d.ti.ri.tj.rj l.DET.det.ADJ.amod A.t1.r1.t2.r2 A.BOS.BOS.DET.det, A.DET.det.ADJ.amod, A.ADJ.amod.NOUN.head, A.NOUN.head.EOS.EOS plus backoff features and H features (not shown). plus backoff features and H features (not shown). • d.ti.ri.tj.rj, d.ti.tj, and d.ri.rj, where d is l (left), m (middle), or r (right) according to whether the head position h satisﬁes i < j < h, i < h < j, or h < i < j. For example, l.nsubj.dobj will ﬁre on SOV clauses. This is a specialization of the previous feature, and is skipped if i = h or j = h. Downloaded from http://www.mitpressjournals.org/doi/pdf/10.1162/tacl_a_00113 by guest on 24 October 2024 4.1 Efﬁcient sampling We use (1) to permute the X nodes of substrate lan- guage S into an order resembling superstrate lan- guage RX. In essence, this applies the RX order- ing model to out-of-domain data, since the X nodes may have rather different sets of dependents in the S treebank than in the RX treebank. We mitigate this issue in two ways. To sample exactly from the distribution pθ,4 we must explicitly compute all n! unnormalized prob- 494 First, our ordering model (1) is designed to be more robust to transfer than, say, a Markov model. The position of each node is inﬂuenced by all n −1 other nodes, not just by the two adjacent nodes. As a result, the burden of explaining the ordering is dis- tributed over more features, and we hope some of these features will transfer to S. For example, sup- pose RX lacks adverbs and yet we wish to use θRX X to permute a sequence of S that contains adverbs. Even though the resulting order must disrupt some familiar non-adverb bigrams by inserting adverbs, other features—which consider non-adjacent tags— will still favor an RX-like order for the non-adverbs. • A.ti.ri.tj.rj and A.ti.tj and A.ri.rj, pro- vided that j = i + 1. These “bigram fea- tures” detect two adjacent nodes. For this fea- ture and the next one, we extend the summa- tion in (1) to allow 0 ≤i < j ≤n + 1, tak- ing t0 = r0 = BOS (“beginning of sequence”) and tn+1 = rn+1 = EOS (“end of sequence”). Thus, a bigram feature such as A.DET.EOS would ﬁre on DET when it falls at the end of the sequence. • A.ti.ri.tj.rj and A.ti.tj and A.ri.rj, pro- vided that j = i + 1. These “bigram fea- tures” detect two adjacent nodes. For this fea- ture and the next one, we extend the summa- tion in (1) to allow 0 ≤i < j ≤n + 1, tak- ing t0 = r0 = BOS (“beginning of sequence”) and tn+1 = rn+1 = EOS (“end of sequence”). Thus, a bigram feature such as A.DET.EOS would ﬁre on DET when it falls at the end of the sequence. The position of each node is inﬂuenced by all n −1 other nodes, not just by the two adjacent nodes. 4.4 Feature Templates DET ADJ NOUN this particular future det amod th f t th t ﬁ DET ADJ NOUN this particular future det amod We write ti for the POS tag of node πi, and ri for the dependency relation of πi to the head node. If πi is itself the head, then necessarily ti = X,5 and we specially deﬁne ri = head. In our feature vector f(π, i, j), the features with the following names have value 1, while all others have value 0: the features that ﬁre are Template Features L.ti.ri L.DET.det, L.ADJ.amod L.ti.ri.tj.rj L.DET.det.ADJ.amod d.ti.ri.tj.rj l.DET.det.ADJ.amod A.t1.r1.t2.r2 A.BOS.BOS.DET.det, A.DET.det.ADJ.amod, A.ADJ.amod.NOUN.head, A.NOUN.head.EOS.EOS plus backoff features and H features (not shown). • L.ti.ri and L.ti and L.ri, provided that rj = head. For example, L.ADJ will ﬁre on each ADJ node to the left of the head. • L.ti.ri.tj.rj and L.ti.tj and L.ri.rj, pro- vided that ri ̸= head, rj ̸= head. These fea- tures detect the relative order of two siblings. plus backoff features and H features (not shown). 5 The Resource R ∈[0, 1] measures the freeness of the language’s word order, as the condi- tional cross-entropy of our trained ordering model pθ rel- ative to that of a uniform distribution: R = H(˜p,pθ) H(˜p,punif) = meanx[−log2 pθ(π∗(x)\|x)] meanx[−log2 1/n(x)!] , where x ranges over all N and V tokens in the dev sentences, n(x) is 1 + the number of dependents of x, and π∗(x) is the observed ordering at x. RV each range over the 37 available languages. (RN = S or RV = S gives “self-permutation”). This yields 37 × 38 × 38 = 53, 428 languages in total. Each language is provided as a directory of 3 ﬁles: training, development, and test treebanks. The directories are systematically named: for example, English[French/N, Hindi/V] can be found in direc- tory en∼fr@N∼hi@V. Our treebanks provide alignment information, to facilitate error analysis as well as work on machine translation. Each word in a synthetic sentence is an- notated with its original position in the substrate sen- tence. Thus, all synthetic treebanks derived from the same substrate treebank are node-to-node aligned to the substrate treebank and hence to one another. parser (Rasooli and Tetreault, 2015), a fast arc-eager transition-based projective dependency parser, with beam size of 8. We train only delexicalized parsers, whose input is the sequence of POS tags. Parsing accuracy is evaluated by the unlabeled attachment score (UAS), that is, the fraction of word tokens in held-out (dev) data that are assigned their correct parent. For language modeling, we train simple tri- gram backoff language models with add-1 smooth- ing, and we measure predictive accuracy as the per- plexity of held-out (dev) data. In addition to the generated data, we also pro- vide the parameters θL X of our ordering models; code for training new ordering models; and code for pro- ducing new synthetic trees and synthetic languages. Our code should produce reproducible results across platforms, thanks to Java’s portability and our stan- dard random number seed of 0. Figures 2–3 show how the parsability and per- plexity of a real training language usually get worse when we permute it. We could have discarded low- parsability synthetic languages, on the functionalist grounds that they would be unlikely to survive as natural languages anywhere in the galaxy. 5 The Resource However, the curves in these ﬁgures show that most synthetic languages have parsability and perplexity within the plausible range of natural languages, so we elected to simply keep all of them in our collection. Downloaded from http://www.mitpressjournals.org/doi/pdf/10.1162/tacl_a_00113 by guest on 24 October 2024 5 The Resource In Galactic Dependencies v1.0, or GD, we release real and synthetic treebanks based on UD v1.2. Each synthetic treebank is a modiﬁed work that is freely licensed under the same CC or GPL license as its substrate treebank. We provide all languages of the form S, S[RV/N], S[RN/V], and S[RN/N, RV/V], where the substrate S and the superstrates RN and 5Recall that for each head POS X of language L, we learn a separate ordering model with parameter vector θL X. 495 lang sents tokens T UAS R ar 4K / 6K 119K / 226K 85% 72% / 69% 0.37 cs 5K / 7K 687K / 1173K 94% 81% / 78% 0.38 de 9K / 14K 136K / 270K 94% 84% / 80% 0.47 es 10K / 14K 211K / 382K 94% 85% / 82% 0.32 fr 8K / 15K 154K / 356K 95% 86% / 84% 0.27 hi 9K / 13K 160K / 281K 96% 82% / 82% 0.20 it 9K / 12K 144K / 249K 95% 87% / 84% 0.30 la itt 7K / 15K 87K / 247K 90% 66% / 58% 0.72 no 11K / 16K 135K / 245K 93% 82% / 79% 0.31 pt 5K / 9K 87K / 202K 96% 86% / 84% 0.32 Table 2: Some statistics on the 10 real training lan- guages. When two numbers are separated by “/”, the sec- ond represents the full UD treebank, and the ﬁrst comes from our GD version, which discards non-projective trees and high-fanout trees (n ≥8). UAS is the language’s parsability: the unlabeled attachment score on its dev sentences after training on its train sentences. T is the percentage of GD tokens that are touched by reordering (namely N, V, and their dependents). R ∈[0, 1] measures the freeness of the language’s word order, as the condi- tional cross-entropy of our trained ordering model pθ rel- ative to that of a uniform distribution: R = H(˜p,pθ) H(˜p,punif) = meanx[−log2 pθ(π∗(x)\|x)] meanx[−log2 1/n(x)!] , where x ranges over all N and V tokens in the dev sentences, n(x) is 1 + the number of dependents of x, and π∗(x) is the observed ordering at x. 5 The Resource lang sents tokens T UAS R ar 4K / 6K 119K / 226K 85% 72% / 69% 0.37 cs 5K / 7K 687K / 1173K 94% 81% / 78% 0.38 de 9K / 14K 136K / 270K 94% 84% / 80% 0.47 es 10K / 14K 211K / 382K 94% 85% / 82% 0.32 fr 8K / 15K 154K / 356K 95% 86% / 84% 0.27 hi 9K / 13K 160K / 281K 96% 82% / 82% 0.20 it 9K / 12K 144K / 249K 95% 87% / 84% 0.30 la itt 7K / 15K 87K / 247K 90% 66% / 58% 0.72 no 11K / 16K 135K / 245K 93% 82% / 79% 0.31 pt 5K / 9K 87K / 202K 96% 86% / 84% 0.32 Table 1: The 37 real UD languages. Following the usual setting of rich-to-poor transfer, we take the 10 largest non-English languages (left column) as our pool of real source languages, which we can combine to synthesize new languages. The remaining languages are our low- resource target languages. We randomly hold out 17 non- English languages (right column) as the test languages for our ﬁnal result table. During development, we studied and graphed performance on the remaining 10 languages (middle column)—including English for interpretability. Table 1: The 37 real UD languages. Following the usual setting of rich-to-poor transfer, we take the 10 largest non-English languages (left column) as our pool of real source languages, which we can combine to synthesize new languages. The remaining languages are our low- resource target languages. We randomly hold out 17 non- English languages (right column) as the test languages for our ﬁnal result table. During development, we studied and graphed performance on the remaining 10 languages (middle column)—including English for interpretability. Table 2: Some statistics on the 10 real training lan- guages. When two numbers are separated by “/”, the sec- ond represents the full UD treebank, and the ﬁrst comes from our GD version, which discards non-projective trees and high-fanout trees (n ≥8). UAS is the language’s parsability: the unlabeled attachment score on its dev sentences after training on its train sentences. T is the percentage of GD tokens that are touched by reordering (namely N, V, and their dependents). 6For each of the 10 real training languages, we sampled 9 synthetic languages: 3 N-permuted, 3 V-permuted and 3 {N, V}- permuted. We also included all 10 training + 10 dev languages. 6 Exploratory Data Analysis How do the synthetic languages compare to the real ones? For analysis and experimentation, we parti- tion the real UD languages into train/dev/test (Ta- ble 1). (This is orthogonal to the train/dev/test split of each language’s treebank.) Table 2 shows some properties of the real training languages. An interesting exception in Figure 2 is Latin In this section and the next, we use the Yara 496 Figure 2: Parsability of real versus synthetic languages (deﬁned as in Table 2). The upper graphs are kernel den- sity estimates. Each lower graph is a 1-dimensional scat- terplot, showing the parsability of some real language S (large dot) and all its permuted versions, including the “self-permuted” languages S[S/N] (diamond), S[S/V] (square), and S[S/N, S/V] (medium dot). de da en fr et got bg grc la_proiel grc_proiel cs pt hi la_itt it ar nl no es fi N-superstrate V-superstrate 2 superstrates real Figure 4: Each point represents a language. The color of a synthetic language is the same as its substrate lan- guage. Dev languages are shown in black. This 2- dimensional embedding was constructed using metric multidimensional scaling (Borg and Groenen, 2005) on a symmetrized version of our dissimilarity matrix (which is not itself a metric). The embedded distances are rea- sonably faithful to the symmetrized dissimilarities: met- ric MDS achieves a low value of 0.20 on its “stress” ob- jective, and we ﬁnd that Kendall’s tau = 0.76, meaning that if one pair of languages is displayed as farther apart than another, then in over 7/8 of cases, that pair is in fact more dissimilar. Among the real languages, note the clustering of Italic languages (pt, es, fr, it), Germanic lan- guages (de, no, en, nl, da), Slavic languages (cs, bg), and Uralic languages (et, ﬁ). Outliers are Arabic (ar), the only Afroasiatic language here, and Hindi (hi), the only SOV language, whose permutations are less outr´e than it is. de da en fr et got bg grc la_proiel grc_proiel cs pt hi la_itt it ar nl no es fi N-superstrate V-superstrate 2 superstrates real Figure 2: Parsability of real versus synthetic languages (deﬁned as in Table 2). The upper graphs are kernel den- sity estimates. Each lower graph is a 1-dimensional scat- terplot, showing the parsability of some real language S (large dot) and all its permuted versions, including the “self-permuted” languages S[S/N] (diamond), S[S/V] (square), and S[S/N, S/V] (medium dot). 6 Exploratory Data Analysis Figure 4: Each point represents a language. The color of a synthetic language is the same as its substrate lan- guage. Dev languages are shown in black. This 2- dimensional embedding was constructed using metric multidimensional scaling (Borg and Groenen, 2005) on a symmetrized version of our dissimilarity matrix (which is not itself a metric). The embedded distances are rea- sonably faithful to the symmetrized dissimilarities: met- ric MDS achieves a low value of 0.20 on its “stress” ob- jective, and we ﬁnd that Kendall’s tau = 0.76, meaning that if one pair of languages is displayed as farther apart than another, then in over 7/8 of cases, that pair is in fact more dissimilar. Among the real languages, note the clustering of Italic languages (pt, es, fr, it), Germanic lan- guages (de, no, en, nl, da), Slavic languages (cs, bg), and Uralic languages (et, ﬁ). Outliers are Arabic (ar), the only Afroasiatic language here, and Hindi (hi), the only SOV language, whose permutations are less outr´e than it is. 1 2 3 4 5 6 7 8 real pos synthetic pos real word synthetic word Figure 3: Perplexity of the POS tag sequence, as well as the word sequence, of real versus synthetic languages. Words with count < 10 are mapped to an OOV symbol. real pos synthetic pos real word synthetic word Figure 3: Perplexity of the POS tag sequence, as well as the word sequence, of real versus synthetic languages. Words with count < 10 are mapped to an OOV symbol. diversity—or too radically different to belong in the galaxy of natural languages. Fortunately, we are at neither extreme. Figure 4 visualizes a small sam- ple of 110 languages from our collection.6 For each ordered pair of languages (S, T), we deﬁned the dis- similarity d(S, T) as the decrease in UAS when we parse the dev data of T using a parser trained on S instead of one trained on T. Small dissimilarity (i.e., good parsing transfer) translates to small distance in the ﬁgure. The ﬁgure shows that the permuta- tions of a substrate language (which share its color) can be radically different from it, as we already saw above. Downloaded from http://www.mitpressjournals.org/doi/pdf/10.1162/tacl_a_00113 by guest on 24 October 2024 7The m −n GD treebanks are comparatively impoverished because—in the current GD release—they include only projec- tive sentences (Table 2). The n UD treebanks are unﬁltered. 8The Yara parser can only produce projective parses. It at- tempts to parse all test sentences of T projectively, but sadly ignores non-projective training sentences of S (as can occur for real S). 6 Exploratory Data Analysis Some may be unnatural, but others are sim- ilar to other real languages, including held-out dev (la itt), whose poor parsability—at least by a delex- icalized parser that does not look at word endings— may be due to its especially free word order (Ta- ble 2). When we impose another language’s more consistent word order on Latin, it becomes more parsable. Elsewhere, permutation generally hurts, perhaps because a real language’s word order is globally optimized to enhance parsability. It even hurts slightly when we randomly “self-permute” S trees to use other word orders that are common in S itself! Presumably this is because the authors of the original S sentences chose, or were required, to or- der each constituent in a way that would enhance its parsability in context: see the last paragraph of §3.2. Synthesizing languages is a balancing act. The synthetic languages are not useful if all of them are too conservatively close to their real sources to add 497 languages. Thus Dutch (nl) and Estonian (et) have close synthetic neighbors within this small sample, although they have no close real neighbors. that is, the fraction of word tokens that were as- signed their correct parent. In these experiments (unlike those of §6), we always evaluate fairly on T’s full dev or test set from UD—not just the sen- tences we kept for its GD version (cf. Table 2).8 7 An Experiment The hope is that a large pool will contain at least one language—real or synthetic—that is “close” to T. We have two ways of trying to select a source S with this property: We now illustrate the use of GD by studying how expanding the set of available treebanks can improve a simple NLP method, related to Figure 4. Supervised selection selects the S whose parser achieves the highest UAS on 100 training sen- tences of language T. This requires 100 good trees for T, which could be obtained with a modest investment—a single annotator attempting to follow the UD annotation standards in a consistent way on 100 sentences of T, without writing out formal T- speciﬁc guidelines. (There is no guarantee that se- lecting a parser on training data will choose well for the test sentences of T. We are using a small amount of data to select among many dubious parsers, many of which achieve similar results on the training sen- tences of T. Furthermore, in the UD treebanks, the test sentences of T are sometimes drawn from a dif- ferent distribution than the training sentences.) 7.1 Single-source transfer Dependency parsing of low-resource languages has been intensively studied for years. A simple method is called “single-source transfer”: parsing a target language T with a parser that was trained on a source language S, where the two languages are syntac- tically similar. Such single-source transfer parsers (Ganchev et al., 2010; McDonald et al., 2011; Ma and Xia, 2014; Guo et al., 2015; Duong et al., 2015; Rasooli and Collins, 2015) are not state-of- the-art, but they have shown substantial improve- ments over fully unsupervised grammar induction systems (Klein and Manning, 2004; Smith and Eis- ner, 2006; Spitkovsky et al., 2013). It is permitted for S and T to have different vo- cabularies. The S parser can nonetheless parse T (as in Figure 4)—provided that it is a “delexicalized” parser that only cares about the POS tags of the input words. In this case, we require only that the target sentences have already been POS tagged using the same tagset as S: in our case, the UD tagset. Unsupervised selection selects the S whose training sentences had the best “coverage” of the POS tag sequences in the actual data from T that we aim to parse. More precisely, we choose the S that maximizes pS(tag sequences from T)—in other words, the maximum-likelihood S—where pS is our trigram language model for the tag sequences of S. This approach is loosely inspired by Søgaard (2011). Downloaded from http://www.mitpressjournals.org/doi/pdf/10.1162/tacl_a_00113 by guest on 24 October 2024 7.3 Results We evaluate single-source transfer when the pool of m source languages consists of n real UD lan- guages, plus m −n synthetic GD languages derived by “remixing” just these real languages.7 We try var- ious values of n and m, where n can be as large as 10 (training languages from Table 1) and m can be as large as n × (n + 1) × (n + 1) ≤1210 (see §5). We evaluate single-source transfer when the pool of m source languages consists of n real UD lan- guages, plus m −n synthetic GD languages derived by “remixing” just these real languages.7 We try var- ious values of n and m, where n can be as large as 10 (training languages from Table 1) and m can be as large as n × (n + 1) × (n + 1) ≤1210 (see §5). Given a real target language T from outside the pool, we select a single source language S from the pool, and try to parse UD sentences of T with a parser trained on S. We evaluate the results on T by measuring the unlabeled attachment score (UAS), Our most complete visualization is Figure 5, which we like to call the “kite graph” for its appearance. We plot the UAS on the development treebank of T as a function of n, m, and the selection method. As Appendix A details, each point on this graph is actu- ally an average over 10,000 experiments that make random choices of T (from the UD development lan- guages), the n real languages (from the UD train- ing languages), and the m −n synthetic languages (from the GD languages derived from the n real lan- Given a real target language T from outside the pool, we select a single source language S from the pool, and try to parse UD sentences of T with a parser trained on S. 7.3 Results 20 21 22 23 24 25 26 27 28 29 210 211 m = number of source languages 35 40 45 50 55 60 65 Average UAS (71.38 supervised) n = 10 real languages n = 4 real languages n = 2 real languages n = 1 real languages 23 24 25 26 27 28 29 210 211 0.0 0.2 0.4 0.6 0.8 1.0 n = 10 real languages 22 23 24 25 26 27 n = 4 real languages 21 22 23 24 25 0.0 0.2 0.4 0.6 0.8 1.0 n = 2 real languages 20 21 22 n = 1 real languages oracle selection supervised selection unsupervised selection m = number of source languages Chance of selecting a helpful synthetic language m = number of source languages m = number of source languages Figure 5: Comprehensive results for single-source trans- fer from a pool of m languages (the horizontal axis) synthesized from n real languages. For each color 1, 2, . . . , n, the upper dashed line shows the UAS achieved by supervised selection; the lower solid line shows unsupervised selection; and the shaded area high- lights the difference. The black dashed and solid lines connect the points where m = n, showing how rapidly UAS increases with n when only real languages are used. Figure 6: Chance that selecting a source from m lan- guages achieves strictly better dev UAS than just select- ing from the n real languages. The “selection graph” in Figure 6 visualizes the same experiments in a different way. Here we ask about the fraction of experiments in which using the full pool of m source languages was strictly bet- ter than using only the n real languages. We ﬁnd that when m has increased to its maximum, the full pool nearly always contains a synthetic source lan- guage that gets better results than anything in the real pool. After all, our generation of “random” lan- guages is a scattershot attempt to hit the target: the more languages we generate, the higher our chances of coming close. However, our selection methods only manage to pick a better language in about 60% of those experiments. Each point is the mean dev UAS over 10,000 exper- iments. We use paler lines in the same color and style to show the considerable variance of these UAS scores. 7.3 Results We evaluate the results on T by measuring the unlabeled attachment score (UAS), 498 20 21 22 23 24 25 26 27 28 29 210 211 m = number of source languages 35 40 45 50 55 60 65 Average UAS (71.38 supervised) n = 10 real languages n = 4 real languages n = 2 real languages n = 1 real languages Figure 5: Comprehensive results for single-source trans- fer from a pool of m languages (the horizontal axis) 23 24 25 26 27 28 29 210 211 0.0 0.2 0.4 0.6 0.8 1.0 n = 10 real languages 22 23 24 25 26 27 n = 4 real languages 21 22 23 24 25 0.0 0.2 0.4 0.6 0.8 1.0 n = 2 real languages 20 21 22 n = 1 real languages oracle selection supervised selection unsupervised selection m = number of source languages Chance of selecting a helpful synthetic language Figure 6: Chance that selecting a source from m lan- guages achieves strictly better dev UAS than just select- 23 24 25 26 27 28 29 210 211 0.0 0.2 0.4 0.6 0.8 1.0 n = 10 real languages 22 23 24 25 26 27 n = 4 real languages 21 22 23 24 25 0.0 0.2 0.4 0.6 0.8 1.0 n = 2 real languages 20 21 22 n = 1 real languages oracle selection supervised selection unsupervised selection m = number of source languages Chance of selecting a helpful synthetic language Figure 6: Chance that selecting a source from m lan- guages achieves strictly better dev UAS than just select- ing from the n real languages. Downloaded from http://www.mitpressjournals.org/doi/pdf/10.1162/tacl_a_00113 by guest on 24 October 2024 7.3 Results 61.25 62.46 62.81 65.13 bg 79.80 74.52 79.80 79.80 nl 58.44 57.94 58.44 57.85 et 68.83 72.21 68.83 74.75 la proiel 48.50 49.66 48.50 49.66 da 71.65 71.65 71.65 70.79 en 63.37 61.37 63.37 65.43 grc 42.59 42.59 46.15 47.84 grc proiel 50.76 51.29 52.04 54.06 ﬁ 51 28 55 21 54 46 55 21 unsupervised (weakly) supervised target real +synthetic real +synthetic el 60.07 65.72 65.87 66.98 he 63.39 60.65 62.86 64.28 la 46.79 51.82 56.62 59.06 hr 68.69 68.89 68.69 69.11 sv 74.96 74.96 74.96 74.96 hu 56.41 64.67 56.72 66.22 fa 53.41 58.37 53.41 60.18 ﬁftb 50.90 55.36 53.03 55.86 cu 54.11 57.89 54.11 59.28 ga 53.55 59.38 57.72 64.72 sl 80.41 80.41 80.41 80.41 eu 47.12 48.97 45.35 52.90 ro 66.33 68.01 71.38 69.19 ja ktc 62.51 54.04 62.51 62.49 id 63.79 61.89 65.36 65.36 pl 75.69 74.63 75.69 73.05 ta 63.15 56.20 63.15 63.15 Test Avg. 61.25 62.46 62.81 65.13 bg 79.80 74.52 79.80 79.80 nl 58.44 57.94 58.44 57.85 et 68.83 72.21 68.83 74.75 la proiel 48.50 49.66 48.50 49.66 da 71.65 71.65 71.65 70.79 en 63.37 61.37 63.37 65.43 grc 42.59 42.59 46.15 47.84 grc proiel 50.76 51.29 52.04 54.06 ﬁ 51.28 55.21 54.46 55.21 got 54.98 57.57 54.98 58.66 All Avg. 60.43 61.33 61.71 63.75 en no de es cs it fr la_itt pt hi ar Substrate Language 35 40 45 50 55 60 65 70 75 80 UAS on en superstrate language fr en pt no de it la_itt ar cs hi es Figure 7: UAS performance of different source parsers when applied to English development sentences. The x axis shows the 10 real training languages S, in de- creasing order of their UAS performance (plotted as large black dots). For each superstrate R, we plot a curve showing—for each substrate S—the best UAS of the lan- guages S[R/N], S[R/V] and S[R/N, R/V]. The points where R = S are specially colored in black; these are in- stances of self-permutation (§5). We also add “cheating results” where English itself is used as the substrate (left column) and/or the superstrate (solid black line at top). Thus, the large black dot at the upper left is a supervised English parser. uate on test sentences. The comparison is similar to the comparison in the selection graph: do the synthetic treebanks add value? 7.3 Results We use our largest source pools, n = 10 and m = 1210. With super- vised selection, selecting the source language from the full pool of m options (not just the n real lan- guages) tends to achieve signiﬁcantly better UAS on the target language, often dramatically so. On av- erage, the UAS on the test languages increases by 2.3 percentage points, and this increase is statisti- cally signiﬁcant across these 17 data points. Even with unsupervised selection, UAS still increases by 1.2 points on average, but this difference could be a chance effect. Table 3: Our ﬁnal comparison on the 17 test languages appears in the upper part of this table. We ask whether single-source transfer to these 17 real target languages is improved by augmenting the source pool of 10 real lan- guages with 1200 synthetic languages. When different languages are selected in these two settings, we boldface the setting with higher test UAS, or both settings if they are not signiﬁcantly different (paired permutation test by sentence, p < 0.05). For completeness, we extend the ta- ble with the 10 development languages. The “Avg.” lines report the average of 17 test or 27 test+dev languages. The two supervised-selection averages are signiﬁcantly different (paired permutation test by language, p < 0.05). The results above use gold POS tag sequences for T. These may not be available if T is a low-resource language; see Appendix B for a further experiment. that we would beneﬁt from ﬁnding ways to gener- ate even more synthetic languages. Diversity of lan- guages seems to be crucial, since adding new real languages improves performance much faster than remixing existing languages. This suggests that we should explore making more extensive changes to 7.3 Results The points where R = S are specially colored in black; these are in- stances of self-permutation (§5). We also add “cheating results” where English itself is used as the substrate (left column) and/or the superstrate (solid black line at top). Thus, the large black dot at the upper left is a supervised English parser. uate on test sentences. The comparison is similar to the comparison in the selection graph: do the synthetic treebanks add value? We use our largest source pools, n = 10 and m = 1210. With super- unsupervised (weakly) supervised target real +synthetic real +synthetic el 60.07 65.72 65.87 66.98 he 63.39 60.65 62.86 64.28 la 46.79 51.82 56.62 59.06 hr 68.69 68.89 68.69 69.11 sv 74.96 74.96 74.96 74.96 hu 56.41 64.67 56.72 66.22 fa 53.41 58.37 53.41 60.18 ﬁftb 50.90 55.36 53.03 55.86 cu 54.11 57.89 54.11 59.28 ga 53.55 59.38 57.72 64.72 sl 80.41 80.41 80.41 80.41 eu 47.12 48.97 45.35 52.90 ro 66.33 68.01 71.38 69.19 ja ktc 62.51 54.04 62.51 62.49 id 63.79 61.89 65.36 65.36 pl 75.69 74.63 75.69 73.05 ta 63.15 56.20 63.15 63.15 Test Avg. 61.25 62.46 62.81 65.13 bg 79.80 74.52 79.80 79.80 nl 58.44 57.94 58.44 57.85 et 68.83 72.21 68.83 74.75 la proiel 48.50 49.66 48.50 49.66 da 71.65 71.65 71.65 70.79 en 63.37 61.37 63.37 65.43 grc 42.59 42.59 46.15 47.84 grc proiel 50.76 51.29 52.04 54.06 ﬁ 51.28 55.21 54.46 55.21 got 54.98 57.57 54.98 58.66 All Avg. 60.43 61.33 61.71 63.75 Table 3: Our ﬁnal comparison on the 17 test languages appears in the upper part of this table. We ask whether en no de es cs it fr la_itt pt hi ar Substrate Language 35 40 45 50 55 60 65 70 75 80 UAS on en superstrate language fr en pt no de it la_itt ar cs hi es Figure 7: UAS performance of different source parsers when applied to English development sentences. The x axis shows the 10 real training languages S, in de- creasing order of their UAS performance (plotted as large black dots). For each superstrate R, we plot a curve showing—for each substrate S—the best UAS of the lan- guages S[R/N], S[R/V] and S[R/N, R/V]. The points where R = S are specially colored in black; these are in- stances of self-permutation (§5). 7.3 Results We also add “cheating results” where English itself is used as the substrate (left column) and/or the superstrate (solid black line at top). Thus, the large black dot at the upper left is a supervised English parser. en no de es cs it fr la_itt pt hi ar Substrate Language 35 40 45 50 55 60 65 70 75 80 UAS on en superstrate language fr en pt no de it la_itt ar cs hi es Figure 7: UAS performance of different source parsers when applied to English development sentences. The x axis shows the 10 real training languages S, in de- creasing order of their UAS performance (plotted as large black dots). For each superstrate R, we plot a curve showing—for each substrate S—the best UAS of the lan- guages S[R/N], S[R/V] and S[R/N, R/V]. The points where R = S are specially colored in black; these are in- stances of self-permutation (§5). We also add “cheating results” where English itself is used as the substrate (left column) and/or the superstrate (solid black line at top). Thus, the large black dot at the upper left is a supervised English parser. unsupervised (weakly) supervised target real +synthetic real +synthetic el 60.07 65.72 65.87 66.98 he 63.39 60.65 62.86 64.28 la 46.79 51.82 56.62 59.06 hr 68.69 68.89 68.69 69.11 sv 74.96 74.96 74.96 74.96 hu 56.41 64.67 56.72 66.22 fa 53.41 58.37 53.41 60.18 ﬁftb 50.90 55.36 53.03 55.86 cu 54.11 57.89 54.11 59.28 ga 53.55 59.38 57.72 64.72 sl 80.41 80.41 80.41 80.41 eu 47.12 48.97 45.35 52.90 ro 66.33 68.01 71.38 69.19 ja ktc 62.51 54.04 62.51 62.49 id 63.79 61.89 65.36 65.36 pl 75.69 74.63 75.69 73.05 ta 63.15 56.20 63.15 63.15 Test Avg. Downloaded from http://www.mitpressjournals.org/doi/pdf/10.1162/tacl_a_00113 by guest on 24 October 2024 7.3 Results These essentially delimit the interdecile range from the 10th to the 90th percentile of UAS score. However, if the plot shows a mean of 57, an interdecile range from 53 to 61 actually means that the middle 80% of experiments were within ±4 percentage points of the mean UAS for their target language. (In other words, before computing this range, we adjust each UAS score for target T by sub- tracting the mean UAS from the experiments with target T, and adding back the mean UAS from all 10,000 exper- iments (e.g., 57).) Figure 7 offers a ﬁne-grained look at which real and synthetic source languages S succeeded best when T = English. Each curve shows a differ- ent superstrate, with the x-axis ranging over sub- strates. (The ﬁgure omits the hundreds of synthetic source languages that use two distinct superstrates, RV ̸= RN.) Real languages are shown as solid black dots, and are often beaten by synthetic languages. For comparison, this graph also plots results that “cheat” by using English supervision. Notice that on the n = 10 curve, there is no variation among experiments either at the minimum m (where the pool always consists of all 10 real languages) or at the maximum m (where the pool always consists of all 1210 galactic languages). guages). We see from the black lines that increas- ing the number of real languages n is most beneﬁ- cial. But crucially, when n is ﬁxed in practice, grad- ually increasing m by remixing the real languages does lead to meaningful improvements. This is true for both selection methods. Supervised selection is markedly better than unsupervised. The above graphs are evaluated on development sentences in development languages. 7.3 Results For our ﬁnal results, Table 3, we ﬁnally allow ourselves to try transferring to the UD test languages, and we eval- 499 unsupervised (weakly) supervised target real +synthetic real +synthetic el 60.07 65.72 65.87 66.98 he 63.39 60.65 62.86 64.28 la 46.79 51.82 56.62 59.06 hr 68.69 68.89 68.69 69.11 sv 74.96 74.96 74.96 74.96 hu 56.41 64.67 56.72 66.22 fa 53.41 58.37 53.41 60.18 ﬁftb 50.90 55.36 53.03 55.86 cu 54.11 57.89 54.11 59.28 ga 53.55 59.38 57.72 64.72 sl 80.41 80.41 80.41 80.41 eu 47.12 48.97 45.35 52.90 ro 66.33 68.01 71.38 69.19 ja ktc 62.51 54.04 62.51 62.49 id 63.79 61.89 65.36 65.36 pl 75.69 74.63 75.69 73.05 ta 63.15 56.20 63.15 63.15 Test Avg. 61.25 62.46 62.81 65.13 bg 79.80 74.52 79.80 79.80 nl 58.44 57.94 58.44 57.85 et 68.83 72.21 68.83 74.75 la proiel 48.50 49.66 48.50 49.66 da 71.65 71.65 71.65 70.79 en 63.37 61.37 63.37 65.43 grc 42.59 42.59 46.15 47.84 grc proiel 50.76 51.29 52.04 54.06 ﬁ 51.28 55.21 54.46 55.21 got 54.98 57.57 54.98 58.66 All Avg. 60.43 61.33 61.71 63.75 Table 3: Our ﬁnal comparison on the 17 test languages appears in the upper part of this table. We ask whether single-source transfer to these 17 real target languages is improved by augmenting the source pool of 10 real lan- guages with 1200 synthetic languages. When different languages are selected in these two settings, we boldface the setting with higher test UAS, or both settings if they are not signiﬁcantly different (paired permutation test by sentence, p < 0.05). For completeness, we extend the ta- ble with the 10 development languages. The “Avg.” lines report the average of 17 test or 27 test+dev languages. The two supervised-selection averages are signiﬁcantly different (paired permutation test by language, p < 0.05). en no de es cs it fr la_itt pt hi ar Substrate Language 35 40 45 50 55 60 65 70 75 80 UAS on en superstrate language fr en pt no de it la_itt ar cs hi es Figure 7: UAS performance of different source parsers when applied to English development sentences. The x axis shows the 10 real training languages S, in de- creasing order of their UAS performance (plotted as large black dots). For each superstrate R, we plot a curve showing—for each substrate S—the best UAS of the lan- guages S[R/N], S[R/V] and S[R/N, R/V]. 9Our current handling of punctuation produces unnatural re- sults, and not merely because we treat all tokens with tag PUNCT as interchangeable. Proper handling of punctuation and capital- ization would require more than just reordering. For example, “Jane loves her dog, Lexie.” should reorder into “Her dog, Lexie, Jane loves.”, which has an extra comma and an extra capital. Accomplishing this would require ﬁrst recovering a richer tree for the original sentence, in which the appositive Lexie is bracketed by a pair of commas and the name Jane is doubly capitalized. These extra tokens were not apparent in the original sentence’s surface form because the ﬁ- nal comma was absorbed into the adjacent period, and the start- of-sentence capitalization was absorbed into the intrinsic capi- talization of Jane (Nunberg, 1990). The tokenization provided by the UD treebanks unfortunately does not attempt to undo these orthographic processes, even though it undoes some mor- phological processes such as contraction. 7.4 Discussion We should extend it to allow non-projective trees as well—for exam- ple, by pseudo-projectivizing the substrate treebank (Nivre and Nilsson, 2005) and then deprojectivizing it after reordering. 2. Currently, our reordering method only gener- ates projective dependency trees. We should extend it to allow non-projective trees as well—for exam- ple, by pseudo-projectivizing the substrate treebank (Nivre and Nilsson, 2005) and then deprojectivizing it after reordering. 3. The treebanks of real languages can typically be augmented with larger unannotated corpora in those languages (Majliˇs, 2011), which can be used to train word embeddings and language models, and can also be used for self-training and bootstrapping methods. We plan to release comparable unanno- tated corpora for our synthetic languages, by au- 3. The treebanks of real languages can typically be augmented with larger unannotated corpora in those languages (Majliˇs, 2011), which can be used to train word embeddings and language models, and can also be used for self-training and bootstrapping methods. We plan to release comparable unanno- tated corpora for our synthetic languages, by au- Downloaded from http://www.mitpressjournals.org/doi/pdf/10.1162/tacl_a_00113 by guest on 24 October 2024 7.4 Discussion Many of the curves in Figures 5–6 still seem to be increasing steadily at maximum m, which suggests 500 Downloaded from http://www.mitpressjournals.org/doi/pdf/10.1162/tacl_a_00113 by guest on 24 October 2024 the UD treebanks (see §8). the UD treebanks (see §8). the UD treebanks (see §8). does not increase the diversity of the pool as much as when we add new real languages. Thus, we are particularly interested in generating a wider range of synthetic languages. We could condition reorderings on the surrounding tree structure, as noted in §3.2. We could choose reordering parameters θX more adventurously than by drawing them from a single known superstrate language. We could go beyond reordering, to systematically choose what function words (determiners, prepositions, particles), func- tion morphemes, or punctuation symbols9 should appear in the synthetic tree, or to otherwise alter the structure of the tree (Dorr, 1993). These options may produce implausible languages. To mitigate this, we could ﬁlter or reweight our sample of syn- thetic languages—via rejection sampling or impor- tance sampling—so that they are distributed more like real languages, as measured by their parsabili- ties, dependency lengths, and estimated WALS fea- tures (Dryer and Haspelmath, 2013). Surprisingly, Figures 5–6 show improvements even when n = 1. Evidently, self-permutation of a single language introduces some useful variety, per- haps by transporting specialized word orders (e.g., English still allows some limited V2 constructions) into contexts where the source language would not ordinarily allow them but the target language does. Figure 5 shows why unsupervised selection is considerably worse on average than supervised se- lection. Its 90th percentile is comparable, but at the 10th percentile—presumably representing ex- periments where no good sources are available—the unsupervised heuristic has more trouble at choos- ing among the mediocre options. The supervised method can actually test these options using the true loss function. Figure 7 is interesting to inspect. English is es- sentially a Germanic language with French inﬂuence due to the Norman conquest, so it is reassuring that German and French substrates can each be improved by using the other as a superstrate. We also see that Arabic and Hindi are the worst source languages for English, but that Hindi[Arabic/V] is considerably better. This is because Hindi is reasonably similar to English once we correct its SOV word order to SVO (via almost any superstrate). 2. Currently, our reordering method only gener- ates projective dependency trees. 8 Conclusions and Future Work , L′′ n′} 10: m ←\|P\| 11: Dsup ←Dsup ∪ {(n, m, UASsup(P, T))} 12: Dunsup ←Dunsup ∪ {(n, m, UASunsup(P, T))} 13: return (Dsup, Dunsup) 4. At present, all languages derived from an En- glish substrate use the English vocabulary. In the future, we plan to encipher that vocabulary sepa- rately for each synthetic language, perhaps choosing a cipher so that the result loosely conforms to the realistic phonotactics and/or orthography of some superstrate language. This would let multilingual methods exploit lexical features without danger of overﬁtting to speciﬁc lexical items that appear in many synthetic training languages. Alphabetic ci- phers can preserve features of words that are poten- tially informative for linguistic structure discovery: their cooccurrence statistics, their length and phono- logical shape, and the sharing of substrings among morphologically related words. 5. Finally, we note that this paper has focused on generating a broadly reusable collection of synthetic treebanks. For some applications (including single- source transfer), one might wish to tailor a synthetic language on demand, e.g., starting with one of our treebanks but modifying it further to more closely match the surface statistics of a given target lan- guage (Dorr et al., 2002). In our setup, this would involve actively searching the space of reordering parameters, using algorithms such as gradient ascent or simulated annealing. B Experiment with Noisy Tags Table 4 repeats the single-source transfer experiment using noisy automatic POS tags for T for both parser input and unsupervised selection. We obtained the tags using RDRPOSTagger (Nguyen et al., 2014) trained on just 100 gold-tagged sentences (the same set used for supervised selection). The low tagging accuracy does considerably degrade UAS and mud- dies the usefulness of the synthetic sources. We conclude by revisiting our opening point. Un- supervised discovery of linguistic structure is difﬁ- cult. We often do not know quite what function to maximize, or how to globally maximize it. If we could make labeled languages as plentiful as labeled images, then we could treat linguistic structure dis- covery as a problem of supervised prediction—one that need not succeed on all formal languages, but which should generalize at least to the domain of possible human languages. tag unsupervised (weakly) superv. target real +synth real +synth bg 78.33 53.24 55.08 53.24 53.24 nl 71.70 39.40 38.99 42.42 42.75 et 72.88 45.19 54.81 56.07 55.09 la proiel 71.83 37.25 38.26 37.25 38.10 da 78.04 47.98 43.40 47.98 45.89 en 77.33 48.29 44.40 48.29 48.15 grc 68.80 32.15 32.15 33.52 34.36 grc proiel 72.93 42.46 41.39 43.49 44.19 ﬁ 65.65 29.59 28.81 36.85 36.90 got 76.66 44.77 44.05 44.77 46.83 Avg. 73.42 42.03 42.13 44.39 44.55 Downloaded from http://www.mitpressjournals.org/doi/pdf/10.1162/tacl_a_00113 by guest on 24 October 2024 8 Conclusions and Future Work This paper is the ﬁrst release of a novel resource, the Galactic Dependencies treebank collection, that may unlock a wide variety of research opportuni- ties (discussed in §1). Our empirical studies show that the synthetic languages in this collection remain somewhat natural while improving the diversity of the collection. As a simplistic but illustrative use of the resource, we carefully evaluated its impact on the naive technique of single-source transfer pars- ing. We found that performance could consistently be improved by adding synthetic languages to the pool of sources, assuming gold POS tags. There are several non-trivial opportunities for im- proving and extending our treebank collection in fu- ture releases. 1. Our current method is fairly conservative, only synthesizing languages with word orders already at- tested in our small collection of real languages. This 1. Our current method is fairly conservative, only synthesizing languages with word orders already at- tested in our small collection of real languages. This 501 tomatically parsing and permuting the unnanotated corpora of their substrate languages. Algorithm 1 Data collection for one graph Algorithm 1 Data collection for one graph Input: Sets T (target languages), S (real source lan- guages), S′ (synthetic source languages) Output: Sets of data points Dsup, Dunsup 1: procedure COLLECTDATA 2: D ←∅ 3: Sample a target language T from T 4: L ←random.shufﬂe(S −{T}) 5: L′ ←random.shufﬂe(S′) 6: for n = 1 to \|L\| do 7: L′′ ←a ﬁltered version of L′ that excludes languages with substrates or super- strates outside {L1, . . . , Ln} 8: for n′ = 1 to \|L′′\| do 9: P ←{L1, . . . , Ln, L′′ 1, . . . , L′′ n′} 10: m ←\|P\| 11: Dsup ←Dsup ∪ {(n, m, UASsup(P, T))} 12: Dunsup ←Dunsup ∪ {(n, m, UASunsup(P, T))} 13: return (Dsup, Dunsup) Input: Sets T (target languages), S (real source lan- guages), S′ (synthetic source languages) Output: Sets of data points Dsup, Dunsup 1: procedure COLLECTDATA 2: D ←∅ 3: Sample a target language T from T 4: L ←random.shufﬂe(S −{T}) 5: L′ ←random.shufﬂe(S′) 6: for n = 1 to \|L\| do 7: L′′ ←a ﬁltered version of L′ that excludes languages with substrates or super- strates outside {L1, . . . , Ln} 8: for n′ = 1 to \|L′′\| do 9: P ←{L1, . . . , Ln, L′′ 1, . . . 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https://openalex.org/W2041285308	https://europepmc.org/articles/pmc3654458?pdf=render	English	null	Influenza evolution navigates stability valleys	eLife	2,013	cc-by	2,112	Influenza evolution navigates stability valleys By reconstructing how an influenza protein collected in 1968 might have evolved into one collected in 2007, researchers have obtained new insights into the interactions between genetic mutations. MARY M RORICK AND MERCEDES PASCUAL should be highly contingent on the genomic con text in which they occur, and they described these predictions using the concept of a fitness land scape. They observed that when the selective effect of a point mutation is highly dependent on a protein’s genotype, the fitness landscape is not smooth with a single peak; rather, it is rough and contains various local peaks, with valleys in between. Because natural selection avoids these valleys and instead climbs local peaks in the fit ness landscape, a rough terrain implies path dependency: not all evolutionary trajectories lead to the same place. Out of this work came the important prediction that evolution proceeds in a way that is strongly constrained by the order in which mutations occur (Maynard Smith, 1970; Kimura, 1985). Related research article Gong LI, Suchard MA, Bloom JD. 2013. Stability-mediated epistasis constrains the evolution of an influenza protein. eLife 2:e00631. doi: 10.7554/eLife.00631 Image Reconstructing the evolutionary path of an influenza protein Related research article Gong LI, Suchard MA, Bloom JD. 2013. Stability-mediated epistasis constrains the evolution of an influenza protein. eLife 2:e00631. doi: 10.7554/eLife.00631 Image Reconstructing the evolutionary path of an influenza protein Related research article Gong LI, Suchard MA, Bloom JD. 2013. Stability-mediated epistasis constrains the evolution of an influenza protein. eLife 2:e00631. doi: 10.7554/eLife.00631 Related research article Gong LI, Suchard MA, Bloom JD. 2013. Stability-mediated epistasis constrains the evolution of an influenza protein. eLife 2:e00631. doi: 10.7554/eLife.00631 A A n individual’s phenotype is shaped by a multitude of interactions: those among genes and those with the environment. This means that a mutation’s fitness, and likeli hood of evolutionary success, is largely a function of the specific genetic background and environ ment in which it resides. For example, a mutation that is advantageous within one genome may be detrimental or even lethal in another. This occurs because the effects of a gene on an organism’s phenotype often depend on the actions of many other genes—a phenomenon known as epistasis. Complex epistatic interactions limit the pathways along which evolution can proceed by natural selection. Copyright Rorick and Pascual. This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited. elife.elifesciences.org elife.elifesciences.org INSIGHT elife.elifesciences.org PROTEIN EVOLUTION Influenza evolution navigates stability valleys This lends support to the classic models for how evolution should occur when the fitness landscape contains various valleys and peaks (Figure 1; Maynard Smith, 1970; Kimura, 1985). In other words, mutations that increase the stability of the influ enza nucleoprotein enable it to tolerate other mutations that decrease its stability, and this phe nomenon appears to account for most of the epistasis they observe. Figure 1. Protein stability determines the accessibility of multiple possible evolutionary paths separating two protein variants. The results of Gong et al. suggest that increasing the stability of a protein (y axis) above a certain threshold has no effect on fitness, which means that protein evolution can occur along multiple alternative routes. However, fitness decreases rapidly when stability drops below the threshold (red region), in which case evolution will select for mutations that increase stability. Four sites within a protein are shown, with two possible states for each site, indicated by the presence/absence of the asterisk. Single point mutations are indicated by solid arrows, double mutations are indicated by dashed arrows. Consider the case where a* and c* are mutations that help a protein to evade the immune system and, like the great majority of random mutations, they reduce the stability of the protein relative to the parent protein (abcd). However, mutations that increase stability, such as b* and d, can compensate for mutations that reduce stability, which means that several proteins containing a and/or c* can remain above the stability threshold required for optimal fitness. The results also suggest that once the protein reaches a threshold level of stability, mutations that increase the stability further have no effect on fitness. This is in contrast to the results of a related study (DePristo et al., 2005), and it suggests that extra stability might accumulate through neutral evolutionary processes. Epistatically constrained mutations also seem to have a disproportionately large role in helping proteins escape detection by the immune system. This is suggested by the fact that the three epistatically constrained mutations in influenza nucleoprotein occur more frequently in immune system targets than do the other muta tions in the evolutionary path. The results of Gong et al. paint the following picture of nucleoprotein evolution: natural selec tion maintains stability above the minimum thresh old required for a protein to be viable, but evolution allows for occasional mutations that increase the stability well above this threshold. Influenza evolution navigates stability valleys Now, in eLife, Lizhi Gong, Marc Suchard and Jesse Bloom—at the Fred Hutchinson Cancer Research Center and the University of California Los Angeles—provide new insights into a remark ably simple mechanism that can explain many of the interactions that appear to be shaping the evolutionary path of a human influenza virus pro tein (Gong et al., 2013). Recently, it has become possible to study these processes experimentally. Data sets of genetic sequences for fast-evolving viruses sampled for many time points make it possible to recapitu late with confidence the chain of mutations that separates any two protein variants. Moreover, advances in site-directed mutagenesis allow his torical protein variants to be recreated, so that their fitness can be compared with that of existing proteins. Gong et al. use computational modelling to infer the most likely evolutionary paths between two variants of the influenza nucleoprotein: one collected in 1968 and the other in 2007. In most cases, they are also able to determine the order in which the mutations occurred. Next, they intro duce each of these mutations, one at a time, into the original protein sequence. They also recreate Several decades ago, John Maynard Smith and others predicted that mutation fitness effects Rorick and Pascual. eLife 2013;2:e00842. DOI: 10.7554/eLife.00842 1 of 3 Protein Insight Protein evolution \| Influenza evolution navigates stability valleys can be explained in terms of the individual effect of each mutation on the stability of the protein. Gong et al. show that all three of the epi statically constrained mutations reduce the sta bility of the protein, and that all were preceded by, or occurred almost concurrently with, one or more mutations that increase stability. This lends support to the classic models for how evolution should occur when the fitness landscape contains various valleys and peaks (Figure 1; Maynard Smith, 1970; Kimura, 1985). In other words, mutations that increase the stability of the influ enza nucleoprotein enable it to tolerate other mutations that decrease its stability, and this phe nomenon appears to account for most of the epistasis they observe. can be explained in terms of the individual effect of each mutation on the stability of the protein. Gong et al. show that all three of the epi statically constrained mutations reduce the sta bility of the protein, and that all were preceded by, or occurred almost concurrently with, one or more mutations that increase stability. Rorick and Pascual. eLife 2013;2:e00842. DOI: 10.7554/eLife.00842 Influenza evolution navigates stability valleys While this extra stability has no immediate conse quences for fitness, it does make a wide range of destabilizing mutations newly accessible to the protein, some of which could potentially contrib ute to immune escape. Selection will favour these variants if the stability of the protein remains above the threshold. However, if protein sta bility falls below the threshold, selection for sta bilizing mutations will occur. the intermediate proteins that arose at each stage of the evolutionary path. By comparing the fitness of each mutated protein with that of the corresponding natural intermediate in which the mutation first appeared, they manage to identify three mutations that would have been delete rious for the original protein, but have no adverse impacts on the natural intermediates: such muta tions are said to be epistatically constrained. Prior studies have anecdotally demonstrated the existence of such mutations in natural protein evolutionary paths. However, Gong et al. go fur ther by investigating nearly all naturally occurring mutations along a relatively long evolutionary path (consisting of 39 steps), and by examining the causes of these epistatic interactions. They determine the stability of each nucleoprotein var iant by measuring its melting temperature, with higher melting temperatures corresponding to more stable proteins (Figure 1). They find that— with one exception—the combined effect of a group of mutations on the fitness of a protein The work of Gong, Suchard and Bloom helps us move toward a better understanding of how epistasis shapes protein evolution. The insights gained from empirical studies such as these will aid the development of conceptual and compu tational models of evolution. This information will be particularly relevant for rapidly evolving viruses in the new field of ‘phylodynamics’, at the inter face between phylogenetics and epidemiology (Grenfell et al., 2004). A tantalizing possibility is that mechanisms similar to the ones presented by Rorick and Pascual. eLife 2013;2:e00842. DOI: 10.7554/eLife.00842 2 of 3 Insight Insight Protein evolution \| Influenza evolution navigates stability valleys Gong et al. underlie the immune escape of the protein hemagglutinin—a surface antigen for influenza subtype H3N2 and a major target of the human immune system (Koelle et al., 2006). Rorick and Pascual. eLife 2013;2:e00842. DOI: 10.7554/eLife.00842 Influenza evolution navigates stability valleys Computational models in the field of phylody namics have so far largely focused on mutations that influence immune escape but have little or no impact on other biological functions: there is a clear need to study the evolution of viruses while considering that each mutation may influence both the basic functioning of a protein as well as its appearance to the immune system. How evo lution mediates such trade-offs is an open area of research that is informed by this study. Mercedes Pascual is at the Department of Ecology and Evolutionary Biology, University of Michigan, Ann Arbor, United States pascual@umich.edu Mercedes Pascual is at the Department of Ecology and Evolutionary Biology, University of Michigan, Ann Arbor, United States pascual@umich.edu Competing interests: The authors declare that no competing interests exist. Published 14 May 2013 References References DePristo MA, Weinreich DM, Hartl DL. 2005. 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https://openalex.org/W2568928876	https://www.mdpi.com/1996-1944/10/1/41/pdf?version=1483690594	English	null	A Study of Dip-Coatable, High-Capacitance Ion Gel Dielectrics for 3D EWOD Device Fabrication	Materials	2,017	cc-by	18,443	A Study of Dip-Coatable, High-Capacitance Ion Gel Dielectrics for 3D EWOD Device Fabrication Carlos E. Clement, Dongyue Jiang, Si Kuan Thio and Sung-Yong Park * Department of Mechanical Engineering, National University of Singapore, Block EA, #07-08, 9 Engineering Drive 1, Singapore 117576, Singapore; mpecec@nus.edu.sg (C.E.C.); mpejd@nus.edu.sg (D.J.); sikuan@u.nus.edu (S.K.T.) * Correspondence: mpeps@nus.edu.sg; Tel.: +65-6601-3405 * Correspondence: mpeps@nus.edu.sg; Tel.: +65-6601-3405 Academic Editor: Silvia Schintke Received: 4 November 2016; Accepted: 26 December 2016; Published: 5 January 2017 Abstract: We present a dip-coatable, high-capacitance ion gel dielectric for scalable fabrication of three-dimensional (3D) electrowetting-on-dielectric (EWOD) devices such as an n × n liquid prism array. Due to the formation of a nanometer-thick electric double layer (EDL) capacitor, an ion gel dielectric offers two to three orders higher speciﬁc capacitance (c ≈10 µF/cm2) than that of conventional dielectrics such as SiO2. However, the previous spin-coating method used for gel layer deposition poses several issues for 3D EWOD device fabrication, particularly when assembling multiple modules. Not only does the spin-coating process require multiple repetitions per module, but the ion gel layer also comes in risks of damage or contamination due to handling errors caused during assembly. In addition, it was observed that the chemical formulation previously used for the spin-coating method causes the surface defects on the dip-coated gel layers and thus leads to poor EWOD performance. In this paper, we alternatively propose a dip-coating method with modiﬁed gel solutions to obtain defect-free, functional ion gel layers without the issues arising from the spin-coating method for 3D device fabrication. A dip-coating approach offers a single-step coating solution with the beneﬁts of simplicity, scalability, and high throughput for deposition of high-capacitance gel layers on non-planar EWOD devices. An ion gel solution was prepared by combining the [EMIM][TFSI] ionic liquid and the [P(VDF-HFP)] copolymer at various wt % ratios in acetone solvent. Experimental studies were conducted to fully understand the effects of chemical composition ratios in the gel solution and how varying thicknesses of ion gel and Teﬂon layers affects EWOD performance. The effectiveness and potentiality of dip-coatable gel layers for 3D EWOD devices have been demonstrated through fabricating 5 × 1 arrayed liquid prisms using a single-step dip-coating method. Each prism module has been individually controlled to achieve spatial beam steering without the need for bulky mechanical moving parts. Keywords: electrowetting-on-dielectric; dip-coating; ion gels; liquid prism; high capacitance materials materials materials 1. Ion Gel Dielectrics for EWOD Electrowetting-on-dielectric (EWOD) is an emerging small-scale liquid handling technology [1,2]. With an electric potential applied between a liquid droplet and a solid electrode, the charge redistribution modiﬁes the surface energy and thus decreases the contact angle on the solid surface [3–5]. The Young-Lippmann equation mathematically describes the relationship between the applied voltage (V) and the resultant contact angle (θ) as [6]: cos θ = cos θ0 + 1 2γcV2 (1) (1) Materials 2017, 10, 41; doi:10.3390/ma10010041 www.mdpi.com/journal/materials Materials 2017, 10, 41 2 of 14 where θ0 represents the initial contact angle of the droplet at zero potential, γ is the interfacial tension between two immiscible liquids, and c is the capacitance per unit area. For many practical EWOD applications, the use of high-capacitance dielectrics is critical to induce large surface tension modulation at a given voltage input. To meet this requirement, various dielectrics such as silicon dioxide (SiO2), aluminum oxide (Al2O3), and tantalum pentoxide (Ta2O5) have been used [7–10]. However, these dielectric materials are fabricated by conventional integrated circuit (IC) processes such as plasma enhanced chemical vapor deposition (PECVD), atomic layer deposition (ALD) and sputtering; they are typically time-consuming and require complex and expensive laboratory setups such as high vacuum facilities. To avoid the need of such IC processes, polymer-based dielectric materials such as PDMS and SU-8 have been alternatively used via a low-cost, spin-coating method [11–13]. Nonetheless, not only do these materials provide a lower permittivity than that of SiO2, but using spin coating method would produce a thick dielectric layer in the order of tens or hundreds of micrometers. Consequently, a dielectric layer with such thickness and low permittivity would result in a very low capacitance and thus poor EWOD performance. To overcome these issues arising from conventional dielectrics commonly used for EWOD applications, our group recently reported an ion gel dielectric that provides two to three orders higher speciﬁc capacitance (c ≈10 µF/cm2) than that of conventional dielectrics, while being simply fabricated by a spin-coating method [4]. Such a high capacitance of the ion gel results from the nanometer-thick electric double layer (EDL) capacitor formed by free counter-ions compactly accumulated at the interface [14–17]. With the spin-coated gel layers, we had demonstrated full contact angle modulation at a lower voltage than that of conventional dielectrics such as SiO2 and Al2O3 [4]. 1. Ion Gel Dielectrics for EWOD Despite of our study on ion gel dielectrics showing low-voltage EWOD performance, the spin-coating method was not suited for 3D devices with non-planar structures. The following section discusses the problems of the previous spin-coating methodology, particularly for 3D EWOD applications such as n × n arrayed liquid prisms. 2. Ion Gel Layer Coating for 3D EWOD Device Fabrication A rectangular prism is fabricated by assembling four conductive sidewalls coated with dielectric and hydrophobic layers and ﬁlled with two immiscible liquids. The electrowetting effect is used to adjust the prism apex angle ϕ by applying bias voltages (VL and VR) to the left and right sidewalls, separately. Beam steering of the incoming light can be achieved at the interface of two media whose refractive indices are different (nair ̸= n1 ̸= n2). The left ﬁgure shows the assembled prism device with a 10 × 10 mm2 aperture area before two liquids are placed. modules to form 3D structures, the previous spin-coated method poses several issues. Figure 2a illustrates the spin-coating procedure used to fabricate a single prism as an example of 3D EWOD devices coated with the gel layers. Considering that only a single substrate can be spin-coated at a time, the gel coating process has to be individually repeated for each of the four sidewalls. Next, they are carefully assembled into a hollow rectangular structure with four sidewalls whereby only the inner surfaces are coated with gel layers. However, during their assembly, the gel layer is at risk of damage or contamination from handling errors, which is detrimental to device performance and reliability. While such issues are already present for a single prism module, they are further compounded when fabricating multiple modules such as the case for n × n prism arrays (Figure 2b). The spin-coating step must be repeated four times per module, i.e., 4n2, and the more complicated assembling procedure increases the likelihood of ion gel damages. In addition, the chemical formulation (i.e., high concentrations of ionic liquids in gel solutions) previously used for a spin- coating method [4] causes the surface defects for the thin dip-coated layers, which led to poor EWOD performance. To obtain defect-free, robust gel layers without the issues arising from the spin-coating method aforementioned, we propose a dip-coating approach that not only offers a single-step coating solution to cover the gel layer over the entire surfaces of 3D structures, but also fully eliminates the risk of gel layer damage or contamination brought about during assembly. For the arrayed prism fabrication, substrates with finger-like protrusions are first assembled to form the hollow n × n arrayed structure as shown in Figure 2c. 2. Ion Gel Layer Coating for 3D EWOD Device Fabrication The whole assembly is then dip-coated to achieve the gel layer coating across the entire exposed surface area without requiring any additional steps as compared to the spin-coating method (Figure 2d,e). In this paper, a dip-coatable high-capacitance ion gel dielectric is presented for scalable fabrication of 3D EWOD devices. We first investigated the effect of the chemical composition in gel solutions on EWOD performance. It was found that the high concentration of ionic liquids, which was typically used for the previous spin-coating method, causes the surface defects on the dip-coated While the fabrication of such arrayed EWOD devices typically requires the assembly of multiple modules to form 3D structures, the previous spin-coated method poses several issues. Figure 2a illustrates the spin-coating procedure used to fabricate a single prism as an example of 3D EWOD devices coated with the gel layers. Considering that only a single substrate can be spin-coated at a time, the gel coating process has to be individually repeated for each of the four sidewalls. Next, they are carefully assembled into a hollow rectangular structure with four sidewalls whereby only the inner surfaces are coated with gel layers. However, during their assembly, the gel layer is at risk of damage or contamination from handling errors, which is detrimental to device performance and reliability. While such issues are already present for a single prism module, they are further compounded when fabricating multiple modules such as the case for n × n prism arrays (Figure 2b). The spin-coating step must be repeated four times per module, i.e., 4n2, and the more complicated assembling procedure increases the likelihood of ion gel damages. In addition, the chemical formulation (i.e., high concentrations of ionic liquids in gel solutions) previously used for a spin-coating method [4] causes the surface defects for the thin dip-coated layers, which led to poor EWOD performance. To obtain defect-free, robust gel layers without the issues arising from the spin-coating method aforementioned, we propose a dip-coating approach that not only offers a single-step coating solution to cover the gel layer over the entire surfaces of 3D structures, but also fully eliminates the risk of gel layer damage or contamination brought about during assembly. For the arrayed prism fabrication, substrates with ﬁnger-like protrusions are ﬁrst assembled to form the hollow n × n arrayed structure as shown in Figure 2c. 2. Ion Gel Layer Coating for 3D EWOD Device Fabrication With technical advances in EWOD over the years, recent attention has been extended to 3D devices, as they provide more ﬂexibility and functionality than conventional 2D planar ones. For example, Fan et al. reported a wearable wristband assembled by four ﬂexible EWOD modules where droplets were spatially manipulated on a curved wristband surface [12]. This lab-on-a-wristband (LOW) concept highlights the potential of 3D EWOD technology for point-of-care (POC) applications. Another example of the 3D EWOD devices popularly studied in recent years is the liquid prism that enables spatial beam steering [18–23]. EWOD-driven liquid prism technology has been used in diverse applications including sunlight beam steering for solar energy harvesting [24–26] and optical beam control to achieve a Fresnel lens [23]. By using ﬂuids for light control, one advantage is the smooth interface of ﬂuids formed due to the liquid’s tendency to minimize its surface energy. Such optical-grade smoothness of ﬂuidic interfaces is very useful and cost-effective as it eliminates the need of high-precision fabrication or polishing processes typically required for solid optics [27]. In addition, the shape and position of liquid interfaces can be actively controlled using EWOD without bulky and complex mechanical moving parts [28]. This feature offers more reconﬁgurable prism devices for controlling their optical performances. A prism is fabricated by assembling four conductive sidewalls coated with a hydrophobic and a dielectric layer (Figure 1). Two immiscible liquids are ﬁlled up and enclosed by top and bottom transparent plates. Bias voltages (VL and VR) are subsequently applied to the left and right sidewalls to induce the electrowetting effect to essentially control the prism apex angle (ϕ) [19,21,22]. In several previous studies, beam steering performance achieved by a single prism has been well described as a function of the refractive indices (n1 ̸= n2) of each medium as well as the apex angle (ϕ) [22–24]. Our group recently achieved the highest beam steering angle ever demonstrated up to β = 19.06◦using a double-stacked prism conﬁguration [22]. However, the aperture area is typically limited in a single prism due to surface tension modulation. This can be addressed by proposing an n × n arrayed prisms, which will be advantageous for solar energy 3 of 14 Materials 2017, 10, 41 applications [24]. 2. Ion Gel Layer Coating for 3D EWOD Device Fabrication Single prism modules can be joined to form an array, effectively increasing the overall aperture area where a large number of solar beams can be steered and focused onto a solar receiver with high concentrations reaching up to 2032× [24]. Materials 2017, 10, 41 3 of 14 the overall aperture area where a large number of solar beams can be steered and focused onto a solar receiver with high concentrations reaching up to 2032× [24]. Figure 1. A rectangular prism is fabricated by assembling four conductive sidewalls coated with dielectric and hydrophobic layers and filled with two immiscible liquids. The electrowetting effect is used to adjust the prism apex angle ϕ by applying bias voltages (VL and VR) to the left and right sidewalls, separately. Beam steering of the incoming light can be achieved at the interface of two media whose refractive indices are different (nair ≠ n1 ≠ n2). The left figure shows the assembled prism device with a 10 × 10 mm2 aperture area before two liquids are placed. While the fab i atio of u h a ayed EWOD de i e ty i ally e ui e the a e bly of ulti le Figure 1. A rectangular prism is fabricated by assembling four conductive sidewalls coated with dielectric and hydrophobic layers and ﬁlled with two immiscible liquids. The electrowetting effect is used to adjust the prism apex angle ϕ by applying bias voltages (VL and VR) to the left and right sidewalls, separately. Beam steering of the incoming light can be achieved at the interface of two media whose refractive indices are different (nair ̸= n1 ̸= n2). The left ﬁgure shows the assembled prism device with a 10 × 10 mm2 aperture area before two liquids are placed. Figure 1. A rectangular prism is fabricated by assembling four conductive sidewalls coated with dielectric and hydrophobic layers and filled with two immiscible liquids. The electrowetting effect is used to adjust the prism apex angle ϕ by applying bias voltages (VL and VR) to the left and right sidewalls, separately. Beam steering of the incoming light can be achieved at the interface of two media whose refractive indices are different (nair ≠ n1 ≠ n2). The left figure shows the assembled prism device with a 10 × 10 mm2 aperture area before two liquids are placed. Figure 1. 2. Ion Gel Layer Coating for 3D EWOD Device Fabrication Static EWOD experiments based on the improved fabrication process of employing dip-coating method together with a new chemical formulation were further conducted to understand the thicknesses effect of ion gel and Teﬂon layers on EWOD performance. The effectiveness of such dip-coatable gel layers for 3D device fabrication was demonstrated using 5 × 1 arrayed prisms with a 50 × 7 mm2 aperture area. A dip-coating approach offers a single-step solution with the beneﬁts of simplicity, scalability, and high throughput for deposition of high-capacitance gel layers on 3D EWOD devices. Materials 2017, 10, 41 4 of 14 to understand the thicknesses effect of ion gel and Teflon layers on EWOD performance. The effectiveness of such dip-coatable gel layers for 3D device fabrication was demonstrated using 5 × 1 arrayed prisms with a 50 × 7 mm2 aperture area. A dip-coating approach offers a single-step solution with the benefits of simplicity, scalability, and high throughput for deposition of high-capacitance gel layers on 3D EWOD devices. (a) (b) (c) (d) (e) Figure 2. Schematics of typical spin- and dip-coating processes for fabricating arrayed liquid prisms as an example of 3D electrowetting-on-dielectric (EWOD) devices. (a) Four sidewall modules are separately spin-coated for gel layer deposition and then assembled to form a hollow 3D structure before being filled with two immiscible liquids; (b) For fabrication of n × n prism arrays, the spin- coating process needs to be repeated four times per module, i.e., 4n2, with the gel layers more prone to damage during assembly; (c) A dip-coating process completes a single-step coating of the gel layer over the entire surfaces of the 3D assembled structures. For example, substrates with finger-like protrusions are first assembled to form the arrayed 3D structure; (d) The whole assembly is then dip- coated for the gel layer coating on the entire surface area; (e) A dip-coating approach offers a single- step solution with the benefits of simplicity, scalability, and high throughput for deposition of high- capacitance gel layers on 3D EWOD devices, while fully eliminating the risk of layer damage or contamination that may occur during the assembling processes. 3. Materials and Methods Figure 2. Schematics of typical spin- and dip-coating processes for fabricating arrayed liquid prisms as an example of 3D electrowetting-on-dielectric (EWOD) devices. For the dip-coatab methylimidazolium bi 3. Materials and Methods y ( y y ) ) [ ( )] p y (poly(vinylidene fluoride-co-hexafluoropropylene)), which are the same materials as the ones used in the previous spin-coating studies [4,14,15]. To prepare the ion gel solution, ionic liquid and copolymer were mixed at different weight ratios and further diluted in acetone to vary the solution viscosity, which is one of the variables to determine the thicknesses of the dip-coated gel layer [29]. For static EWOD studies, indium tin oxide (ITO)-coated substrates were dip-coated at various withdrawal speeds ranging from 100 to 800 mm/min, after which the samples were left to dry at room temperature for 24 h and cured at 70 °C for 4 h to purge all remaining acetone solvent. After full curing of the ion gel layer, the second hydrophobic layer was subsequently coated using a 6% Teflon AF solution (DuPont) through a dip-coating method at various withdrawal speeds and cured at 110 °C for 16 h. The thickness of both ion gel and Teflon layers were characterized using an optical profiler (ZYGO NewView 5032). Figure 3 shows the experimental setup for our static EWOD studies. A 7 μL water droplet was placed on the ITO substrate dip-coated with the ion gel and Teflon layers. A platinum (Pt) probe was then connected to the droplet as the ground electrode. The droplet contact angles were measured as the voltage input was increased in 5 V increments to understand the effects of chemical For the dip-coatable ion gel studies, we selected the [EMIM][TFSI] ionic liquid (1-ethyl-3-methylimidazolium bis(triﬂuoromethylsulfonyl)imide) and the [P(VDF-HFP)] copolymer (poly(vinylidene ﬂuoride-co-hexaﬂuoropropylene)), which are the same materials as the ones used in the previous spin-coating studies [4,14,15]. To prepare the ion gel solution, ionic liquid and copolymer were mixed at different weight ratios and further diluted in acetone to vary the solution viscosity, which is one of the variables to determine the thicknesses of the dip-coated gel layer [29]. For static EWOD studies, indium tin oxide (ITO)-coated substrates were dip-coated at various withdrawal speeds ranging from 100 to 800 mm/min, after which the samples were left to dry at room temperature for 24 h and cured at 70 ◦C for 4 h to purge all remaining acetone solvent. 2. Ion Gel Layer Coating for 3D EWOD Device Fabrication (a) Four sidewall modules are separately spin-coated for gel layer deposition and then assembled to form a hollow 3D structure before being ﬁlled with two immiscible liquids; (b) For fabrication of n × n prism arrays, the spin-coating process needs to be repeated four times per module, i.e., 4n2, with the gel layers more prone to damage during assembly; (c) A dip-coating process completes a single-step coating of the gel layer over the entire surfaces of the 3D assembled structures. For example, substrates with ﬁnger-like protrusions are ﬁrst assembled to form the arrayed 3D structure; (d) The whole assembly is then dip-coated for the gel layer coating on the entire surface area; (e) A dip-coating approach offers a single-step solution with the beneﬁts of simplicity, scalability, and high throughput for deposition of high-capacitance gel layers on 3D EWOD devices, while fully eliminating the risk of layer damage or contamination that may occur during the assembling processes. (b) (a) (b) (e) (b) (a) (a) (d) (c) (e) (c) (e) (d) Figure 2. Schematics of typical spin- and dip-coating processes for fabricating arrayed liquid prisms as an example of 3D electrowetting-on-dielectric (EWOD) devices. (a) Four sidewall modules are separately spin-coated for gel layer deposition and then assembled to form a hollow 3D structure before being filled with two immiscible liquids; (b) For fabrication of n × n prism arrays, the spin- coating process needs to be repeated four times per module, i.e., 4n2, with the gel layers more prone to damage during assembly; (c) A dip-coating process completes a single-step coating of the gel layer over the entire surfaces of the 3D assembled structures. For example, substrates with finger-like protrusions are first assembled to form the arrayed 3D structure; (d) The whole assembly is then dip- coated for the gel layer coating on the entire surface area; (e) A dip-coating approach offers a single- step solution with the benefits of simplicity, scalability, and high throughput for deposition of high- capacitance gel layers on 3D EWOD devices, while fully eliminating the risk of layer damage or contamination that may occur during the assembling processes. 3. Materials and Methods Figure 2. Schematics of typical spin- and dip-coating processes for fabricating arrayed liquid prisms as an example of 3D electrowetting-on-dielectric (EWOD) devices. 2. Ion Gel Layer Coating for 3D EWOD Device Fabrication (a) Four sidewall modules are separately spin-coated for gel layer deposition and then assembled to form a hollow 3D structure before being ﬁlled with two immiscible liquids; (b) For fabrication of n × n prism arrays, the spin-coating process needs to be repeated four times per module, i.e., 4n2, with the gel layers more prone to damage during assembly; (c) A dip-coating process completes a single-step coating of the gel layer over the entire surfaces of the 3D assembled structures. For example, substrates with ﬁnger-like protrusions are ﬁrst assembled to form the arrayed 3D structure; (d) The whole assembly is then dip-coated for the gel layer coating on the entire surface area; (e) A dip-coating approach offers a single-step solution with the beneﬁts of simplicity, scalability, and high throughput for deposition of high-capacitance gel layers on 3D EWOD devices, while fully eliminating the risk of layer damage or contamination that may occur during the assembling processes. 2. Ion Gel Layer Coating for 3D EWOD Device Fabrication The whole assembly is then dip-coated to achieve the gel layer coating across the entire exposed surface area without requiring any additional steps as compared to the spin-coating method (Figure 2d,e). was typically used for the previous spin coating method, causes the surface defects on the dip coated gel layers. Large contents of copolymers (or a small amount of ion liquids) in gel solutions helps to remove the surface defects by increasing the melting point of the gel layers over the curing temperature of Teflon at 110 °C. Static EWOD experiments based on the improved fabrication process of employing dip-coating method together with a new chemical formulation were further conducted g In this paper, a dip-coatable high-capacitance ion gel dielectric is presented for scalable fabrication of 3D EWOD devices. We ﬁrst investigated the effect of the chemical composition in gel solutions on EWOD performance. It was found that the high concentration of ionic liquids, which was typically used for the previous spin-coating method, causes the surface defects on the dip-coated gel layers. Large contents of copolymers (or a small amount of ion liquids) in gel solutions helps to remove the surface defects by increasing the melting point of the gel layers over the curing temperature of 4 of 14 Materials 2017, 10, 41 Teﬂon at 110 ◦C. Static EWOD experiments based on the improved fabrication process of employing dip-coating method together with a new chemical formulation were further conducted to understand the thicknesses effect of ion gel and Teﬂon layers on EWOD performance. The effectiveness of such dip-coatable gel layers for 3D device fabrication was demonstrated using 5 × 1 arrayed prisms with a 50 × 7 mm2 aperture area. A dip-coating approach offers a single-step solution with the beneﬁts of simplicity, scalability, and high throughput for deposition of high-capacitance gel layers on 3D EWOD devices. Materials 2017, 10, 41 4 of 14 to understand the thicknesses effect of ion gel and Teflon layers on EWOD performance. The effectiveness of such dip-coatable gel layers for 3D device fabrication was demonstrated using 5 × 1 arrayed prisms with a 50 × 7 mm2 aperture area. A dip-coating approach offers a single-step solution with the benefits of simplicity, scalability, and high throughput for deposition of high-capacitance gel layers on 3D EWOD devices. Teﬂon at 110 ◦C. p Since an ion gel la 4. Experimental Results (CP), it presents the features of both constituents such as the high capacitance contributed by the IL and the gel-like mechanical structure from the CP. Hence, the mixing ratio between two constituents has an important influence on the characteristics of the gel layer fabricated. For applications typically used for gating transistors, thin-film gel layers are fabricated by spin-coating gel solutions which are prepared at high concentrations of the IL ranging from 4:1 to 13:1 weight ratios with the CP [14,30,31]. High concentrations of the IL are critical to obtaining the ion gel layers with high ionic conductivity and thus faster switching speeds needed for gating transistors. Meanwhile, only a small amount of the CP would be sufficient to achieve gelation for mechanical integrity [15,32,33]. For our dip-coatable ion gel studies, we initially followed the previous spin-coating method [4,14,15,34] to prepare the ion gel solution, which included a high concentration of the IL at a 4:1 Since an ion gel layer is fabricated by combining an ionic liquid (IL) and a structuring copolymer (CP), it presents the features of both constituents such as the high capacitance contributed by the IL and the gel-like mechanical structure from the CP. Hence, the mixing ratio between two constituents has an important inﬂuence on the characteristics of the gel layer fabricated. For applications typically used for gating transistors, thin-ﬁlm gel layers are fabricated by spin-coating gel solutions which are prepared at high concentrations of the IL ranging from 4:1 to 13:1 weight ratios with the CP [14,30,31]. High concentrations of the IL are critical to obtaining the ion gel layers with high ionic conductivity and thus faster switching speeds needed for gating transistors. Meanwhile, only a small amount of the CP would be sufﬁcient to achieve gelation for mechanical integrity [15,32,33]. weight ratio with the CP. The gel solution was then dip-coated on an ITO substrate and cured at 70 °C, followed by the Teflon layer at 110 °C, respectively. Static EWOD tests were conducted to evaluate EWOD performance of the dip-coated gel layer. However, we soon realized that the previous spin- coating method including high concentrations of the IL in the gel solutions showed poor EWOD performance with the dip-coated gel layers. Bubbles generated by electrolysis had been consistently observed without any appreciable contact angle change. p Since an ion gel la 4. Experimental Results In the following sections, we discuss how the chemical formulation of the ion gel solution is key to obtaining robust, functional dip-coated gel layers. Using the gel solutions newly prepared, the thickness effects of both ion gel and Teflon layers were investigated on EWOD performance. Finally, the viability of the dip-coatable gel layers for 3D device fabrication was demonstrated by showing the 5 × 1 prism arrays whose apex angles were individually controlled. 4.1. Manifestation of Surface Defects on the Ion Gel Layer Dip-Coated To understand how such poor EWOD performance occurred, we inspected the surface quality f h h d ff l d d h l l d b l For our dip-coatable ion gel studies, we initially followed the previous spin-coating method [4,14,15,34] to prepare the ion gel solution, which included a high concentration of the IL at a 4:1 weight ratio with the CP. The gel solution was then dip-coated on an ITO substrate and cured at 70 ◦C, followed by the Teﬂon layer at 110 ◦C, respectively. Static EWOD tests were conducted to evaluate EWOD performance of the dip-coated gel layer. However, we soon realized that the previous spin-coating method including high concentrations of the IL in the gel solutions showed poor EWOD performance with the dip-coated gel layers. Bubbles generated by electrolysis had been consistently observed without any appreciable contact angle change. In the following sections, we discuss how the chemical formulation of the ion gel solution is key to obtaining robust, functional dip-coated gel layers. Using the gel solutions newly prepared, the thickness effects of both ion gel and Teﬂon layers were investigated on EWOD performance. Finally, the viability of the dip-coatable gel layers for 3D device fabrication was demonstrated by showing the 5 × 1 prism arrays whose apex angles were individually controlled. For the dip-coatab methylimidazolium bi 3. Materials and Methods After full curing of the ion gel layer, the second hydrophobic layer was subsequently coated using a 6% Teﬂon AF solution (DuPont) through a dip-coating method at various withdrawal speeds and cured at 110 ◦C for 16 h. The thickness of both ion gel and Teﬂon layers were characterized using an optical proﬁler (ZYGO NewView 5032). the voltage input was increased in 5 V increments to understand the effects of chemical concentrations in the gel solution and thickness of each layer on EWOD performance. Figure 3 shows the experimental setup for our static EWOD studies. A 7 µL water droplet was placed on the ITO substrate dip-coated with the ion gel and Teﬂon layers. A platinum (Pt) probe was then connected to the droplet as the ground electrode. The droplet contact angles were measured as 5 of 14 Materials 2017, 10, 41 the voltage input was increased in 5 V increments to understand the effects of chemical concentrations in the gel solution and thickness of each layer on EWOD performance. Materials 2017, 10, 41 5 of 14 Figure 3. Experimental setup for static EWOD tests. A water droplet is placed on the ITO substrate dip-coated with the dielectric (ion gel) and hydrophobic (Teflon AF) layers. 4. Experimental Results Figure 3. Experimental setup for static EWOD tests. A water droplet is placed on the ITO substrate dip-coated with the dielectric (ion gel) and hydrophobic (Teﬂon AF) layers. Figure 3. Experimental setup for static EWOD tests. A water droplet is placed on the ITO substrate dip-coated with the dielectric (ion gel) and hydrophobic (Teflon AF) layers. E i l R l Figure 3. Experimental setup for static EWOD tests. A water droplet is placed on the ITO substrate dip-coated with the dielectric (ion gel) and hydrophobic (Teﬂon AF) layers. p p ( ) p g layer baked at 70 °C over which an additional Teflon layer was cure l b k d t 110 °C Fi 4 h th i i i f th 4.1. Manifestation of Surface Defects on the Ion Gel Layer Dip-Coated layer baked at 110 °C. Figure 4 shows the microscopic images of these three samples. The ion gel layer was first dip-coated using a gel solution prepared with a high concentration of IL at a 4:1 weight ratio with the CP and cured at 70 °C. A uniform surface quality is observed for the pure ion gel layer (Figure 4a). However, when a Teflon layer was subsequently dip-coated over the gel layer and cured at 110 °C, abundant surface defects were clearly observed (Figure 4b). This was then compared to the case of a pure Teflon layer dip-coated and baked at 110 °C, where no surface defects were observed (Figure 4c). Based on our observations shown in Figure 4, it becomes evident that the surface defects manifested on the dip-coated gel layer only after the Teflon was dip-coated over it and cured at 110 °C, resulting in poor EWOD performance. Such a surface degradation phenomenon of the ion gel layer can be further reinforced by several previous studies. Jansen et al. experimentally showed that To understand how such poor EWOD performance occurred, we inspected the surface quality of the three different samples dip-coated with (a) a pure ion gel layer cured at 70 ◦C; (b) an ion gel layer baked at 70 ◦C over which an additional Teﬂon layer was cured at 110 ◦C; and (c) a pure Teﬂon layer baked at 110 ◦C. Figure 4 shows the microscopic images of these three samples. The ion gel layer was ﬁrst dip-coated using a gel solution prepared with a high concentration of IL at a 4:1 weight ratio with the CP and cured at 70 ◦C. A uniform surface quality is observed for the pure ion gel layer (Figure 4a). However, when a Teﬂon layer was subsequently dip-coated over the gel layer and cured at 110 ◦C, abundant surface defects were clearly observed (Figure 4b). This was then compared to the case of a 6 of 14 Materials 2017, 10, 41 pure Teﬂon layer dip-coated and baked at 110 ◦C, where no surface defects were observed (Figure 4c). Based on our observations shown in Figure 4, it becomes evident that the surface defects manifested on the dip-coated gel layer only after the Teﬂon was dip-coated over it and cured at 110 ◦C, resulting in poor EWOD performance. p p ( ) p g layer baked at 70 °C over which an additional Teflon layer was cure l b k d t 110 °C Fi 4 h th i i i f th 4.1. Manifestation of Surface Defects on the Ion Gel Layer Dip-Coated Such a surface degradation phenomenon of the ion gel layer can be further reinforced by several previous studies. Jansen et al. experimentally showed that the melting point of the ion gel layer is signiﬁcantly lowered when increasing the concentration of the [EMIM][TFSI] ionic liquid in the gel solution [35]. They observed that the neat [P(VDF-HFP)] copolymer has a melting point around 140 ◦C, which consequently drops down to 90 ◦C when the gel layer is made at a high weight ratio of 4:1 between the IL and the CP. This is because large contents of the CP (or a small amount of the IL) in the gel layer increases the crystallinity of the gelated layer and such large crystalline regions require more heat to melt than the amorphous regions [35]. In contrast, the solutions made with highly concentrated IL create less crystalline regions in the gel layer, resulting in an observed melting point depression. It was also reported that, when thin-ﬁlm polymer layers are treated at a temperature higher than their melting points, the surface tension variations caused by phase separation induce surface deformation and roughness within the thin ﬁlms [36]. In our experiments using high concentration of the IL at a 4:1 weight ratio with the CP, the ion gel layer underneath the Teﬂon layer experienced melting and migration during the Teﬂon curing period at 110 ◦C, which is above the 90 ◦C melting point of the gel layer [35,36]. This led to the formation of tiny aggregations, causing the surface defects as shown in Figure 4b. Hence, it was critical to understand how IL concentrations inﬂuence on EWOD performance of the dip-coated gel layers. Such is discussed in the next section. Materials 2017, 10, 41 6 of 14 the melting point of the ion gel layer is significantly lowered when increasing the concentration of the [EMIM][TFSI] ionic liquid in the gel solution [35]. They observed that the neat [P(VDF-HFP)] copolymer has a melting point around 140 °C, which consequently drops down to 90 °C when the gel layer is made at a high weight ratio of 4:1 between the IL and the CP. This is because large contents of the CP (or a small amount of the IL) in the gel layer increases the crystallinity of the gelated layer and such large crystalline regions require more heat to melt than the amorphous regions [35]. ff f q For this study, several ion gel solutions wer 4.2. Effect of Ionic Liquid Concentrations on EWOD 0.25:1 between the IL and the CP. These mixtures were further diluted in the same amount of acetone at 9%. Dip-coating and curing processes were implemented for both ion gel and Teflon layers, respectively. We inspected the surface quality of the samples dip-coated with the gel solutions prepared at various weight ratios and their microscopic images are shown in Figure 5. As discussed above, the surface defects were evident for the gel layer dip-coated with the solution containing a high concentration of the IL at a 4:1 weight ratio with the CP (Figure 5a). Such surface defects decrease, as the IL content is lowered relative to the CP (e.g., the layer fabricated at a 2.5:1 weight ratio shown in Figure 5b). For the solutions with low IL concentrations at 2:1 (Figure 5c) and 0.25:1 (Figure 5d), the surface defects are no longer observed. This is due to the effective increase of the gel layer’s melting point over the Teflon baking temperature at 110 °C as IL concentrations are lowered beyond the 2:1 ratio [35]. This results in the improvement of surface quality, as surface defects no longer arise from gel layer phase change. These samples shown in Figure 5 were subsequently tested for static EWOD experiments to examine the effect of the IL concentrations on EWOD performance. The graphs in Figure 6 present the droplet contact angles measured on the samples fabricated with four different weight ratios between the IL and the CP. As expected from the results in Figure 5, the ion gel samples fabricated at high IL weight ratios of 4:1 and 2.5:1 with the CP provided poor EWOD For this study, several ion gel solutions were prepared by varying the weight ratios from 4:1 to 0.25:1 between the IL and the CP. These mixtures were further diluted in the same amount of acetone at 9%. Dip-coating and curing processes were implemented for both ion gel and Teﬂon layers, respectively. We inspected the surface quality of the samples dip-coated with the gel solutions prepared at various weight ratios and their microscopic images are shown in Figure 5. As discussed above, the surface defects were evident for the gel layer dip-coated with the solution containing a high concentration of the IL at a 4:1 weight ratio with the CP (Figure 5a). p p ( ) p g layer baked at 70 °C over which an additional Teflon layer was cure l b k d t 110 °C Fi 4 h th i i i f th 4.1. Manifestation of Surface Defects on the Ion Gel Layer Dip-Coated In contrast, the solutions made with highly concentrated IL create less crystalline regions in the gel layer, resulting in an observed melting point depression. It was also reported that, when thin-film polymer layers are treated at a temperature higher than their melting points, the surface tension variations caused by phase separation induce surface deformation and roughness within the thin films [36]. In our experiments using high concentration of the IL at a 4:1 weight ratio with the CP, the ion gel layer underneath the Teflon layer experienced melting and migration during the Teflon curing period at 110 °C, which is above the 90 °C melting point of the gel layer [35,36]. This led to the formation of tiny aggregations, causing the surface defects as shown in Figure 4b. Hence, it was critical to understand how IL concentrations influence on EWOD performance of the dip-coated gel layers. Such is discussed in the next section. Figure 4. Microscopic images of the three different dip-coated samples. (a) An ion gel layer was dip- coated with the gel solution prepared with a high concentration of the ionic liquid (IL) at a 4:1 weight ratio with the copolymer (CP) and cured at 70 °C. A uniform surface quality is observed; (b) A Teflon layer is subsequently dip-coated over the gel layer and cured at 110 °C. Serious surface defects are observed; (c) For comparison, a pure Teflon layer was separately dip-coated and cured at 110 °C. No surface defects are observed. The scale bar represents a 0.5 mm length. 4.2. Effect of Ionic Liquid Concentrations on EWOD Figure 4. Microscopic images of the three different dip-coated samples. (a) An ion gel layer was dip-coated with the gel solution prepared with a high concentration of the ionic liquid (IL) at a 4:1 weight ratio with the copolymer (CP) and cured at 70 ◦C. A uniform surface quality is observed; (b) A Teﬂon layer is subsequently dip-coated over the gel layer and cured at 110 ◦C. Serious surface defects are observed; (c) For comparison, a pure Teﬂon layer was separately dip-coated and cured at 110 ◦C. No surface defects are observed. The scale bar represents a 0.5 mm length. Figure 4. Microscopic images of the three different dip-coated samples. p p ( ) p g layer baked at 70 °C over which an additional Teflon layer was cure l b k d t 110 °C Fi 4 h th i i i f th 4.1. Manifestation of Surface Defects on the Ion Gel Layer Dip-Coated (a) An ion gel layer was dip- coated with the gel solution prepared with a high concentration of the ionic liquid (IL) at a 4:1 weight ratio with the copolymer (CP) and cured at 70 °C. A uniform surface quality is observed; (b) A Teflon layer is subsequently dip-coated over the gel layer and cured at 110 °C. Serious surface defects are observed; (c) For comparison, a pure Teflon layer was separately dip-coated and cured at 110 °C. No surface defects are observed. The scale bar represents a 0.5 mm length. 4 2 Effect of Ionic Liquid Concentrations on EWOD Figure 4. Microscopic images of the three different dip-coated samples. (a) An ion gel layer was dip-coated with the gel solution prepared with a high concentration of the ionic liquid (IL) at a 4:1 weight ratio with the copolymer (CP) and cured at 70 ◦C. A uniform surface quality is observed; (b) A Teﬂon layer is subsequently dip-coated over the gel layer and cured at 110 ◦C. Serious surface defects are observed; (c) For comparison, a pure Teﬂon layer was separately dip-coated and cured at 110 ◦C. No surface defects are observed. The scale bar represents a 0.5 mm length. ff f q For this study, several ion gel solutions wer 4.2. Effect of Ionic Liquid Concentrations on EWOD Such surface defects decrease, as the IL content is lowered relative to the CP (e.g., the layer fabricated at a 2.5:1 weight ratio shown in Figure 5b). For the solutions with low IL concentrations at 2:1 (Figure 5c) and 0.25:1 (Figure 5d), the surface defects are no longer observed. This is due to the effective increase of the gel layer’s melting point over the Teﬂon baking temperature at 110 ◦C as IL concentrations are lowered beyond the 2:1 ratio [35]. This results in the improvement of surface quality, as surface defects no longer arise from gel layer phase change. These samples shown in Figure 5 were subsequently tested for static EWOD experiments to examine the effect of the IL concentrations on EWOD performance. The graphs in Figure 6 present the droplet 7 of 14 Materials 2017, 10, 41 contact angles measured on the samples fabricated with four different weight ratios between the IL and the CP. As expected from the results in Figure 5, the ion gel samples fabricated at high IL weight ratios of 4:1 and 2.5:1 with the CP provided poor EWOD performance where bubbles were caused by electrolysis as a sign of dielectric failure (see Figure 6a,b). For the other cases with the low IL contents (e.g., 2:1 and 0.25:1 weight ratios), full contact angle modulation was achieved without electrolysis until reaching the contact angle saturation around 58◦(see Figure 6c). From the experimental results in Figure 6, it is understood that the gel solution where the IL content is at least lower than the 2:1 weight ratio with the CP is required to obtain stable and robust dip-coated ion gel layers that enable full contact angle modulation without electrolysis. For all following experiments, samples were prepared using solutions with a low IL:CP ratio of 0.25:1. Materials 2017, 10, 41 7 of 14 (see Figure 6c). From the experimental results in Figure 6, it is understood that the gel solution where the IL content is at least lower than the 2:1 weight ratio with the CP is required to obtain stable and robust dip-coated ion gel layers that enable full contact angle modulation without electrolysis. For all following experiments, samples were prepared using solutions with a low IL:CP ratio of 0.25:1. Materials 2017, 10, 41 7 of 14 (see Figure 6c). ff f q For this study, several ion gel solutions wer 4.2. Effect of Ionic Liquid Concentrations on EWOD The gel layers were dip-coated with the solutions made of the IL and the CP at various weight ratios of (a) 4:1; (b) 2.5:1; (c) 2:1; and (d) 0.25:1 and cured at 70 °C. The Teflon layer was subsequently dip-coated and baked at 110 °C. It is clearly observed that the surface defects decrease as the IL content is lowered relative to the CP in the mixture solution. The surface quality of the ion gel layer at the 2:1 and 0.25:1 weight ratios is greatly improved. The same solution concentration with respect to acetone was used for all the cases. The figure (a) is reprinted from Figure 3b The scale bar represents a 0 5 mm length Figure 6. Effect of the IL concentrations in the gel solution on EWOD performance. The initial contact angle was similarly measured to be 115° on the Teflon surface for all the cases. EWOD performance was studied by measuring the contact angles with varying input voltages. Full contact angle modulation without electrolysis was achieved for the ion gel solutions prepared with low IL contents at 2:1 and 0.25:1 weight ratios with the CP. The other two cases at 4:1 and 2.5:1 showed poor EWOD performance. The insets (a,b) show the onset of bubbles generated by electrolysis (indicated by red arrows); while the inset (c) shows full contact angle modulation up to 58° achieved without electrolysis. All samples were prepared in the same experimental conditions, such as constant solution concentration, withdrawal speed, and a Teflon layer of 670 nm thickness. 4.3. Thickness Control of the Dip-Coated Gel Layers According to the Landau-Levich derivation [29], the thickness of dip-coated layers is mainly dete i ed by olutio i o ity a d ithd a al eed To o t ol the el laye thi k e by di Figure 6. Effect of the IL concentrations in the gel solution on EWOD performance. The initial contact angle was similarly measured to be 115° on the Teflon surface for all the cases. EWOD performance was studied by measuring the contact angles with varying input voltages. Full contact angle modulation without electrolysis was achieved for the ion gel solutions prepared with low IL contents at 2:1 and 0.25:1 weight ratios with the CP. The other two cases at 4:1 and 2.5:1 showed poor EWOD performance. ff f q For this study, several ion gel solutions wer 4.2. Effect of Ionic Liquid Concentrations on EWOD From the experimental results in Figure 6, it is understood that the gel solution where the IL content is at least lower than the 2:1 weight ratio with the CP is required to obtain stable and Figure 5. Microscopic images of the samples fabricated with various mixing ratios between the IL and the CP in the gel solution. The gel layers were dip-coated with the solutions made of the IL and the CP at various weight ratios of (a) 4:1; (b) 2.5:1; (c) 2:1; and (d) 0.25:1 and cured at 70 °C. The Teflon layer was subsequently dip-coated and baked at 110 °C. It is clearly observed that the surface defects decrease as the IL content is lowered relative to the CP in the mixture solution. The surface quality of the ion gel layer at the 2:1 and 0.25:1 weight ratios is greatly improved. The same solution concentration with respect to acetone was used for all the cases. The figure (a) is reprinted from Figure 3b. The scale bar represents a 0.5 mm length. Figure 5. Microscopic images of the samples fabricated with various mixing ratios between the IL and the CP in the gel solution. The gel layers were dip-coated with the solutions made of the IL and the CP at various weight ratios of (a) 4:1; (b) 2.5:1; (c) 2:1; and (d) 0.25:1 and cured at 70 ◦C. The Teﬂon layer was subsequently dip-coated and baked at 110 ◦C. It is clearly observed that the surface defects decrease as the IL content is lowered relative to the CP in the mixture solution. The surface quality of the ion gel layer at the 2:1 and 0.25:1 weight ratios is greatly improved. The same solution concentration with respect to acetone was used for all the cases. The ﬁgure (a) is reprinted from Figure 3b. The scale bar represents a 0.5 mm length. robust dip-coated ion gel layers that enable full contact angle modulation without electrolysis. For all following experiments, samples were prepared using solutions with a low IL:CP ratio of 0.25:1. Figure 5. Microscopic images of the samples fabricated with various mixing ratios between the IL and the CP in the gel solution. The gel layers were dip-coated with the solutions made of the IL and the CP at various weight ratios of (a) 4:1; (b) 2.5:1; (c) 2:1; and (d) 0.25:1 and cured at 70 °C. ff f q For this study, several ion gel solutions wer 4.2. Effect of Ionic Liquid Concentrations on EWOD The Teflon layer was subsequently dip-coated and baked at 110 °C. It is clearly observed that the surface defects decrease as the IL content is lowered relative to the CP in the mixture solution. The surface quality of the ion gel layer at the 2:1 and 0.25:1 weight ratios is greatly improved. The same solution concentration with respect to acetone was used for all the cases. The figure (a) is reprinted from Figure 3b. The scale bar represents a 0.5 mm length. Figure 5. Microscopic images of the samples fabricated with various mixing ratios between the IL and the CP in the gel solution. The gel layers were dip-coated with the solutions made of the IL and the CP at various weight ratios of (a) 4:1; (b) 2.5:1; (c) 2:1; and (d) 0.25:1 and cured at 70 °C. The Teflon layer was subsequently dip-coated and baked at 110 °C. It is clearly observed that the surface defects decrease as the IL content is lowered relative to the CP in the mixture solution. The surface quality of the ion gel layer at the 2:1 and 0.25:1 weight ratios is greatly improved. The same solution concentration with respect to acetone was used for all the cases. The figure (a) is reprinted from Figure 3b. The scale bar represents a 0.5 mm length. Figure 5. Microscopic images of the samples fabricated with various mixing ratios between the IL and the CP in the gel solution. The gel layers were dip-coated with the solutions made of the IL and the CP at various weight ratios of (a) 4:1; (b) 2.5:1; (c) 2:1; and (d) 0.25:1 and cured at 70 ◦C. The Teﬂon layer was subsequently dip-coated and baked at 110 ◦C. It is clearly observed that the surface defects decrease as the IL content is lowered relative to the CP in the mixture solution. The surface quality of the ion gel layer at the 2:1 and 0.25:1 weight ratios is greatly improved. The same solution concentration with respect to acetone was used for all the cases. The ﬁgure (a) is reprinted from Figure 3b. The scale bar represents a 0.5 mm length. Figure 5. Microscopic images of the samples fabricated with various mixing ratios between the IL and the CP in the gel solution. ff f q For this study, several ion gel solutions wer 4.2. Effect of Ionic Liquid Concentrations on EWOD The insets (a,b) show the onset of bubbles generated by electrolysis (indicated by red arrows); while the inset (c) shows full contact angle modulation up to 58° achieved without electrolysis. All samples were prepared in the same experimental conditions, such as constant solution concentration, withdrawal speed, and a Teflon layer of 670 nm thickness. 4.3. Thickness Control of the Dip-Coated Gel Layers Figure 6. Effect of the IL concentrations in the gel solution on EWOD performance. The initial contact angle was similarly measured to be 115◦on the Teﬂon surface for all the cases. EWOD performance was studied by measuring the contact angles with varying input voltages. Full contact angle modulation without electrolysis was achieved for the ion gel solutions prepared with low IL contents at 2:1 and 0.25:1 weight ratios with the CP. The other two cases at 4:1 and 2.5:1 showed poor EWOD performance. The insets (a,b) show the onset of bubbles generated by electrolysis (indicated by red arrows); while the inset (c) shows full contact angle modulation up to 58◦achieved without electrolysis. All samples were prepared in the same experimental conditions, such as constant solution concentration, withdrawal speed, and a Teﬂon layer of 670 nm thickness. IL concentrations in the gel solution on EWOD performan measured to be 115° on the Teflon surface for all the cases was studied by measuring the contact angles with varying input voltages. Full contact angle modulation without electrolysis was achieved for the ion gel solutions prepared with low IL contents at 2:1 and 0.25:1 weight ratios with the CP. The other two cases at 4:1 and 2.5:1 showed poor EWOD performance. The insets (a,b) show the onset of bubbles generated by electrolysis (indicated by red arrows); while the inset (c) shows full contact angle modulation up to 58° achieved without electrolysis. All samples were prepared in the same experimental conditions, such as constant solution concentration, withdrawal speed, and a Teflon layer of 670 nm thickness. 4.3. Thickness Control of the Dip-Coated Gel Layers According to the Landau-Levich derivation [29], the thickness of dip-coated layers is mainly Figure 6. Effect of the IL concentrations in the gel solution on EWOD performance. The initial contact angle was similarly measured to be 115° on the Teflon surface for all the cases. EWOD performance was studied by measuring the contact angles with varying input voltages. 4.3. Thickness Control of the Dip-Coated Gel Layers 4.3. Thickness Control of the Dip-Coated Gel Layers 4.3. Thickness Control of the Dip-Coated Gel Layers According to the Landau-Levich derivation [29], the thickness of dip-coated layers is mainly determined by solution viscosity and withdrawal speed. To control the gel layer thickness by dip-coating, the gel solution was prepared at the ﬁxed 0.25:1 weight ratio between the IL and CP, while overall solution concentrations (i.e., both IL and CP) with respect to an acetone solvent and withdrawal speeds were varied. Figure 7 presents the measured thicknesses of the dip-coated layers. Gel solutions with higher concentrations of the IL and the CP (e.g., 9%) in an acetone solvent are more viscous, consequently forming thicker layers. Likewise, dip coating at faster withdrawal speeds gives the wet ﬁlm layer less time to develop, thus depositing thicker layers. It is worth noting that, for the given mixture ratio at 0.25:1, the solution viscosity was signiﬁcantly high even at 9% and close to solute saturation. For the solutions at concentrations higher than 9%, the dip-coated samples showed immediate gelation and highly uneven surfaces and hence poor EWOD performance with electrolysis. This indicates that there is a limit to the thickness of functional ion gel layers to be produced by the dip-coating method. By adjusting the acetone concentrations and withdrawal speeds, we were able to vary the thicknesses of the dip-coated gel layer from 40 nm to 2.21 µm, which are a few orders thinner than the ones fabricated by the previous spin-coating method [4,14,15,34]. Materials 2017, 10, 41 8 of 14 the wet film layer less time to develop, thus depositing thicker layers. It is worth noting that, for the given mixture ratio at 0.25:1, the solution viscosity was significantly high even at 9% and close to solute saturation. For the solutions at concentrations higher than 9%, the dip-coated samples showed immediate gelation and highly uneven surfaces and hence poor EWOD performance with electrolysis. This indicates that there is a limit to the thickness of functional ion gel layers to be produced by the dip-coating method. By adjusting the acetone concentrations and withdrawal speeds, we were able to vary the thicknesses of the dip-coated gel layer from 40 nm to 2.21 μm, which are a few orders thinner than the ones fabricated by the previous spin-coating method [4,14,15,34]. Figure 7. 4.4. Thickness Effect of the Dip-Coated Gel Layers 4.4. Thickness Effect of the Dip-Coated Gel Layers It is known that ion gel layers provide a high specific capacitance (c ≈ 10 μF/cm2) as a result of the nanometer-thick electric double layer (EDL) capacitor formed by free counter-ions of ionic liquids compactly accumulated at the interface with an applied electric field [16,17,34]. This indicates that the intermediate gel layer constructed by structuring copolymers does not contribute to its capacitance [4,14,32], which is hence thickness independent, unlike conventional dielectrics whose capacitance is inversely proportional to their thickness. This thickness independence on capacitance was only verified for the thick ion gel layers (in the range of a few tens of micrometers) spin-coated with the gel solutions including high concentrations of the IL [4,14,15,34]. However, the dip-coated gel layers in our study not only are fabricated with the modified gel solutions, but also typically produce a few orders thinner gel layers (in the ranges of sub-micrometers) than that of spin coating. Therefore, it is worthwhile for us to study the thickness effect on EWOD performance of the dip- coated gel layers that have a different fabrication technique as compared to the previous spin-coating approach. Fo this study a io el solutio as p epa ed at the lo co ce t atio of the IL i ed ith It is known that ion gel layers provide a high speciﬁc capacitance (c ≈10 µF/cm2) as a result of the nanometer-thick electric double layer (EDL) capacitor formed by free counter-ions of ionic liquids compactly accumulated at the interface with an applied electric ﬁeld [16,17,34]. This indicates that the intermediate gel layer constructed by structuring copolymers does not contribute to its capacitance [4,14,32], which is hence thickness independent, unlike conventional dielectrics whose capacitance is inversely proportional to their thickness. This thickness independence on capacitance was only veriﬁed for the thick ion gel layers (in the range of a few tens of micrometers) spin-coated with the gel solutions including high concentrations of the IL [4,14,15,34]. However, the dip-coated gel layers in our study not only are fabricated with the modiﬁed gel solutions, but also typically produce a few orders thinner gel layers (in the ranges of sub-micrometers) than that of spin coating. Therefore, it is worthwhile for us to study the thickness effect on EWOD performance of the dip-coated gel layers that have a different fabrication technique as compared to the previous spin-coating approach. ff f q For this study, several ion gel solutions wer 4.2. Effect of Ionic Liquid Concentrations on EWOD Full contact angle modulation without electrolysis was achieved for the ion gel solutions prepared with low IL contents at 2:1 and 0.25:1 weight ratios with the CP. The other two cases at 4:1 and 2.5:1 showed poor EWOD performance. The insets (a,b) show the onset of bubbles generated by electrolysis (indicated by red arrows); while the inset (c) shows full contact angle modulation up to 58° achieved without electrolysis. All samples were prepared in the same experimental conditions, such as constant solution concentration, withdrawal speed, and a Teflon layer of 670 nm thickness. 4 3 Thickness Control of the Dip-Coated Gel Layers Figure 6. Effect of the IL concentrations in the gel solution on EWOD performance. The initial contact angle was similarly measured to be 115◦on the Teﬂon surface for all the cases. EWOD performance was studied by measuring the contact angles with varying input voltages. Full contact angle modulation without electrolysis was achieved for the ion gel solutions prepared with low IL contents at 2:1 and 0.25:1 weight ratios with the CP. The other two cases at 4:1 and 2.5:1 showed poor EWOD performance. The insets (a,b) show the onset of bubbles generated by electrolysis (indicated by red arrows); while the inset (c) shows full contact angle modulation up to 58◦achieved without electrolysis. All samples were prepared in the same experimental conditions, such as constant solution concentration, withdrawal speed, and a Teﬂon layer of 670 nm thickness. Materials 2017, 10, 41 8 of 14 4.3. Thickness Control of the Dip-Coated Gel Layers The weight ratio between the IL and the CP was fixed at 0.25:1, while the amounts of acetone and withdrawal speeds were varied to control the layer thickness. The legend indicates the concentrations of the IL and the CP with respect to the acetone solvent. Figure 7. By adjusting the acetone concentrations and withdrawal speeds, we were able to vary the thicknesses of the dip-coated gel layer from 40 nm to 2.21 µm, which are a few orders thinner than the ones fabricated by the spin-coating method. The weight ratio between the IL and the CP was ﬁxed at 0.25:1, while the amounts of acetone and withdrawal speeds were varied to control the layer thickness. The legend indicates the concentrations of the IL and the CP with respect to the acetone solvent. 4.3. Thickness Control of the Dip-Coated Gel Layers By adjusting the acetone concentrations and withdrawal speeds, we were able to vary the thicknesses of the dip-coated gel layer from 40 nm to 2.21 μm, which are a few orders thinner than the ones fabricated by the spin-coating method. The weight ratio between the IL and the CP was fixed at 0.25:1, while the amounts of acetone and withdrawal speeds were varied to control the layer thickness. The legend indicates the concentrations of the IL and the CP with respect to the acetone solvent. Figure 7. By adjusting the acetone concentrations and withdrawal speeds, we were able to vary the thicknesses of the dip-coated gel layer from 40 nm to 2.21 µm, which are a few orders thinner than the ones fabricated by the spin-coating method. The weight ratio between the IL and the CP was ﬁxed at 0.25:1, while the amounts of acetone and withdrawal speeds were varied to control the layer thickness. The legend indicates the concentrations of the IL and the CP with respect to the acetone solvent. Figure 7. By adjusting the acetone concentrations and withdrawal speeds, we were able to vary the thicknesses of the dip-coated gel layer from 40 nm to 2.21 μm, which are a few orders thinner than the ones fabricated by the spin-coating method. The weight ratio between the IL and the CP was fixed at 0.25:1, while the amounts of acetone and withdrawal speeds were varied to control the layer thickness. The legend indicates the concentrations of the IL and the CP with respect to the acetone solvent. Figure 7. By adjusting the acetone concentrations and withdrawal speeds, we were able to vary the thicknesses of the dip-coated gel layer from 40 nm to 2.21 µm, which are a few orders thinner than the ones fabricated by the spin-coating method. The weight ratio between the IL and the CP was ﬁxed at 0.25:1, while the amounts of acetone and withdrawal speeds were varied to control the layer thickness. The legend indicates the concentrations of the IL and the CP with respect to the acetone solvent. Figure 7. By adjusting the acetone concentrations and withdrawal speeds, we were able to vary the thicknesses of the dip-coated gel layer from 40 nm to 2.21 μm, which are a few orders thinner than the ones fabricated by the spin-coating method. Since ion gel layers are fabricated by a n 4.5. Thickness Effect of the Dip-Coated Teﬂon Layers understand the thickness effect of the dip-coated Teflon layer on EWOD performance. With the gel layer fixed at 1.19 μm, the thickness of the Teflon layer was varied from 120 to 1010 nm. The contact angles were measured at varying voltage inputs and the results are shown in Figure 9. EWOD performance improved as decreasing the thickness of the Teflon layer. For example, full contact angle modulation was achieved around 60° at 50 V for the 220 nm thick Teflon layer, while the contact angle was around 92° for the 1010 nm thick layer at the same voltage input. These observations are very consistent with our general understanding of EWOD performance, i.e., the capacitance is inversely proportional to the layer thickness and thus a thin Teflon layer can achieve more contact angle modulation than that of a thick layer at the same bias voltage. However, for very thin Teflon layers such as the case of 120 nm, electrolysis takes place at 30 V before full contact angle modulation (see Figure 9a). Since the capacitance of the ion gel is a few orders higher than that of the Teflon, nearly 99% of the voltage input is distributed across the hydrophobic layer when the two capacitors of the ion gel and Teflon layers are connected in series, as shown in Figure 3. The theoretical breakdown voltage for the 120 nm thick Teflon layer was estimated as 24 V [9]. Nevertheless, the droplet contact angle was further modulated even at the voltage of 30 V exceeding this breakdown threshold. This observation can be explained by several previous studies [37,38], reporting that, although the voltage distributed across the Teflon layer exceeds its breakdown voltage, the device performs continuously while also slowly degrading. Since ion gel layers are fabricated by a new dip-coating method, it is also important to understand the thickness effect of the dip-coated Teﬂon layer on EWOD performance. With the gel layer ﬁxed at 1.19 µm, the thickness of the Teﬂon layer was varied from 120 to 1010 nm. The contact angles were measured at varying voltage inputs and the results are shown in Figure 9. EWOD performance improved as decreasing the thickness of the Teﬂon layer. 4.4. Thickness Effect of the Dip-Coated Gel Layers 4.4. Thickness Effect of the Dip-Coated Gel Layers For this study, an ion gel solution was prepared at the low concentration of the IL mixed with the CP at a 0.25:1 weight ratio. Then, the dip-coated gel samples were fabricated with various thicknesses ranging from 80 nm to 1.19 μm, while the Teflon layer was fixed at 670 nm atop of each gel layer. Static EWOD experiments were again conducted on these samples to see the thickness effect q p p p g pp For this study, an ion gel solution was prepared at the low concentration of the IL mixed with the CP at a 0.25:1 weight ratio. Then, the dip-coated gel samples were fabricated with various thicknesses ranging from 80 nm to 1.19 µm, while the Teﬂon layer was ﬁxed at 670 nm atop of each gel layer. 9 of 14 Materials 2017, 10, 41 Static EWOD experiments were again conducted on these samples to see the thickness effect on the capacitance. Figure 8 shows the very similar contact angle changes regardless of the gel layer thicknesses. All the gel layers were capable of achieving full contact angle modulation without the practical issues of EWOD such as electrolysis and dielectric breakdown, with the contact angles saturating around 58◦at 75 V. From the experimental results, the thickness independence of the ion gel’s capacitance holds true even for the thin dip-coated gel layers, which were fabricated with the gel solutions including low content of the IL. As a comparative study, a pure 670 nm thick Teﬂon layer over an ITO plate was tested and showed much poorer performance than that of the Teﬂon and ion gel stack, when it is used alone as a dielectric layer. Our study here demonstrated that a new dip-coating method using solutions with low IL concentrations (differently fabricated from the previous spin-coating method) is capable of producing defect-free, reliable dielectric layers as thin as 80 nm, thickness comparative with other conventional dielectrics commonly used for EWOD studies, by simply fabricating with a single-step dip-coating process. Materials 2017, 10, 41 9 of 14 layer over an ITO plate was tested and showed much poorer performance than that of the Teflon and ion gel stack, when it is used alone as a dielectric layer. 4.4. Thickness Effect of the Dip-Coated Gel Layers 4.4. Thickness Effect of the Dip-Coated Gel Layers For comparison, data from a bare Teﬂon layer of the same thickness is added to highlight the signiﬁcant contribution of the dip-coated gel layer. 4.4. Thickness Effect of the Dip-Coated Gel Layers 4.4. Thickness Effect of the Dip-Coated Gel Layers Our study here demonstrated that a new dip- coating method using solutions with low IL concentrations (differently fabricated from the previous spin-coating method) is capable of producing defect-free, reliable dielectric layers as thin as 80 nm, thickness comparative with other conventional dielectrics commonly used for EWOD studies, by simply fabricating with a single-step dip-coating process. Figure 8. Thickness effect of the dip-coated gel layer on static EWOD performance. The gel layer thickness varies from 80 nm to 1.19 μm, while the Teflon layer was fixed at 670 nm for all experiments. The droplet contact angles were measured at various input voltages. A negligible difference in EWOD performance was observed for the gel layers of varying thicknesses. For comparison, data from a bare Teflon layer of the same thickness is added to highlight the significant contribution of the dip-coated gel layer. 4 5 Thickness Effect of the Dip-Coated Teflon Layers Figure 8. Thickness effect of the dip-coated gel layer on static EWOD performance. The gel layer thickness varies from 80 nm to 1.19 µm, while the Teﬂon layer was ﬁxed at 670 nm for all experiments. The droplet contact angles were measured at various input voltages. A negligible difference in EWOD performance was observed for the gel layers of varying thicknesses. For comparison, data from a bare Teﬂon layer of the same thickness is added to highlight the signiﬁcant contribution of the dip-coated gel layer. Figure 8. Thickness effect of the dip-coated gel layer on static EWOD performance. The gel layer thickness varies from 80 nm to 1.19 μm, while the Teflon layer was fixed at 670 nm for all experiments. The droplet contact angles were measured at various input voltages. A negligible difference in EWOD performance was observed for the gel layers of varying thicknesses. For comparison, data from a bare Teflon layer of the same thickness is added to highlight the significant contribution of the dip-coated gel layer. 4 5 Thi k Eff t f th Di C t d T fl L Figure 8. Thickness effect of the dip-coated gel layer on static EWOD performance. The gel layer thickness varies from 80 nm to 1.19 µm, while the Teﬂon layer was ﬁxed at 670 nm for all experiments. The droplet contact angles were measured at various input voltages. A negligible difference in EWOD performance was observed for the gel layers of varying thicknesses. f p f y p f To demonstrate the viability of our dip-coated ion gel for 3D EWOD devices, a 5 × 1 liquid prism 4.6. Demonstration of the Dip-Coatable Ion Gel for an Arrayed Prism as an Example of 3D EWOD Devices array were fabricated with an aperture area of 50 × 7 mm2 as an example of 3D devices. Multiple sidewalls were first assembled to form five adjacent hollow structures (Figure 10a). Copper wires were connected on each sidewall using a conductive adhesive. The gel solution was prepared at a 0.25:1 weight ratio between the [EMIM][TFSI] ionic liquid and the [P(VDF-HFP)] copolymer, which was further diluted in acetone at 3%. A single-step dip-coating procedure was conducted to deposit the ion gel dielectric layer over the arrayed 3D hollow structures, which offers the benefits of simplicity, scalability, and high throughput over the spin-coating method as discussed in Section 2. After curing the gel layer coated on the 3D device surfaces at 70 °C, another dip-coating and baking processes at 110 °C were carried out to provide a hydrophobic (Teflon) layer. Figure 10b presents the fully assembled 3D structure of the arrayed prisms where an additional ITO base plate serves as the common ground electrode. Two immiscible liquids, water and silicone oil, were then filled in each of the prisms. To improve interfacial stability, a 0.05 wt % sodium dodecyl sulfate (SDS) surfactant was added to water [39]. Due to Laplace pressure difference, its initial contact angle is measured at 167°, as illustrated in Figure 10c. For operation of the liquid prism array fabricated, we built a LabView- programmed multi-channel power supply (Keysight N6700B) which can separately control the bias voltages applied to all ten inner sidewalls to achieve individual control of the five prisms. Figure 10d–f show the versatility of our prism arrays that are formed with the apex angles, ranging −20° ≤ ϕ ≤ 20° by varying the applied voltages at each of the prism sidewalls. By using two liquids with different refractive indices (nair ≠ n1 ≠ n2), the previous studies have demonstrated beam steering of the incoming light that can be effectively achieved without the need of bulky mechanical moving parts [22–24]. Figure 11a shows an experimental demonstration of beam steering using a single liquid prism filled with high-reflective-index oil 1-bromonapthalene (liquid 1) and water (liquid 2). Beam steering angles of β = −8.82°, 0°, 8.40° were achieved when voltages were applied due to respective prism apex angle modulations as a result of the large difference in refractive indices between the two liquids [22]. f p f y p f To demonstrate the viability of our dip-coated ion gel for 3D EWOD devices, a 5 × 1 liquid prism 4.6. Demonstration of the Dip-Coatable Ion Gel for an Arrayed Prism as an Example of 3D EWOD Devices Our group recently achieved the highest beam steering angle ever demonstrated up to β = 19.06° using a double-stacked prism configuration [22]. Supplementary to the double-stacked prism configuration, we have experimentally demonstrated spatial focal tuning using 3 × 1 linearly arrayed liquid prisms, as illustrated in Figure 11b. Each prism in the array can be individually controlled to replicate the subsections of a conventional Fresnel lens. Incoming light can then be steered accordingly to vary the focal length of the lens along both the longitudinal and lateral directions, thus making x- and z-axis spatial tuning possible [23]. To demonstrate the viability of our dip-coated ion gel for 3D EWOD devices, a 5 × 1 liquid prism array were fabricated with an aperture area of 50 × 7 mm2 as an example of 3D devices. Multiple sidewalls were ﬁrst assembled to form ﬁve adjacent hollow structures (Figure 10a). Copper wires were connected on each sidewall using a conductive adhesive. The gel solution was prepared at a 0.25:1 weight ratio between the [EMIM][TFSI] ionic liquid and the [P(VDF-HFP)] copolymer, which was further diluted in acetone at 3%. A single-step dip-coating procedure was conducted to deposit the ion gel dielectric layer over the arrayed 3D hollow structures, which offers the beneﬁts of simplicity, scalability, and high throughput over the spin-coating method as discussed in Section 2. After curing the gel layer coated on the 3D device surfaces at 70 ◦C, another dip-coating and baking processes at 110 ◦C were carried out to provide a hydrophobic (Teﬂon) layer. Figure 10b presents the fully assembled 3D structure of the arrayed prisms where an additional ITO base plate serves as the common ground electrode. Two immiscible liquids, water and silicone oil, were then ﬁlled in each of the prisms. To improve interfacial stability, a 0.05 wt % sodium dodecyl sulfate (SDS) surfactant was added to water [39]. Due to Laplace pressure difference, its initial contact angle is measured at 167◦, as illustrated in Figure 10c. For operation of the liquid prism array fabricated, we built a LabView-programmed multi-channel power supply (Keysight N6700B) which can separately control the bias voltages applied to all ten inner sidewalls to achieve individual control of the ﬁve prisms. Figure 10d–f show the versatility of our prism arrays that are formed with the apex angles, ranging −20◦≤ϕ ≤20◦by varying the applied voltages at each of the prism sidewalls. Since ion gel layers are fabricated by a n 4.5. Thickness Effect of the Dip-Coated Teﬂon Layers For example, full contact angle modulation was achieved around 60◦at 50 V for the 220 nm thick Teﬂon layer, while the contact angle was around 92◦for the 1010 nm thick layer at the same voltage input. These observations are very consistent with our general understanding of EWOD performance, i.e., the capacitance is inversely proportional to the layer thickness and thus a thin Teﬂon layer can achieve more contact angle modulation than that of a thick layer at the same bias voltage. However, for very thin Teﬂon layers such as the case of 120 nm, electrolysis takes place at 30 V before full contact angle modulation (see Figure 9a). Since the capacitance of the ion gel is a few orders higher than that of the Teﬂon, nearly 99% of the voltage input is distributed across the hydrophobic layer when the two capacitors of the ion gel and Teﬂon layers are connected in series, as shown in Figure 3. The theoretical breakdown voltage for the 120 nm thick Teﬂon layer was estimated as 24 V [9]. Nevertheless, the droplet contact angle was further modulated even at the voltage of 30 V exceeding this breakdown threshold. This observation can be explained by 10 of 14 Materials 2017, 10, 41 several previous studies [37,38], reporting that, although the voltage distributed across the Teﬂon layer exceeds its breakdown voltage, the device performs continuously while also slowly degrading. Materials 2017, 10, 41 10 of 14 Figure 9. Thickness effect of the dip-coated Teflon layer on static EWOD performance. For all experiments, the gel layer was held fixed at 1.19 μm, while varying the Teflon layer thickness. A thin Teflon layer allows a high capacitance and thus the same angle modulation is achieved at a lower voltage than other thick layers. The inset shows bubble generation due to electrolysis when the Teflon layer is 120 nm thin at 30 V. 4 6 Demonstration of the Dip Coatable Ion Gel for an Arrayed Prism as an Example of 3D EWOD Devices Figure 9. Thickness effect of the dip-coated Teﬂon layer on static EWOD performance. For all experiments, the gel layer was held ﬁxed at 1.19 µm, while varying the Teﬂon layer thickness. A thin Teﬂon layer allows a high capacitance and thus the same angle modulation is achieved at a lower voltage than other thick layers. Since ion gel layers are fabricated by a n 4.5. Thickness Effect of the Dip-Coated Teﬂon Layers The inset shows bubble generation due to electrolysis when the Teﬂon layer is 120 nm thin at 30 V. Figure 9. Thickness effect of the dip-coated Teflon layer on static EWOD performance. For all experiments the gel layer was held fixed at 1 19 μm while varying the Teflon layer thickness A thin Figure 9. Thickness effect of the dip-coated Teﬂon layer on static EWOD performance. For all Figure 9. Thickness effect of the dip-coated Teflon layer on static EWOD performance. For all experiments, the gel layer was held fixed at 1.19 μm, while varying the Teflon layer thickness. A thin Teflon layer allows a high capacitance and thus the same angle modulation is achieved at a lower voltage than other thick layers. The inset shows bubble generation due to electrolysis when the Teflon layer is 120 nm thin at 30 V. f h bl I l f A d E l f E O Figure 9. Thickness effect of the dip-coated Teﬂon layer on static EWOD performance. For all experiments, the gel layer was held ﬁxed at 1.19 µm, while varying the Teﬂon layer thickness. A thin Teﬂon layer allows a high capacitance and thus the same angle modulation is achieved at a lower voltage than other thick layers. The inset shows bubble generation due to electrolysis when the Teﬂon layer is 120 nm thin at 30 V. Figure 9. Thickness effect of the dip-coated Teflon layer on static EWOD performance. For all experiments, the gel layer was held fixed at 1.19 μm, while varying the Teflon layer thickness. A thin Teflon layer allows a high capacitance and thus the same angle modulation is achieved at a lower voltage than other thick layers. The inset shows bubble generation due to electrolysis when the Teflon layer is 120 nm thin at 30 V. Figure 9. Thickness effect of the dip-coated Teﬂon layer on static EWOD performance. For all experiments, the gel layer was held ﬁxed at 1.19 µm, while varying the Teﬂon layer thickness. A thin Teﬂon layer allows a high capacitance and thus the same angle modulation is achieved at a lower voltage than other thick layers. The inset shows bubble generation due to electrolysis when the Teﬂon layer is 120 nm thin at 30 V. f p f y p f To demonstrate the viability of our dip-coated ion gel for 3D EWOD devices, a 5 × 1 liquid prism 4.6. Demonstration of the Dip-Coatable Ion Gel for an Arrayed Prism as an Example of 3D EWOD Devices E pe i e a esu s e o s a e a e p opose ip coa i g e o e ec i e y p o i es a single-step coating solution to provide defect-free, functional gel layers for 3D EWOD device fabrication, which is much simpler and more convenient than spin coating. Although we have shown arrayed prisms as examples of 3D EWOD devices, the advantages of our proposed method wil become significantly more evident when fabricating devices with more complex geometries such a n × n arrayed prism panels and other non-planar structures. Figure 10. Demonstration of the dip-coatable ion gel fabrication for 5 × 1 arrayed liquid prisms as an example of 3D EWOD devices. (a) Five adjacent hollow structures were first assembled with multiple sidewalls modules; (b) The depositions of the ion gel and Teflon layers were completed through a single-step dip-coating method, which offers the benefits of simplicity, scalability, and high throughput over spin coating. The sidewalls were connected to a multi-channel power supply for individual voltage control, while a base substrate served as a ground electrode; (c) The arrayed prisms were filled with silicone oil and water added with a 0.05% SDS surfactant. The curved meniscus was initially formed with the contact angle of 167° at the interfaces between the two immiscible liquids; (d) When external bias voltages were equally applied to the sidewall (VL = VR = 43 V), the apex angles were set to ϕ = 0° and all five prisms were able to achieve a flat interface; (e) The interface was controlled with the apex angle of ϕ = 20° when VL = 30 V and VR = 90 V; (f) Similarly, the interface was titled to ϕ = −20° when VL = 90 V and VR = 30 V. Figure 10. Demonstration of the dip-coatable ion gel fabrication for 5 × 1 arrayed liquid prisms as an example of 3D EWOD devices. (a) Five adjacent hollow structures were ﬁrst assembled with multiple sidewalls modules; (b) The depositions of the ion gel and Teﬂon layers were completed through a single-step dip-coating method, which offers the beneﬁts of simplicity, scalability, and high throughput over spin coating. f p f y p f To demonstrate the viability of our dip-coated ion gel for 3D EWOD devices, a 5 × 1 liquid prism 4.6. Demonstration of the Dip-Coatable Ion Gel for an Arrayed Prism as an Example of 3D EWOD Devices By using two liquids with different refractive indices (nair ̸= n1 ̸= n2), the previous studies have demonstrated beam steering of the incoming light that can be effectively achieved without the need of bulky mechanical moving parts [22–24]. Figure 11a shows an experimental demonstration of beam steering using a single liquid prism ﬁlled with high-reﬂective-index oil 1-bromonapthalene (liquid 1) and water (liquid 2). Beam steering angles of β = −8.82◦, 0◦, 8.40◦were achieved when voltages were applied due to respective prism apex angle modulations as a result of the large difference in refractive indices between the two liquids [22]. Our group recently achieved the highest beam steering angle ever demonstrated up to β = 19.06◦using a double-stacked prism conﬁguration [22]. Supplementary to the double-stacked prism conﬁguration, we have experimentally demonstrated spatial focal tuning Materials 2017, 10, 41 11 of 14 using 3 × 1 linearly arrayed liquid prisms, as illustrated in Figure 11b. Each prism in the array can be individually controlled to replicate the subsections of a conventional Fresnel lens. Incoming light can then be steered accordingly to vary the focal length of the lens along both the longitudinal and lateral directions, thus making x- and z-axis spatial tuning possible [23]. Materials 2017, 10, 41 11 of 14 Materials 2017, 10, 41 11 of 1 Experimental results demonstrate that the proposed dip-coating method effectively provides Experimental results demonstrate that the proposed dip-coating method effectively provides a single-step coating solution to provide defect-free, functional gel layers for 3D EWOD device fabrication, which is much simpler and more convenient than spin coating. Although we have shown arrayed prisms as examples of 3D EWOD devices, the advantages of our proposed method will become signiﬁcantly more evident when fabricating devices with more complex geometries such as n × n arrayed prism panels and other non-planar structures. Experimental results demonstrate that the proposed dip-coating method effectively provides a single-step coating solution to provide defect-free, functional gel layers for 3D EWOD device fabrication, which is much simpler and more convenient than spin coating. Although we have shown arrayed prisms as examples of 3D EWOD devices, the advantages of our proposed method will become significantly more evident when fabricating devices with more complex geometries such as n × n arrayed prism panels and other non-planar structures. f p f y p f To demonstrate the viability of our dip-coated ion gel for 3D EWOD devices, a 5 × 1 liquid prism 4.6. Demonstration of the Dip-Coatable Ion Gel for an Arrayed Prism as an Example of 3D EWOD Devices The sidewalls were connected to a multi-channel power supply for individual voltage control, while a base substrate served as a ground electrode; (c) The arrayed prisms were ﬁlled with silicone oil and water added with a 0.05% SDS surfactant. The curved meniscus was initially formed with the contact angle of 167◦at the interfaces between the two immiscible liquids; (d) When external bias voltages were equally applied to the sidewall (VL = VR = 43 V), the apex angles were set to ϕ = 0◦and all ﬁve prisms were able to achieve a ﬂat interface; (e) The interface was controlled with the apex angle of ϕ = 20◦when VL = 30 V and VR = 90 V; (f) Similarly, the interface was titled to ϕ = −20◦when VL = 90 V and VR = 30 V. Figure 10. Demonstration of the dip-coatable ion gel fabrication for 5 × 1 arrayed liquid prisms as an example of 3D EWOD devices. (a) Five adjacent hollow structures were first assembled with multipl sidewalls modules; (b) The depositions of the ion gel and Teflon layers were completed through single-step dip-coating method, which offers the benefits of simplicity, scalability, and high throughput over spin coating. The sidewalls were connected to a multi-channel power supply fo individual voltage control, while a base substrate served as a ground electrode; (c) The arrayed prism were filled with silicone oil and water added with a 0.05% SDS surfactant. The curved meniscus wa initially formed with the contact angle of 167° at the interfaces between the two immiscible liquids (d) When external bias voltages were equally applied to the sidewall (VL = VR = 43 V), the apex angle were set to ϕ = 0° and all five prisms were able to achieve a flat interface; (e) The interface wa controlled with the apex angle of ϕ = 20° when VL = 30 V and VR = 90 V; (f) Similarly, the interface wa titled to ϕ = −20° when VL = 90 V and VR = 30 V. Figure 10. Demonstration of the dip-coatable ion gel fabrication for 5 × 1 arrayed liquid prisms as an example of 3D EWOD devices. f p f y p f To demonstrate the viability of our dip-coated ion gel for 3D EWOD devices, a 5 × 1 liquid prism 4.6. Demonstration of the Dip-Coatable Ion Gel for an Arrayed Prism as an Example of 3D EWOD Devices (a) Five adjacent hollow structures were first assembled with multiple sidewalls modules; (b) The depositions of the ion gel and Teflon layers were completed through a single-step dip-coating method, which offers the benefits of simplicity, scalability, and high throughput over spin coating. The sidewalls were connected to a multi-channel power supply for individual voltage control, while a base substrate served as a ground electrode; (c) The arrayed prisms were filled with silicone oil and water added with a 0.05% SDS surfactant. The curved meniscus was initially formed with the contact angle of 167° at the interfaces between the two immiscible liquids; (d) When external bias voltages were equally applied to the sidewall (VL = VR = 43 V), the apex angles were set to ϕ = 0° and all five prisms were able to achieve a flat interface; (e) The interface was controlled with the apex angle of ϕ = 20° when VL = 30 V and VR = 90 V; (f) Similarly, the interface was titled to ϕ = −20° when VL = 90 V and VR = 30 V. Figure 10. Demonstration of the dip-coatable ion gel fabrication for 5 × 1 arrayed liquid prisms as an example of 3D EWOD devices. (a) Five adjacent hollow structures were ﬁrst assembled with multiple sidewalls modules; (b) The depositions of the ion gel and Teﬂon layers were completed through a single-step dip-coating method, which offers the beneﬁts of simplicity, scalability, and high throughput over spin coating. The sidewalls were connected to a multi-channel power supply for individual voltage control, while a base substrate served as a ground electrode; (c) The arrayed prisms were ﬁlled with silicone oil and water added with a 0.05% SDS surfactant. The curved meniscus was initially formed with the contact angle of 167◦at the interfaces between the two immiscible liquids; (d) When external bias voltages were equally applied to the sidewall (VL = VR = 43 V), the apex angles were set to ϕ = 0◦and all ﬁve prisms were able to achieve a ﬂat interface; (e) The interface was controlled with the apex angle of ϕ = 20◦when VL = 30 V and VR = 90 V; (f) Similarly, the interface was titled to ϕ = −20◦when VL = 90 V and VR = 30 V. Figure 10. 12 of 14 12 f 14 (B) The bottom images present arrayed liquid prims that enable 3D focal tuning by controlling arrayed prisms; (a) When all steering angles are zero, βa1 = βa2 = βa3 = 0◦, the Fresnel lens does not perform as a concentrating element (i.e., f a = ∞); (b) When the outer prisms are modulated such that the steering is βb1 = −3.2◦, βb3 = 3.3◦and βb2 remains at 0◦, the beams converge and the lens is then said to have a focal length at ƒb ≈525 mm; (c) Further manipulation at higher angles of βc1 = −6.2◦, βc2 = 0◦, and βc3 = 6.4◦allows the focal point to reduce from ƒb ≈525 mm to ƒc ≈263 mm; (d) By controlling the arrayed prisms (βd1 = −7.3◦, βd2 = −4.0◦, and βd3 = 0◦) non-symmetrically, spatial focal tuning has been achieved in both longitudinal and lateral directions at ƒd ≈444 mm and ƒlat = 30 mm (reprinted from [21] with the permission). Figure 11. Experimental demonstrations of beam steering with the dip-coatable 3D liquid prisms. (A) The top images show the beam deflection using a single prism; (a) At ϕ = −25°, the beam steering was achieved as large as β = −8.82°; (b) At ϕ = 0°, a laser beam passes perpendicularly through the interface; (c) Similarly, we can achieve beam steering of β = 8.40° at φ = 24° (reprinted from the Reference [20] with the permission). 5. Conclusions 5. Conclusions This work presents a dip-coatable high-capacitance ion gel dielectric for 3D EWOD device fabrication that typically requires the assembly of multiple modules to form non-planar structures. A dip-coating approach offers a single-step solution with the benefits of simplicity, scalability, and high throughput for deposition of high-capacitance gel layers on 3D EWOD devices. With the dip- coated ion gel layers fabricated from an [EMIM][TFSI] ionic liquid and a [P(VDF-HFP)] copolymer, our experimental tests showed that the previous spin-coating method for preparation of the ion gel solution cannot be used for EWOD due to the surface defects that manifested when the gels were subject to the high temperatures needed for curing the hydrophobic (Teflon) layer. A new formulation lowering the content of ionic liquids in the gel solution was developed to obtain defect- free robust gel layers. The following experimental studies further showed the thickness effects of the ion gel and Teflon layers on EWOD performance. It was found to be highly dependent on the thickness of the hydrophobic layer, yet largely independent of the dip-coated gel thickness. Finally, we demonstrated the viability of our proposed dip-coating method using the 5 × 1 arrayed prisms as an example of 3D EWOD devices. With the dip-coated ion gels, our prism array was able to achieve individual prism control with the apex angle steering of up to ±20°. h b f h d h l d l bl EWO f h l This work presents a dip-coatable high-capacitance ion gel dielectric for 3D EWOD device fabrication that typically requires the assembly of multiple modules to form non-planar structures. A dip-coating approach offers a single-step solution with the beneﬁts of simplicity, scalability, and high throughput for deposition of high-capacitance gel layers on 3D EWOD devices. With the dip-coated ion gel layers fabricated from an [EMIM][TFSI] ionic liquid and a [P(VDF-HFP)] copolymer, our experimental tests showed that the previous spin-coating method for preparation of the ion gel solution cannot be used for EWOD due to the surface defects that manifested when the gels were subject to the high temperatures needed for curing the hydrophobic (Teﬂon) layer. A new formulation lowering the content of ionic liquids in the gel solution was developed to obtain defect-free robust gel layers. The following experimental studies further showed the thickness effects of the ion gel and Teﬂon layers on EWOD performance. 12 of 14 12 f 14 12 of 14 12 f 14 Materials 2017, 10, 41 M t i l 2017 10 41 terials 2017, 10, 41 12 o Figure 11. Experimental demonstrations of beam steering with the dip-coatable 3D liquid prisms. (A) The top images show the beam deflection using a single prism; (a) At ϕ = −25°, the beam steering was achieved as large as β = −8.82°; (b) At ϕ = 0°, a laser beam passes perpendicularly through the interface; (c) Similarly, we can achieve beam steering of β = 8.40° at φ = 24° (reprinted from the Reference [20] with the permission). (B) The bottom images present arrayed liquid prims that enable 3D focal tuning by controlling arrayed prisms; (a) When all steering angles are zero, βa1 = βa2 = βa3 = 0°, the Fresnel lens does not perform as a concentrating element (i.e., fa = ∞); (b) When the outer prisms are modulated such that the steering is βb1 = −3.2°, βb3 = 3.3° and βb2 remains at 0°, the beams converge and the lens is then said to have a focal length at ƒb ≈ 525 mm; (c) Further manipulation at higher angles of βc1 = −6.2°, βc2 = 0°, and βc3 = 6.4° allows the focal point to reduce from ƒb ≈ 525 mm to ƒc ≈ 263 mm; (d) By controlling the arrayed prisms (βd1 = −7.3°, βd2 = −4.0°, and βd3 = 0°) non-symmetrically, spatial focal tuning has been achieved in both longitudinal and lateral directions at ƒd ≈ 444 mm and ƒlat = 30 mm (reprinted from [21] with the permission). Figure 11. Experimental demonstrations of beam steering with the dip-coatable 3D liquid prisms. (A) The top images show the beam deﬂection using a single prism; (a) At ϕ = −25◦, the beam steering was achieved as large as β = −8.82◦; (b) At ϕ = 0◦, a laser beam passes perpendicularly through the interface; (c) Similarly, we can achieve beam steering of β = 8.40◦at ϕ = 24◦(reprinted from the Reference [20] with the permission). 12 of 14 12 f 14 (B) The bottom images present arrayed liquid prims that enable 3D focal tuning by controlling arrayed prisms; (a) When all steering angles are zero, βa1 = βa2 = βa3 = 0°, the Fresnel lens does not perform as a concentrating element (i.e., fa = ∞); (b) When the outer prisms are modulated such that the steering is βb1 = −3.2°, βb3 = 3.3° and βb2 remains at 0°, the beams converge and the lens is then said to have a focal length at ƒb ≈ 525 mm; (c) Further manipulation at higher angles of βc1 = −6.2°, βc2 = 0°, and βc3 = 6.4° allows the focal point to reduce from ƒb ≈ 525 mm to ƒc ≈ 263 mm; (d) By controlling the arrayed prisms (βd1 = −7.3°, βd2 = −4.0°, and βd3 = 0°) non-symmetrically, spatial focal tuning has been achieved in both longitudinal and lateral directions at ƒd ≈ 444 mm and ƒlat = 30 mm (reprinted from [21] with the permission). Figure 11. Experimental demonstrations of beam steering with the dip-coatable 3D liquid prisms. (A) The top images show the beam deﬂection using a single prism; (a) At ϕ = −25◦, the beam steering was achieved as large as β = −8.82◦; (b) At ϕ = 0◦, a laser beam passes perpendicularly through the interface; (c) Similarly, we can achieve beam steering of β = 8.40◦at ϕ = 24◦(reprinted from the Reference [20] with the permission). (B) The bottom images present arrayed liquid prims that enable 3D focal tuning by controlling arrayed prisms; (a) When all steering angles are zero, βa1 = βa2 = βa3 = 0◦, the Fresnel lens does not perform as a concentrating element (i.e., f a = ∞); (b) When the outer prisms are modulated such that the steering is βb1 = −3.2◦, βb3 = 3.3◦and βb2 remains at 0◦, the beams converge and the lens is then said to have a focal length at ƒb ≈525 mm; (c) Further manipulation at higher angles of βc1 = −6.2◦, βc2 = 0◦, and βc3 = 6.4◦allows the focal point to reduce from ƒb ≈525 mm to ƒc ≈263 mm; (d) By controlling the arrayed prisms (βd1 = −7.3◦, βd2 = −4.0◦, and βd3 = 0◦) non-symmetrically, spatial focal tuning has been achieved in both longitudinal and lateral directions at ƒd ≈444 mm and ƒlat = 30 mm (reprinted from [21] with the permission). f p f y p f To demonstrate the viability of our dip-coated ion gel for 3D EWOD devices, a 5 × 1 liquid prism 4.6. Demonstration of the Dip-Coatable Ion Gel for an Arrayed Prism as an Example of 3D EWOD Devices Demonstration of the dip-coatable ion gel fabrication for 5 × 1 arrayed liquid prisms as an example of 3D EWOD devices. (a) Five adjacent hollow structures were first assembled with multipl sidewalls modules; (b) The depositions of the ion gel and Teflon layers were completed through single-step dip-coating method, which offers the benefits of simplicity, scalability, and high throughput over spin coating. The sidewalls were connected to a multi-channel power supply fo individual voltage control, while a base substrate served as a ground electrode; (c) The arrayed prism were filled with silicone oil and water added with a 0.05% SDS surfactant. The curved meniscus wa initially formed with the contact angle of 167° at the interfaces between the two immiscible liquids (d) When external bias voltages were equally applied to the sidewall (VL = VR = 43 V), the apex angle were set to ϕ = 0° and all five prisms were able to achieve a flat interface; (e) The interface wa controlled with the apex angle of ϕ = 20° when VL = 30 V and VR = 90 V; (f) Similarly, the interface wa titled to ϕ = −20° when VL = 90 V and VR = 30 V. Figure 11. Cont. Figure 11. Cont. 12 of 14 12 f 14 References 1. Wheeler, A.R. Putting electrowetting to work. Science 2008, 322, 539–540. [CrossRef] [PubMed] 2. Park, S.-Y.; Teitell, M.A.; Chiou, E.P.Y. Single-sided continuous optoelectrowetting (scoew) for droplet manipulation with light patterns. Lab Chip 2010, 10, 1655–1661. [CrossRef] [PubMed] 2. Park, S.-Y.; Teitell, M.A.; Chiou, E.P.Y. Single-sided continuous optoelectrowetting (scoew) for droplet manipulation with light patterns. 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With the dip-coated ion gels, our prism array was able to achieve individual prism control with the apex angle steering of up to ±20◦. The beauty of the proposed approach lies in providing reliable EWOD performance with simple and scalable processes. The single-step dip-coating process becomes greatly advantageous when fabricating 3D devices with more complex geometries such as n × n arrayed liquid prism panels and other non-planar structures. Our study here using the dip-coatable high-capacitance ion gel dielectric will have numerous potential uses in the growing field of 3D EWOD devices. The beauty of the proposed approach lies in providing reliable EWOD performance with simple and scalable processes. The single-step dip-coating process becomes greatly advantageous when fabricating 3D devices with more complex geometries such as n × n arrayed liquid prism panels and other non-planar structures. 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https://openalex.org/W2138346579	http://www.scielo.br/pdf/reeusp/v48n6/0080-6234-reeusp-48-06-1093.pdf	English	null	Educação superior em Enfermagem: o processo de trabalho docente em diferentes contextos institucionais	null	2,014	cc-by	7,356	RESUMO ABSTRACT Objective: To analyze the characteristics of faculty work in nursing higher educa tion. Method: An exploratory qualitative study with a theoretical-methodological framework of dialectical and historical materialism. The faculty work process was adopted as the analytical category, groun ded on conceptions of work and profes sionalism. Semi-structured interviews were conducted with 24 faculty members from three higher education institutions in the city of São Paulo, classified according to the typology of institutional contexts. Results: The faculty members at these higher education institutions are a hete rogeneous group, under different working conditions. Intensification and precarious conditions of the faculty work is common to all three contexts, although there are important distinctions in the practices re lated to teaching, research and extension. Conclusion: Faculty professionalization can be the starting point for analyzing and coping with such a distinct reality of facul ty work and practice. RESUMEN Objetivo: Analizar las características del trabajo docente en la educación superior en Enfermería. Método: Estudio explorato rio y cualitativo cuyo marco de referencia teórico-metodológico fue el materialismo histórico y dialético. Como categoría analí tica, se adoptó el proceso de trabajo docen te, anclado en las concepciones de trabajo y profesionalidad. Se realizaron entrevistas semiestructuradas con 24 docentes de tres instituciones de educación superior de la ciudad de São Paulo, clasificadas según la tipología de contextos institucionales. Resultados: Se revelaron que los docentes de dichas instituciones de educación supe rior constituyen un grupo heterogéneo, so metido a distintas condiciones laborales. La intensificación y la precarización del traba jo docente son comunes a los tres marcos, aunque existan diferencias importantes en las prácticas didácticas relacionadas con la enseñanza, la investigación y la extensión. Conclusión: La profesionalización docente puede ser el punto de partida para el análi sis y el enfrentamiento de una realidad tan distinta de trabajo y práctica docente. Objetivo: Analisar as características do trabalho docente no ensino superior em Enfermagem. Método: Estudo explorató rio e qualitativo cujo referencial teórico- metodológico foi o materialismo históri co e dialético. Como categoria analítica, adotou-se o processo de trabalho docente, ancorado nas concepções de trabalho e profissionalidade. Foram realizadas entre vistas semiestruturadas com 24 docentes de três instituições de ensino superior da cidade de São Paulo, classificadas segun do a tipologia de contextos institucionais. Resultados: Revelaram que os docentes dessas instituições de ensino superior constituem um grupo heterogêneo, sub metido a diferentes condições de trabalho. DOI: 10.1590/S0080-623420140000700018 DOI: 10.1590/S0080-623420140000700018 Original Article EDUCAÇÃO SUPERIOR EM ENFERMAGEM: O PROCESSO DE TRABALHO DOCENTE EM DIFERENTES CONTEXTOS INSTITUCIONAIS EDUCACIÓN SUPERIOR EN ENFERMERÍA: EL PROCESO DE TRABAJO DOCENTE EN DISTINTOS MARCOS INSTITUCIONALES Valéria Marli Leonello1, Maria Amélia de Campos Oliveira2 1 PhD Professor, Professional Orientation Department, School of Nursing, São Paulo University, São Paulo, SP, Brazil. 2 Full Professor, Department of Nursing in Collective Health, School of Nursing, São Paulo University, São Paulo, SP, Brazil. METHOD In Brazil, higher education has undergone a number of changes over the last two decades, especially after the promulgation of the Lei de Diretrizes e Bases (Law of Directives and Bases, LDB) in 1996, with the diversifica tion and expansion at all levels and modalities deserving note(1). The LDB determined that higher education can be conducted at public or private higher educational institu tions (HEIs), which may be universities, colleges or univer sity centers(2). Dialectical and historical materialism was adopted as the theoretical and methodological framework, and the faculty work process was adopted as an analytical category, grounded in conceptions of work(7) and professionalism(8). When seeking to understand the main aspects of the differ ent levels in which faculty work and professionalism operate, the historical materialist and dialectical analysis performs an articulated movement within the dynamics of social reality. These changes resulted in a strong expansion of the system, mainly in the private sector, with the support of government policies. As a consequence of the adopted policy, the growth was marked by unequal geographic distribution of courses, concentrated in the southeastern and southern regions. An assumption is made that faculty work is a way to in tervene in the social reality; therefore, it is a social practice. As such, it can only be understood and comprehended if its relationship with the institutional context in which it is in serted is analyzed(3). Professionalism, in turn, refers to what is specific to the faculty action, referring to the behaviors, skills, attitudes and values of being a faculty member(8). Studies show that the scenario of higher education ex pansion and diversification directly affects faculty work, which confronts the consequences of policy changes estab lished in Brazil. Therefore, confronting this reality and work process are distinctly and directly related to the institutional format in which the faculty members are located(3-4). Considering work and professionalism as analytical categories implies an unveiling of the heterogeneity of faculty work in institutional formats existing in the current scenario of higher education in Brazil, assuming that this scenario determines different working conditions, practices and professional development. In health education, particularly in nursing education, there is a gap in the scientific literature on the subject. Higher education in nursing: the faculty work process in different institutional contexts Leonello VM, Oliveira MAC INTRODUCTION contexts? Under what conditions is it performed? What are the practices of these faculty members? Higher education has a strategic position in the socioeco nomic development of a country, given its relationship with the training and qualification of a workforce able to opera tionalize and implement modernization and improvement processes within the society. Therefore, the object of this study is the nursing faculty work process in different institutional contexts, and its objec tive is to analyze the work process of nursing faculty mem bers in HEIs, in the city of São Paulo. RESUMO A intensificação e a precarização do traba lho docente é comum aos três contextos, embora haja distinções importantes nas práticas didáticas relacionadas a ensino, pesquisa e extensão. Conclusão: A profis sionalização docente pode ser o ponto de partida para a análise e o enfrentamento de uma realidade tão distinta de trabalho e prática docente. DESCRIPTORS Education higher Education nursing Faculty Nursing DESCRIPTORES Educación superior Educación en enfermería Docentes de Enfermería Docencia DESCRITORES Educação superior Educação em enfermagem Docentes de enfermagem DESCRITORES Educação superior Educação em enfermagem Docentes de enfermagem DESCRIPTORES Educación superior Educación en enfermería Docentes de Enfermería Docencia 1 PhD Professor, Professional Orientation Department, School of Nursing, São Paulo University, São Paulo, SP, Brazil. 2 Full Professor, Department of Nursing in Collective Health, School of Nursing, São Paulo University, São Paulo, SP, Brazil. 1090 Rev Esc Enferm USP 2014; 48(6):1091-9 www.ee.usp.br/reeusp/ Higher education in nursing: the faculty work process in different institutional contexts Leonello VM, Oliveira MAC Received: 13/05/2014 Approved: 18/09/2014 Português / Inglês www.scielo.br/reeusp Received: 13/05/2014 Approved: 18/09/2014 1090 Rev Esc Enferm USP 2014; 48(6):1091-9 www.ee.usp.br/reeusp/ Rev Esc Enferm USP 2014; 48(6):1091-9 www.ee.usp.br/reeusp/ Rev Esc Enferm USP 2014; 48(6):1091-9 www.ee.usp.br/reeusp/ INTRODUCTION METHOD Table 1 − Distribution of higher education institutions with nur sing courses in the state of São Paulo, according to the typology of institutional contexts - São Paulo, 2010 Table 1 − Distribution of higher education institutions with nur sing courses in the state of São Paulo, according to the typology of institutional contexts - São Paulo, 2010 Considering the general context of higher education in the country, in nursing education in particular, and in order to contribute to the understanding of this multifaceted reality of faculty work in different institutional contexts, this study sought to answer the following questions: What is the work process of nursing faculty members in different institutional Of the total of 114 HEIs with nursing courses in the state of São Paulo in 2010, five (4.5%) were public univer sities, and all were classified as academic contexts, which have a high proportion of full-time or exclusive dedication of doctorally prepared faculty members. However, most (108 HEIs, 94.5%) were classified as business contexts, with a small proportion of doctorally prepared faculty members and full-time contracts. No HEI was classified as a regional context. One institution (1.0%) did not fit in any of the contexts provided by the typology, because of the high proportion of doctorally prepared faculty members and low proportion of tenured faculty members. Because it had characteristics of both of these two contexts, it was classified as a mixed context: a high proportion of doctor ally prepared faculty members, characteristic of academic contexts, and a high proportion of part-time faculty mem bers, a characteristic of business contexts. infrastructure. The didactic practices refer to the set of aspects related to the daily work of faculty members in different contexts, related, for example, to teaching, re search and extension (Figure 1). Institutional contexts in higher education business context organizational practices didactic practices mixed context organizational practices didactic practices academic context organizational practices didactic practices Figure 1 – Representation of the nested practices system(8) adap ted to the higher education system. Institutional contexts in higher education After classification, three HEIs were selected through convenience sampling, one in each context. Because these institutions allowed the researchers to meet the faculty members, semi-structured interviews were con ducted with 24 faculty members: nine from the business context, seven from the academic context, and eight from the mixed context. METHOD In general, studies address the characteristics of faculty work in limited institutional contexts, according to the adminis trative or academic nature of the HEIs, not taking as a start ing point the faculty work process in different contexts, but rather using specific approaches, such as quality of life(5) and job satisfaction(6). In order to classify the HEIs, a typology of institutional contexts(9) was used, which classifies higher education institu tions into three major types: business contexts, presenting a small proportion of doctorally prepared faculty members and those faculty members with full-time contracts; regional con texts, which have a small proportion of doctorally prepared faculty members and a high proportion of full-time faculty members; and academic contexts, with a high proportion of full-time or exclusive dedication of doctorally prepared fac ulty members. The data required for the classification were obtained through a request to the Instituto Nacional de Estu dos e Pesquisas Educacionais Anísio Teixeira (Anísio Teixeira National Institute for Educational Studies, NIEE) and the clas sification of the contexts is shown in Table 1. According to the Marxian conception(7), work is conceived as a process, a transformation of a given object by means of the intentional action of an agent, who, in order to achieve this purpose, employs material or intellectual instruments. With regard to the nursing faculty work process, the agents are faculty members in nursing higher education, in other words, whose working object is another human being, the nursing student, more specifically his knowledge, skills and attitudes. The instruments represent the entire arsenal, physical or intellectual, which faculty members use to inter vene/act with this individual. RESULTS In all three contexts, business, mixed and academic, results showed that there is a process of intensification and precarious conditions of faculty member work that impacts on organizational and didactic practices, but with important distinctions in different contexts. METHOD For the interviews, a script was constructed with objective questions for the characteris tics of subjects, and guiding questions to achieve the re search objectives. In the characterization step, there were eight questions that addressed faculty member titles, the year in which they began to work in the institution, work regimen, type of contract, work in other institutions, and weekly workload. In the next step, the subjects were asked to talk about their daily faculty work, identifying dif ficulties, facilities and challenges of teaching in their insti tutional context. Figure 1 – Representation of the nested practices system(8) adap ted to the higher education system. Higher education in nursing: the faculty work process in different institutional contexts Leonello VM, Oliveira MAC Mixed context In this context, concerning didactic practices, faculty member work focused on research and administrative activities, and on graduate teaching. However, according to some subjects, there was a large institutional effort to accomplish curricular reorientation of undergradu ate education. For the faculty members interviewed, the academic context overvalued administrative and research activities at the expense of undergraduate teaching and extension activities, which were considered undervalued. With regard to teaching practices, faculty members’ work had some aspects of the business context, mixed with others from the academic context. The work was primarily aimed at undergraduate teaching, but there was an institutional effort to conduct graduate research activities. With regard to the theory-practice relationship, the faculty who taught the the ory also accompanied the students into the practical teach ing conducted in the internship, unlike the business context in which the faculty members who taught the theory were not necessarily linked to the clinical practice. In relation to the student profile, there was no mention of great difficulties or challenges to teaching. The difficulties and challenges were related to the need to promote active student participation and to facing resistance from many faculty members, who valued research over teaching. There were also difficulties in developing practical teaching in the clinical field, given the unpredictability of the actions, due to the complex reality of the services, which created insecurity. Regarding the students’ profile, according to the fac ulty members, the initial selection was more accurate and tuition had a higher value compared to the busi ness environment. There were fewer students per class, with better social integration and less learning difficul ties. The faculty members felt that they taught a more select group, with differentiated income and level of instruction. This profile, with an institutional policy of small class sizes, facilitates the teaching practice, making teaching more individualized for the student and more satisfying for the faculty. Regarding research activities, a great concern and even anxiety was reported by the faculty members to achieve institutional goals of scientific production and publication, with the value of quantity over quality of the articles being mentioned; competitiveness among peers for research fund ing; publishing in reputable journals in the field; difficulty managing funded projects, with intense activity reporting and accountability to funding agencies; and, the need for an international curriculum, which required work in collabora tion with research groups from foreign institutions. Higher education in nursing: the faculty work process in different institutional contexts Leonello VM, Oliveira MAC Business context With regard to didactic practices, the faculty members in the business context state that their work is primarily directed towards undergraduate classroom teaching, with little integration into administrative, research, extension and graduate teaching activities. The research project was approved by the Ethics Com mittee of the School of Nursing, São Paulo University (Process #876/2009). The HEIs were invited by the nurs ing course coordinators, who authorized the study. All the nursing faculty members were invited through an invita tion letter sent via email. Those who agreed to participate were informed about the study objectives and signed a consent form. Faculty members with different back grounds, titles, and work regimens were interviewed. Research activities were not included in faculty mem ber work, therefore, they were not paid. They were performed sporadically in undergraduate education, in course completion studies, and scientific initiation. Exten sion activities were also sporadic and were not considered part of faculty member work, and therefore were unpaid. Although they had these difficulties in research and exten sion, the faculty members recognized them as activities that enhanced the quality of education. The empirical material was submitted to the technique of discourse analysis(10), which enabled the construction of a set of themes which, in turn, were gathered into empiri cal categories, arranged according to the nested practices system(8), adapted to this study. In the nested practices system, there is a set of interconnected and nested prac tices that characterize faculty work. The faculty members also faced difficulties related to the profile of the students, who had already been in the labor market, namely, they are student-workers, arriving to the classroom already tired. The challenges concern, for example, the choice of teaching strategies and appro priate assessment of these students. The results were organized into two main sets of prac tices: didactic and organizational. Organizational practices refer to the aspects of the structure of the organizational context that reflect on the teaching work, such as, for ex ample, the career plan, work regimen, and institutional With regard to organizational practices, the career plan, although formal, is not effectively operated. Regard ing the work system, they cited financial instability because the payment is made per hour/class, so the faculty salary is 2 Rev Esc Enferm USP 2014; 48(6):1091-9 www.ee.usp.br/reeusp/ 1092 Rev Esc Enferm USP 2014; 48(6):1091-9 www.ee.usp.br/reeusp/ mentioned, were sporadic and occasional, through cours es, events and presentations of students’ work. Business context mentioned, were sporadic and occasional, through cours es, events and presentations of students’ work. dependent on the number of classes/disciplines assigned to them every six months, and there may be changes according to the number of classes each semester. The faculty mem bers in this context had other jobs, some in health care, to complement the salary and ensure financial stability. As for the organizational practices, there was a career plan that followed some institutional criteria (publication, title, time on the job), although it was mentioned that the salary was not compatible with the workload. The labor regimen was predominantly partial and offered some stability against the hourly regimen, characteristic of the business environment. Insufficient institutional infrastructure - with scarce material resources and inadequate facilities - was identi fied as an aspect that hindered faculty work. Communi cation difficulties between the professors and the admin istrative structure were also mentioned. However, in this HEI, the coordination of the nursing area offered support, dialogue, respect, autonomy and freedom to faculty mem bers. That allowed professors, for example, to exclusively teach disciplines in their area of interest, which, according to the interviewees, does not always occur at HEIs linked to institutional contexts with business characteristics. Despite these difficulties, the faculty members shared the perception that the HEI valued the quality of education, aiming not only at profit, but at the quality of education of its students. They exemplified this fact by citing the good acceptance of graduates in the labor market in the area. Nursing faculty working conditions in times of work precariousness and flexibility The precariousness and flexibility of faculty work man ifested itself in different ways in different contexts, and the main aspects observed were related to the work regi men, career, and constraints in structural, material and human infrastructure. Regarding organizational practices, the full-time work regimen prevailed, with exclusive dedication, in which the fac ulty profession was explicit and the criteria for advancement was institutionally validated. With regard to the administra tive activities, the main complaint was that they consumed a substantial part of the work, due to the bureaucracy in insti tutional management processes that led to more work. These aspects indicate the new configuration of the working relationships established in the states that adopt neoliberalism as a political-economic basis, where unem ployment and dispossession of historically constructed labor rights are seen increasingly. In this reconfiguration, a new profile of employee arises, increasingly flexible to perform multitasking, proactive and aware of his/her need for constant updating and training in the face of de mands from the labor market(11-12). Faculty autonomy, characteristic of this context, was identified as a generator of great responsibility, related to the difficulty in establishing rules and consensus among the other professional colleagues. It was unanimous among the respondents that the context gives financial stability and the career is institutionally formalized and ensured, although some report a discrepancy between the work performed and the remuneration received. In the case of faculty work, especially in nursing, this profile is expressed in work overload, intensifica tion of tasks, and self-penalization for not achieving the many front lines. Even in contexts in which the form of hiring is more stable, faculty members complain about the intensification of activities and little time to per form them(13). In summary, faculty work within the three contexts showed similar features, for example, lack of time to per form the different activities and the need for improved or ganizational infrastructure, according to the expectations and needs of the work covered in each context. In the business and mixed contexts, healthcare experi ence in nursing was seen as an important aspect of faculty practice, being a highly valued differential by students and faculty members themselves. In these contexts, some facul ty members mentioned the need for a second job for wage supplementation. Nursing faculty working conditions in times of work precariousness and flexibility In the academic context, and to some ex tent in the mixed context, given the institutional value for research, the faculty members reported feeling pressured to achieve institutional goals of academic productivity. When contracts are unstable, faculty members accu mulate jobs in teaching or health care to maintain a rea sonable level of financial stability, overloading themselves with activities and often surpassing the 40 hour-week es tablished by labor legislation. A market relationship is es tablished between the work performed by the professor and the HEIs, which purchase the work, making it increas ingly precarious, through precarious contracts and finan cially unstable work regimens(14). DISCUSSION The international scenario also shows signs of re duced quality of the faculty work force in higher educa tion. A study conducted in Australia in 2011 showed that, although faculty members felt motivated and considered their careers rewarding, the increased number of stu dents, not accompanied by new faculty positons, has in creased their workload(15). The analysis of faculty work in different institutional contexts enabled the construction of four major themes of analysis, also arranged according to the organizational and didactic practices,(8) and adapted for this study (Figure 2). Organizational practices Didactic practices Nursing faculty working conditions in times of work precariousness and fexibility Teaching in the classroom and clinical practice: realities and challenges of nursing faculty practice From overvaluation to the shortage of research: efects on faculty work and the quality of nursing education The teaching-research-extension articulation: the need for a reframing of nursing extension activities Figure 2 – Analysis themes according to the nested practices system Organizational practices in the potential of extension, in conjunction with teaching and research, they mentioned that this was undervalued within the institutional context, which was expressed by the low weight assigned to it in faculty evaluation. Nursing faculty working conditions in times of work precariousness and flexibility Higher education in nursing: the faculty work process in different institutional contexts Leonello VM, Oliveira MAC Mixed context The main difficulties and challenges of the faculty work in the mixed context were also about undergraduate teaching, especially in practical teaching in clinical prac tice, considered by the subjects as a generator of physi cal and mental strain, given the responsibility to monitor students, manage tensions and reach consensus between teaching and caregiving activities in the fields of practice. In graduate teaching, according to the subjects, both internal and external assessments directed faculty partici pation to research and the advising of masters and doc toral students, and the faculty members that were not part of this group felt less valued institutionally. Another relevant aspect of this context was noted in remarks by the faculty members that other areas of knowledge were more renowned and valued than nursing. Regarding research activities, although hired as part- time faculty members, they conducted research activities due to an institutional requirement, and they dissemi nated their findings in publications and paper presenta tions at events. They mentioned that sometimes there was insufficient time to conduct such activities, which led them to perform them outside of working hours. As in the business context, the research activity was also focused on undergraduate education. Extension activities, seldom The faculty members also stated that the extension ac tivities were subsumed into research and inclusion of facul ty members in administrative positions. Since they believed Rev Esc Enferm USP 2014; 48(6):1091-9 www.ee.usp.br/reeusp/ Rev Esc Enferm USP 2014; 48(6):1091-9 www.ee.usp.br/reeusp/ 1093 Organizational practices Teaching in the classroom and clinical practice: realities and challenges of the nursing faculty practice Teaching in the classroom and clinical practice: realities and challenges of the nursing faculty practice The challenges are reflected in the didactic practice of faculty members that develop teaching, research and ex tension in different ways, depending on the institutional context. In the theoretical and clinical practice teaching, the challenges range from lack of practical training in the field, in the business context, to the mental and physical wear from this activity, given the unpredictability of the actions in the fields of clinical practice in mixed and aca demic contexts. The availability of nursing courses grew by 340% in the period between 1991-2004, a strong growth in the private sector, as is the case of many institutions in the business context(18). The growth has a direct impact on faculty practice: the higher the number of students, the more challenging the pedagogical work. In the mixed con text in which the students’ profile is different, with a more rigorous initial selection and the highest monthly value as compared to the business environment, there are few stu dents per class, with better social integration and minor learning difficulties. In the academic context, the faculty members did not mention the student’s profile, because the students go through a rigorous entrance exam, to de vote to a full-time course. Studies show that the clinical practice overwhelms the faculty due to the accumulation of activities, such as prior contact with health professionals, recognizing clini cal practice and its rules and routines, supervision of stu dents, and concern about the care of the service user(16). The faculty feels so responsible for activities that can be seen as a catalyst for different demands encountered in the field, taking on more responsibilities. Although exhausting, clinical teaching is deemed nec essary to promote student contact with the reality of health services and the health problems of the assisted population. The unpredictability in practice settings, al though generating stress, is seen as an opportunity for student and faculty members to exercise listening to the needs of users of health services, respecting the decisions of the people around them(17). This distinction in the students’ profile in different con texts shows that students’ access to higher education has also changed after the LDB. The legislation gives freedom to the HEIs to formulate entrance exams, which results in easy admission of students with different profiles(22). Higher education in nursing: the faculty work process in different institutional contexts Leonello VM, Oliveira MAC Teaching in the classroom and clinical practice: realities and challenges of the nursing faculty practice Regarding the students’ profile, mentioned in the cor porate context as an aspect that complicates the choice of teaching strategies and assessment, the results support a study about the trajectory of nursing courses in the pe riod from 1991-2004, which identified that most students in higher nursing education were already integrated into the labor market. This double work was also an important characteristic of nursing students, mostly from private schools, where working students predominate(18). Another aspect present in business contexts is the lack of preparation for teaching, with few opportunities to par ticipate in professionalization processes, more evident in the academic context. Brazilian researchers warn about the fact that most of the stricto sensu graduate faculty training programs do not prepare faculty members for teaching, stressing that train ing for research does not equate to pedagogical training(3). Another characteristic is related to the social integra tion of the students who come from families where the level of parental education is medium. This fact, coupled with the fact that they attended elementary and second ary education in public schools, deserves attention from nursing courses, which must seek diversified strategies in order to promote student learning. A nursing study showed that deficiency in the theoretical basis of the newly admitted students in higher education has become an obstacle to the conduct of the teaching-learning pro cess and therefore to faculty practice(19). Internationally, since 2004, the European Trade Union Committee for Education recommends three great ar eas for priority actions for faculty training: improving the quality of initial training, recruitment and retention of qualified faculty members, and ensuring that professional development is a right of faculty members and is part of their work process(23). From overvaluation to the shortage of research: effects on faculty work and the quality of nursing education This concern has also been implicated in a global con text, according to the report from the 2013 European Union Study Group on the Modernization of Higher Edu cation, which recommends that the preponderance of re search in relation to teaching in the definition of academic merit needs to be revised(27). In the business context, the extension activities are mentioned little by faculty members. Some even say that they are not developed, while others cite its epi sodic nature. As the labor regimen, in this context, is per hour/lesson, extension activities are not an institu tional requirement. On the other hand, there are institutional contexts in which faculty work in research activity is scarce. In mixed and business contexts, research is seen only as a mandato ry activity for the training of students, limited to the com pletion of course work. Research activities not articulated in this work are scarce. Nonetheless, faculty members feel that the mixed context should develop research, although they are hired on a part-time basis, with the workload di rected strongly toward classroom teaching. Teachers in the business environment, in turn, develop a research ac tivity, voluntarily, outside of work hours. Sometimes, the research activity in the form of completion of course work is offered in a discipline, and a faculty member is assigned to this activity and the amount paid by the hour/class is assigned by the discipline. In the mixed context, extension is understood as ac tivities performed in work leagues or as an extension of the academic activities in the form of dissemination of work by students. Therefore, there are few activities, con ducted by a few faculty members, which is to say that the work process of the faculty members do not incorporate this type of activity. Although extension activities are part of the functions of the university, with the activities of teaching and re search, they are not on an equal position in terms of in stitutional value, which unbalances the desired tripod of university functioning. Recently, a movement of reframing of the concept of university extension has increased the traditional sense of diffusion and dissemination of knowledge to an edu cational, cultural and scientific process that articulates teaching and research with transforming potential(28). From overvaluation to the shortage of research: effects on faculty work and the quality of nursing education According to the National Education Forum 2013, Bra zil still has to face the challenge of making quality, obliga tory secondary education universal. This challenge cer tainly will result in improvements in the quality of higher education(20). In the academic context, research activity is consid ered to be the focus of faculty work, the extent to which faculty assessments value research activity production more than undergraduate teaching and extension. This reality is not unique to Brazil. In the United States, the report from the National Center for Public Policy in Higher Education, in partnership with the National Center for Higher Education Management Systems, identifies the need to improve the quality of secondary education to en sure the best future conditions for access and retention to future professionals(21). The institutional assessment, in turn, follows external parameters such as the Coordenadoria de Aperfeiçoamen to de Pessoal de Nível Superior (Coordination of Improve ment of Higher Education Personnel – CAPES), in that fac ulty work is mainly measured by: time spent on research activities, expressed by the number of articles published in journals indexed with high Qualis (impact factors); re search projects funded by funding agencies; graduate teaching activities; and also, the number of masters and doctoral advisees(24). In addition to the characteristics already mentioned, there is a large number of students per class, which, in the business context, for example, is quite variable, which also can hinder faculty work. It is interesting to note that this movement clearly reflects the increase in enrollment in higher education, especially since the 1990s. The phenomenon is called academic productivity, in which the faculty member is considered productive if he/ she has a significant amount of products, such as advisees, Rev Esc Enferm USP 2014; 48(6):1091-9 www.ee.usp.br/reeusp/ Rev Esc Enferm USP 2014; 48(6):1091-9 www.ee.usp.br/reeusp/ 1095 projects, publications, patents, among others. These as pects lead to increased competitiveness among peers, the professional isolation of faculty members in their research group and, often, alienation at work(25-26). provided as part of the teaching activities, or else institu tionally devalued. Even in the academic context in which it is planned as part of the faculty activities, it is subsumed under the administrative and research activities. From overvaluation to the shortage of research: effects on faculty work and the quality of nursing education Therefore, with regard to research activities, it is seen that faculty work is conditioned to the actual conditions provided by the institutional format, such as work regi men, for example, which directs the work for this activity. In this study, the research activity in faculty work transi tioned from overvaluation in the academic context, to an institutional development effort in the mixed context, to the scarcity of this activity in the business context. One of the examples of programs that stimulate exten sion is the University Extension Program (PROEXT), with the purpose of supporting public HEIs in the development of extension activities that promote greater interaction with the society. It is a program dedicated to public HEIs, because in the private HEIs, extension is the responsibil ity of the institutional context and its value depends on administrative management(28). From the perspective of faculty members, the over valuation of research generates overhead. The institu tional requirement of research, even if limited to graduate teaching, if not combined with a consistent work regimen, also overwhelms and intensifies faculty work. The lack of institutional requirement for the exercise of that activity, in turn, has consequences on faculty professionalization. In fact, both forms of conceiving research exert effects on the quality of education provided. Some experiences show that in private HEIs, it is pos sible to develop extension activities with a positive impact on community healthcare. These studies share the under standing that extension activity is vital for strengthening the role of HEIs in the training of professionals commit ted to the health of people(29-30). This vision enables the development of teaching and learning processes from the everyday practices articulated with research, leading to the confrontation between theory and the real world. The operational difficulties are related to poor institutional value, expressed in forms of internal assessment that pri oritize scientific production at the expense of other activi ties and, consequently, a low financial increment to per form them. Being a recognized researcher, such as in the academ ic context, does not necessarily guarantee an excellent pedagogical performance. It is necessary to put research at the service of the faculty practice itself, causing teach ers to reframe their practice, to review, contextualize and confront it with the conditioning and determining aspects of their work. Higher education in nursing: the faculty work process in different institutional contexts Leonello VM, Oliveira MAC 1096 Rev Esc Enferm USP 2014; 48(6):1091-9 www.ee.usp.br/reeusp/ The teaching-research-extension articulation: the need for a reframing of nursing extension activities Regardless of the institutional format in which they are conducted, extension activities cannot be considered an extra to the training of students. They need to be considered as part of the teaching process, Regarding extension, in all the contexts analyzed, these activities are occasional, sporadic, sometimes not 1096 Rev Esc Enferm USP 2014; 48(6):1091-9 www.ee.usp.br/reeusp/ and articulated with research. Particularly in nursing, extension activities are essential to formation and there fore the teaching practice, because without them, train ing is disconnected from the reality of the problems and health needs of the population. it is necessary to recognize that the social determinants of faculty work cannot preclude the search for coping strat egies towards improvement of working conditions and teaching practices. Rather, it is essential that, through the analysis of this process, higher education faculty members in their different organizational and instructional practices can identify and understand problems and reflect about their work, giving new meaning to it and looking for new opportunities to rebuild their practice. CONCLUSION It was observed that the faculty work process in three distinct contexts assumes formats with regard to orga nizational and didactic practices. However, as common characteristics, intensification and job insecurity were identified. Considering these results, it appears that there are many challenges to faculty work which unfold as ques tions, including: how can change processes in faculty work and, therefore, in practice, be triggered or initiated if the institutional context seems be given, immutable, and not subject to faculty interference? How is this reality to be faced without the action of the characters involved in it, especially the faculty members? This process can be guaranteed by faculty professionalization, as long as it is not limited to the de velopment of specific capabilities of the profession, but includes proposing alternatives that may promote better objective working conditions. Faculty professionalization is an ongoing process of training and reflection on pro fessional practice. It takes into account the critical and reflective recognition about didactic practices, organiza tional practices, and their relation to the structural con text of higher education. This process of social change is complex and involves actions to challenge the reality of teaching, promoted by participation in unions, scientific and student organiza tions, a movement of resistance to a neoliberal proposal, resulting in precarious faculty work. The answers to these challenges are complex. The improvement of structural conditions arising from a pro cess of institutional restructuring, oriented more by edu cational policies towards the quality of higher education than for quantitative expansion of places in this area will positively impact the process of faculty work in different institutional contexts, and therefore quality of education and training. Such a change seems far from the current re ality, which is increasingly grounded on market principles, with little appreciation of education, especially universal access to quality education. Recognizing the diversity of these formats in the faculty work context and practice enables higher edu cation faculty members to be a heterogeneous group, which, although integrated in the same structural reality, perform under different working conditions. Faculty professionalization as a process of social change can be taken as a starting point for proposing coping strategies consistent with such a distinct reality of fac ulty work and practice. However, it is a particular social and historical reality that, in a dialectical process, can also be rebuilt. For that, REFERENCES 6. Lemos MC, Passos JP. Satisfação e frustação no desempenho do trabalho docente em enfermagem. REME Rev Min Enferm. 2012;16(1):48-55. 1. Martins CB. Reconfiguring higher education in Brazil: the participation of private institutions. Análise Soc (Lisboa). 2013;(208):622-58. 2. Brasil. Lei n. 9.394, de 20 de dezembro de 1996. Estabelece as diretrizes e bases da educação nacional. [Internet]. 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W4220685128.txt	https://www.frontiersin.org/articles/10.3389/fphys.2022.858941/pdf	en		The Paraventricular Nucleus of the Hypothalamus in Control of Blood Pressure and Blood Pressure Variability	Frontiers in physiology	2,022	cc-by	13,664	REVIEW published: 16 March 2022 doi: 10.3389/fphys.2022.858941 The Paraventricular Nucleus of the Hypothalamus in Control of Blood Pressure and Blood Pressure Variability Bojana Savić 1, David Murphy 2 and Nina Japundžić-Žigon 1* Laboratory for Cardiovascular Pharmacology and Toxicology, Faculty of Medicine, Institute of Pharmacology, Clinical Pharmacology and Toxicology, University of Belgrade, Belgrade, Serbia, 2 Molecular Neuroendocrinology Research Group, Bristol Medical School, Translational Health Sciences, University of Bristol, Bristol, United Kingdom 1 Edited by: Geoffrey A. Head, Baker Heart and Diabetes Institute, Australia Reviewed by: Stephanie Tjen-A-Looi, University of California, Irvine, United States Hreday Sapru, Rutgers New Jersey Medical School, United States *Correspondence: Nina Japundžić-Žigon nina.zigon@med.bg.ac.rs Specialty section: This article was submitted to Integrative Physiology, a section of the journal Frontiers in Physiology Received: 20 January 2022 Accepted: 15 February 2022 Published: 16 March 2022 Citation: Savić B, Murphy D and Japundžić-Žigon N (2022) The Paraventricular Nucleus of the Hypothalamus in Control of Blood Pressure and Blood Pressure Variability. Front. Physiol. 13:858941. doi: 10.3389/fphys.2022.858941 Frontiers in Physiology \| www.frontiersin.org The paraventricular nucleus (PVN) is a highly organized structure of the hypothalamus that has a key role in regulating cardiovascular and osmotic homeostasis. Functionally, the PVN is divided into autonomic and neuroendocrine (neurosecretory) compartments, both equally important for maintaining blood pressure (BP) and body fluids in the physiological range. Neurosecretory magnocellular neurons (MCNs) of the PVN are the main source of the hormones vasopressin (VP), responsible for water conservation and hydromineral balance, and oxytocin (OT), involved in parturition and milk ejection during lactation. Further, neurosecretory parvocellular neurons (PCNs) take part in modulation of the hypothalamic– pituitary–adrenal axis and stress responses. Additionally, the PVN takes central place in autonomic adjustment of BP to environmental challenges and contributes to its variability (BPV), underpinning the PVN as an autonomic master controller of cardiovascular function. Autonomic PCNs of the PVN modulate sympathetic outflow toward heart, blood vessels and kidneys. These pre-autonomic neurons send projections to the vasomotor nucleus of rostral ventrolateral medulla and to intermediolateral column of the spinal cord, where postganglionic fibers toward target organs arise. Also, PVN PCNs synapse with NTS neurons which are the end-point of baroreceptor primary afferents, thus, enabling the PVN to modify the function of baroreflex. Neuroendocrine and autonomic parts of the PVN are segregated morphologically but they work in concert when the organism is exposed to environmental challenges via somatodendritically released VP and OT by MCNs. The purpose of this overview is to address both neuroendocrine and autonomic PVN roles in BP and BPV regulation. Keywords: blood pressure, blood pressure variability, PVN, vasopressin, oxytocin, baroreflex INTRODUCTION Occupying a small portion of the vertebrate brain (1%), the PVN is a highly organized effector structure (Swanson, 1995; Benarroch, 2005). This hypothalamic nucleus is located bilaterally around the third ventricle (Badoer, 2001). Morphological studies of the PVN reveal different cell populations within its borders, such that the PVN can be divided into at least three 1 March 2022 \| Volume 13 \| Article 858941 Savić et al. PVN in BP Control magnocellular (anterior, posterior and medial subnuclei) and five parvocellular (dorsal, lateral, medial, periventricular and anterior subnucleus) compartments (Swanson and Kuypers, 1980; Sawchenko and Swanson, 1982a; Swanson and Sawchenko, 1983; Badoer, 2001; Pyner, 2009). Two functionally separate areas of the PVN, neuroendocrine and autonomic, subserve its potential to regulate BP, making the PVN a major integrative site of cardiovascular function (Swanson and Kuypers, 1980; Swanson and Sawchenko, 1983; Son et al., 2013; Sladek et al., 2015). Blood pressure, which is defined by peripheral vascular resistance and cardiac output (resultant of heart rate and stroke volume), is modified by both neuroendocrine and autonomic premotor PVN in at least three different effector pathways (Figure 1): neurosecretory magnocellular, neurosecretory parvocellular, and pre-autonomic parvocellular neural pathway (Badoer, 2001; Sladek et al., 2015). MODULATION OF BLOOD PRESSURE BY THE NEUROENDOCRINE PVN Over 30 distinct neurotransmitters and neuromodulators have been identified to be synthesized within the PVN (Swanson and Sawchenko, 1983). Most abundantly expressed are vasopressin (VP) and oxytocin (OT), which are produced by the magnocellular neurons (MCNs) of the PVN (Swanson and Sawchenko, 1980; Sladek et al., 2015). VP is best known for its role in maintaining cardiovascular and body fluid balance, whereas OT takes part in parturition, lactation and accompanying reproductive behaviors (Gimpl and Fahrenholz, 2001; Koshimizu et al., 2006; Stoop, 2014). Additionally, VP and corticotropinreleasing hormone (CRH) expressing parvocellular neurons (PCNs) of the PVN possess a secretory capacity and mediate the central response of the hypothalamic–pituitary–adrenal (HPA) axis to stress (Swanson and Sawchenko, 1980; Sawchenko et al., 1996; Benarroch, 2005; Sladek et al., 2015). Magnocellular and parvocellular neuroendocrine neurons of the PVN initiate a downstream chain of events that dictate changes in BP. FIGURE 1 \| Neuroendocrine and pre-autonomic paraventricular nucleus in cardiovascular regulation. MCNs synthetize VP which is transported via their axons to neurohypophysis for systemic release. Once in the circulation, VP reaches distant targets (kidneys, resistance vessels) to exert its effects. Some portion of VP is released intranuclearly, and modulates the activity of pre-autonomic PCNs. These pre-autonomic neurons have the potential to modulate the autonomic outflow toward heart, kidneys and arterioles. Additionally, VP is co-expressed with CRH alternating the reactivity of HPA axis. VP, vasopressin; CRH, corticotropin-releasing hormone; ACTH, adrenocorticotropic hormone; MCN, Magnocellular neuron; PCNs, parvocellular neuron; SPANs, spinally projecting pre-autonimic neurons; RVLM, rostral ventrolateral medulla; IML, intermediolateral nucleus. posterior subdivision of the PVN, MCNs send their axonal projections to neurohypophysis, from where VP and OT is released into the systemic circulation (Armstrong et al., 1980; Swanson and Sawchenko, 1983; Sladek et al., 2015). Once secreted into blood, these peptide hormones act upon distant targets (Dierickx, 1980; Gutkowska et al., 2000; Japundžić-Žigon, 2013). VP and OT exert their effects through activation of cognate receptors that are expressed both in the brain and periphery (Brinton et al., 1984; Ostrowski et al., 1992; Hirasawa et al., 1994; Adan et al., 1995; Kato et al., 1995). VP receptors (VR) and OT receptors (OTR) are a subfamily of G coupled receptors (G protein-coupled receptors—GPCRs) with high structural homology (85% homology found between V1aR and OTR; Barberis et al., 1998; Thibonnier et al., 2002; Koshimizu et al., 2012). In the periphery, VP mainly engages V1aR and V2R, while in the central nervous system VP action is mostly mediated by V1aR and far less by V1bRs (Ostrowski et al., 1992, 1994; Young et al., 2006; Roper et al., 2011; Russell and Brunton, 2017). The Role of Neurosecretory MCNs in BP Regulation Magnocellular neurons express VP and OT in large quantities (Dierickx, 1980). Located mostly in the medial and the lateral Abbreviations: ACTH, Adrenocorticotropic hormone; ADH, Antidiuretic hormone; ANP, Atrial natriuretic peptide; AP, Area postrema; AQP-2, Aquaporin 2; AT1R, Angiotensin II receptor type 1; AV3V, Anteroventral third ventricle; BHR, Borderline hypertensive rats; BPV, BP variability; BP, Blood pressure; BRS, Baroreflex sensitivity; CRH, Corticotropin-releasing hormone; CRHR2, corticotropin-releasing hormone receptor; DMV, dorsal motor nucleus of vagus; GABA, γaminobutyric acid; GPCRs, G protein-coupled receptors; HF, High frequency; HPA, Hypothalamo–hypophyseal axis; HRV, Heart rate variability; IML, Intermediolateral nucleus; LF, Low frequency; MCNs, Magnocellular neurons; MnPO, Medial preoptic nucleus; NO, Nitrogen oxide; NTS, Nucleus tractus solitarius; OT, Oxytocin; OTR, Oxytocin receptor; OVLT, Organum vasculosum laminae terminalis; PCNs, Parvocelular neuron; PVN, Paraventricular nucleus; RVLM, Rostral ventrolateral medulla; SFO, Circumventricular subfornical organ; SHR, Spontaneously hypertensive rats; SND, Sympathetic nerve discharge; SPANs, Spinally projecting pre-utonimic neurons; VLF, Very low frequency; VLM, Ventrolateral medulla; VP, Vasopressin; VR, Vasopressin receptor. Frontiers in Physiology \| www.frontiersin.org Peripheral VP and OT in Blood Pressure Regulation The strongest stimulus for the MCNs to secret VP into the bloodstream is hyperosmotic change (McKinley et al., 2004). Hyperosmolality of blood is sensed by circumventricular organs 2 March 2022 \| Volume 13 \| Article 858941 Savić et al. PVN in BP Control in the periventricular region of the third ventricle (anteroventral third ventricle—AV3V), which is devoid of the blood brain barrier. The circumventricular subfornical organ (SFO) and organum vasculosum laminae terminalis (OVLT) are richly vascularised, and their neurons easily sense perturbations in blood osmolality (Leng et al., 1989; Cunningham and Sawchenko, 1991; McKinley et al., 2004). From these structures, direct or indirect (via medial preoptic nucleus—MnPO) excitatory axonal projections to MCNs, stimulate the secretion of VP into circulation (Leng et al., 2001; McKinley et al., 2004). Additionally, several other stimuli trigger VP secretion: decreased blood volume and pressure which induce reduction of stretch in low-pressure receptors of venous system and high-pressure baroreceptors respectively and ANG II (Japundžić-Žigon et al., 2020). The best known roles of peripheral VP are water conservation in kidneys and vasoconstriction (Altura and Altura, 1984; Bankir, 2001; Sladek et al., 2015). Indeed, VP is often referred to as antidiuretic hormone (ADH), due to its role in water conservation by the activation of V2Rs in the kidneys (Ostrowski et al., 1993; Gordan et al., 2015). Even small changes in VP concentration in the serum will activate renal V2Rs, with highest affinity to VP, to preserve water (Japundžić-Žigon, 2013). V2Rs are located in the basolateral membrane of the epithelial cells of the collecting ducts of the kidney. Activation of V2R is responsible for a cascade of events that involve phosphorylation of aquaporin 2 (AQP-2) and its translocation into the luminal membrane of epithelial cells, leading to reabsorption of water by the kidneys. VP also affects AQP-2 transcription rates and increases AQP-2 protein abundance (Imbert et al., 1975; Knepper, 1997; Bankir, 2001; Wilson et al., 2013; Jung and Kwon, 2016). The vasoconstrictor effect of VP is mediated by V1aRs located on blood vessels. Although in vitro studies confirm VP as a most potent vasoconstrictor agent, relatively high concentrations of VP are necessary to elevate BP in vivo under basal physiological conditions (Altura and Altura, 1984; Johnston, 1985; Koshimizu et al., 2006) in respect to much less potent vasoconstrictors at molar level such as angiotensin II and noradrenalin. Nevertheless, vascular V1aRs are crucial for BP maintenance during hypovolemia and hypotension (Koshimizu et al., 2012). Also, in some vascular beds, such as in lung, liver and kidneys VP activates V1aR and V2R to produce nitrogen monoxide (NO) dependent vasodilatation (Liard, 1984; Hirsch et al., 1989; Russ and Walker, 1992; Aki et al., 1994; Koshimizu et al., 2006). Circulating VP can circumvent the blood brain barrier and reach centrally located receptors (Landgraf and Neumann, 2004). The most prominent impact of peripheral VP on blood pressure is exerted at the level of area postrema (AP), where it can modify the activity of the baroreflex (Brizzee and Walker, 1990; Hasser et al., 1997). Even though the VP effects on baroreflex are controversial, the majority of studies report enhancement of baroreflex sensitivity (BRS; Brizzee and Walker, 1990; Hasser and Bishop, 1990; Hasser et al., 1997; Koshimizu et al., 2006). Further support is provided by experiments which show that lesions of the AP region disable peripheral VP to modulate baroreflex (Hasser et al., 1997). Also, VP deficient Brattleboro rats exhibit decreased BRS (Imai et al., 1983b). Frontiers in Physiology \| www.frontiersin.org Pharmacological studies suggest that this effect is conveyed by V1aR (Imai et al., 1983a; Hasser and Bishop, 1990; Sampey et al., 1999). The effect of VP on BRS is complex, and when VP is released centrally during stress and exercise, it will act oppositely and reduce BRS via medullary V1aRs (Unger et al., 1986). Afferents that originate from stretch receptors and baroreceptors are tonically active under physiological conditions and inhibit VP secretion from MCNs, while decrease in blood volume and BP leads to disinhibition and consequent release of VP (Bisset and Chowdrey, 1988). Before it reaches the PVN, the information from the baroreceptors is conveyed via the nucleus tractus solitarius (NTS) and ventrolateral medulla (VLM). Afferents arising from the NTS (A2 type neurons) and VLM (A1 cell group) that project to MCNs are primarily noradrenergic (Sawchenko and Swanson, 1982b). Putative inhibitory mechanisms involve NTS residing GABAergic neurons which silence the noradrenergic excitatory pathways directed to the magnocellular subdivision of the PVN (Jhamandas and Renaud, 1987; Bisset and Chowdrey, 1988; Leng et al., 1999). Altogether, it seems that the minor effect of VP on BP maintenance under basal physiological conditions is due to the BRS enhancement via AP and consequent decrease in heart rate which efficiently oppose its vasoconstrictor performance on the periphery (Abboud et al., 1990; Koshimizu et al., 2006). Oxytocin is a nonapeptide hormone best known for inducing uterine contractions during labor and milk ejection during lactation (Dale, 1906; Ott and Scott, 1910; Du Vigneaud, 1954). Apart from these primary roles, OT is involved in a large number of physiological activities, including cardiovascular control (Maier et al., 1998). OT secretion in hypothalamus is not only sex dependent, and it can be modulated by hyperosmotic stimuli, hypovolemia and ANG II (Kadekaro et al., 1992; Gimpl and Fahrenholz, 2001). Mechanisms underlying OT involvement in BP regulation are not yet fully elucidated (Gutkowska et al., 2000). OT activity usually correlates with BP decrease in many species. Experiments with RNA interference report that blocking brain OT RNA leads to an increase in BP (Petersson et al., 1996, 1997; Maier et al., 1998). One of the putative mechanisms behind OT modulation of BP lies in its engagement with electrolyte excretion in the kidneys. It is suggested that OT induced natriuresis is mediated by atrial natriuretic peptide (ANP; Verbalis et al., 1991; Conrad et al., 1993; Gutkowska et al., 2000). However, this natriuretic effect is not confirmed in humans, and it appears that it is present only in certain species, such as rats (Rasmussen et al., 2004). Since VP and OT share very similar primary structures (only differing by 2 amino acids at positions 3 and 8), as well as their receptors, OT can bind with high affinity to V1aRs as well as OTRs (Gimpl and Fahrenholz, 2001; De Bree et al., 2003). OT receptors have a wide distribution in the body (Adan et al., 1995). As well as the reproductive system, OTRs can be found within the brain and heart (Adan et al., 1995; Gutkowska et al., 1997). OT induced peripheral vasoconstriction can be mediated by V1aRs. In vitro studies reveal that the potency of OT to contract blood vessel smooth muscle cells is much less than VP, with the exception of the umbilical 3 March 2022 \| Volume 13 \| Article 858941 Savić et al. PVN in BP Control The Role of PVN Neurosecretory PCNs in BP Regulation artery at term (Altemus et al., 2001). When vascular tone is already increased, OT will produce NO dependent vasodilatation in some vascular beds, like in basilar arteries (Katusic et al., 1986; Russ et al., 1992; Thibonnier et al., 1999). It seems that OT is not responsible for modulating the peripheral resistance in pregnant rats, and does not play a significant role in setting the levels of BP under physiological conditions (Miller et al., 2002). Even though experimental evidence does not support strong involvement of VP and OT in BP regulation under baseline physiological conditions, studies with VP and OT gene knock-out mice provide a contrasting insight. VP knock-out mice exhibit lower basal values of BP, while OT deficient mice demonstrate elevated BP and HR values in comparison to wild type controls (Bernatova et al., 2004; Koshimizu et al., 2006). Apart from being normotensive, VP deficient Brattleboro rats exhibit decreased BRS. However, it should be noted that the complex and unpredictable developmental compensations occurring in global knock-out mice makes the interpretation of adult phenotypes problematic (Valtin, 1982; Imai et al., 1983b; Bohus and de Wied, 1998). Stress is an attributed risk factor for cardiovascular diseases that can trigger bad clinical outcomes (Hjemdahl, 2002; Rosmond, 2005). PVN involvement in the stress response has been documented in spontaneously hypertensive rats (SHRs) and other experimental models (Benarroch, 2005). The PVN promotes several aspects of hemodynamic regulation during stress (Herman et al., 1996; Benarroch, 2005). Anterior and medial PCNs that synthesize CRH, are responsible for activating the HPA axis during exposure to stress (Rivier and Vale, 1983; Swanson et al., 1983; Sawchenko et al., 1996; Sladek et al., 2015). Apart from expressing CRH, PCNs produce VP as secretagogue. It appears that the CRH:VP ratio is dictated by the type of stressor, and is crucial for maintaining responsiveness of the HPA axis during chronic stress (Sawchenko and Swanson, 1982a; Swanson and Sawchenko, 1983; Whitnall et al., 1985; de Goeij et al., 1992; Aguilera, 1994; Sawchenko et al., 1996; Amaya et al., 2001). Both CRH and VP are axonally transported to eminentia mediana and released into the portal circulation. Borne by the portal bloodstream, they reach adenohypophysis, where they act on corticotropic cells. CRH stimulates the release of adrenocorticotropic hormone (ACTH), whereas VP potentiates its release by activating V1bRs (Benarroch, 2005). Once in the systemic circulations (Figure 1), ACTH acts on the cells of zona fasciculata in adrenal gland to release glucocorticoids (Myers et al., 2012; Sladek et al., 2015). Cortisol when in excess, has been shown to contribute to hypertension (Kelly et al., 1998). Additionally, elegant ontogenetic experiments suggest that CRH PCNs can increase BP and heart rate via axonal projections to the NTS (Wang et al., 2019), involving corticotropin-releasing hormone receptor type 2 (CRHR2), also associated with hypertension triggered by intermittent hypoxia (Wang et al., 2019). It has been shown that forced swimming and social confrontation, stressors employed in experimental conditions, can induce somatodendritic release of VP and OT (Wotjak et al., 1996; Engelmann et al., 2001; Ebner et al., 2005), which in turn modifies the activity of the HPA axis. Intranuclear VP has an inhibitory effect on CRH PCNs and consequently reduces secretion of ACTH (Wotjak et al., 1996, 2002; Bosch et al., 2004; Ebner et al., 2005). Extracellular OT exhibits both inhibitory and excitatory influence on the activity of the HPA axis (Neumann et al., 2000; Heinrichs et al., 2002; Neumann, 2002; Landgraf and Neumann, 2004). Intranuclear OT and VP in Blood Pressure Regulation Vasopressin and OT can be synthesized and released from dendrites and soma of MCNs and this can happen without cell depolarization (Scala-Guenot et al., 1987; Moos et al., 1989, 1998; Pow and Morris, 1989; Neumann et al., 1993b; Ludwig et al., 1995, 2002; Hurbin et al., 1998, 2002). VP in the extracellular space can exert both autocrine and paracrine effects (Ludwig and Stern, 2015). Intranuclear VP can activate V1aRs and V1bR expressed on MCNs, or it can spill over through the extracellular space into the cerebrospinal fluid (Landgraf and Neumann, 2004) and reach remote targets (Hurbin et al., 1998, 2002; Son et al., 2013). It has been proposed that intranuclear receptors optimize the firing rate of the entire population of MCNs to best respond to physiological demands (Son et al., 2013). Somatodendritic release depends on the quality and intensity of stimulus and it can be regulated independently from systemic release (Pow and Morris, 1989; Neumann et al., 1993a; Ota et al., 1994; Kendrick et al., 1997; Gouzènes et al., 1998; Landgraf and Neumann, 2004). It has been suggested that the autocontrol of MCN by intranuclear VP can be either inhibitory or excitatory (Inenaga and Yamashita, 1986; Gouzènes et al., 1998; Dayanithi et al., 2000; Landgraf and Neumann, 2004). Additionally, somatodendritically released VP (Figure 1) can activate surrounding silent MCNs (Moos et al., 1998), or depolarize neighboring interneurons and PCNs (Carette and Poulain, 1989; Son et al., 2013). Activation of autonomic PCNs through engagement of V1aRs, especially during hyperosmotic stimulus, is particularly important in terms of integration of neuroendocrine and autonomic regulation of blood pressure (Swanson and Sawchenko, 1980; Son et al., 2013). It has also been reported that intranuclear V1bRs participate in setting sympathetic outflow toward the kidneys (El-Werfali et al., 2015). Frontiers in Physiology \| www.frontiersin.org THE ROLE OF THE “PRE-AUTONOMIC” PVN IN BP REGULATION Apart from being locally regulated, the cardiovascular system is subject to central control by numerous relevant brain areas (Dampney, 1994). It is well established that PVN takes an important central place in such control. Numerous studies show that the PVN is implicated in the heightened sympathetic tone observed in hypertension (Allen, 2002; 4 March 2022 \| Volume 13 \| Article 858941 Savić et al. PVN in BP Control Dampney et al., 2018). The autonomic PVN consists of morphologically and functionally diverse cell populations with a specific topography (Stern, 2001; Dampney et al., 2018). It occupies ventromedial, lateral and dorsal (dorsal cap) subdivision of PVN (Nunn et al., 2011; Sladek et al., 2015). More precisely, these PCNs are pre-autonomic, since they control functionally different sympathetic and parasympathetic centers downstream in the medulla: NTS, dorsal motor nucleus of vagus (DMV) and rostral ventrolateral medulla (RVLM), and the spinal cord: intermediolateral nucleus (IML). Therefore, the “pre-autonomic” premotor PVN is responsible for altering the autonomic output toward the cardiovascular and renal systems (Strack et al., 1989a; Coote et al., 1998; Portillo et al., 1998; Coote, 2005; Dampney et al., 2018). Hence it is not surprising that PVN is commonly referred to as an “autonomic master controller,” a term originally introduced by Loewy (1991). There are at least 3 pathways (Figure 1) through which PVN modulates sympathetic outflow (Pyner, 2009): The first pathway includes PVN pre-sympathetic neurons that have axonal projections that terminate on somata of motor sympathetic preganglionic neurons (SPNs) in the thoraco-lumbal IML. These neurons are usually named as spinally projecting pre-autonomic neurons (SPANs; Dampney, 1994; Badoer, 2001; Nunn et al., 2011). The IML is the final spot of central integration and origin of preganglionic fibers which regulate the activity of blood vessels, heart, kidneys and adrenal gland. Therefore, this is a particularly important target of the PVN pre-autonomic neurons, displaying its enormous potential to directly alternate neurogenic output to the cardiorenal system (Strack et al., 1989a; Dampney, 1994; Badoer, 2001). The second pathway involves pre-sympathetic PVN neurons that exert indirect influence on sympathetic activity. These neurons terminate at the level of the motor pressor nucleus of the RVLM, responsible for setting sympathetic tone. From there, second order neurons arise and project to SPNs in the thoracic and lumbar IML and change sympathetic outflow toward the cardiovascular system and the kidneys (Dampney, 1994; Badoer, 2001). In general, PVN neurons projecting to RVLM are in greater number than PVN neurons projecting monosinaptically to IML. Therfore, RVLM is a structure embedding most of the pre-sympathetic neurons, which are projecting to IML to exert major autonomic cardiovascular control (Badoer, 2001). Some of the sympatho-excitatory effects in the PVN-RVLM pathways are glutamate mediated (Yang and Coote, 1998). Additionally, evidence has emerged, that PVN can interfere with this PVN-RVLM pathway through its glutamatergic synapses within medial subnucleus of the NTS (mNTS; Kawabe et al., 2008). Using anterograde and retrograde tracing, Kawabe working group confirmed the presence of such bilateral glutamatergic projections spanning to mNTS. They showed that unilateral PVN stimulation with N-methyl-D-aspartic acid (NMDA) leads to increase in mean arterial pressure and greater splanchic nerve activity, and that this effect is emphasized by bilateral blockade of glutamate ionotropic receptors within the mNTS. Frontiers in Physiology \| www.frontiersin.org The authors suggested that PVN glutamatergic pathways directed toward NTS, stimulate inhibition of RVLM-mediated cardiovascular overactivity. Therefore, PVN stimulation with NMDA evokes both RVLM and NTS neuronal routes, but the effects are opposing (Kawabe et al., 2008). Further, same authors showed that tachycardia induced by NMDA stimulation of the PVN is a result of conjoined inhibition of vagal (via ionotropic glutamate and GABA receptors in the mNTS) and activation of sympathetic outputs (via spinal ionotropic glutamate) toward heart, without involvement of spinal VRs and OTRs (Kawabe et al., 2009). A third pathway is represented by PVN pre-autonomic neurons that can change sympathetic tone both directly and indirectly. Around 30 % of PVN neurons innervate SPNs in the IML, but send collaterals to the RVLM, thus having a dual regulatory role (Badoer, 2001). The majority of PVN autonomic regulatory function is conveyed by SPANs (Coote, 2007). Since the relevant portion of SPANs is implicated in cardiovascular control (Badoer, 1996; Badoer, 2001; Pyner, 2009; Nunn et al., 2011), this makes them an attractive target for new drug development. Although their exact functions have yet to be elucidated, SPANs have been suggested to regulate blood volume (Lovick et al., 1993; Pyner and Coote, 2000), circadian variations in BP (Cui et al., 2001), stress induced cardiovascular responses (Jansen et al., 1995a) and much more (Nunn et al., 2011). For these reasons SPANs are referred to as “central command neurons” (Jansen et al., 1995a). Discovering their primary physiological function is further complicated by the fact that SPANs express a lot of different neuroactive substances/ neurotransmitters. Indeed, it appears that the majority of these neurons synthesize more than one neurotransmitter (Nunn et al., 2011). The largest portion of SPANs (up to 40 %) is positive for VP and OT (usually co-expressed), as well as dynorphin (Hallbeck and Blomqvist, 1999; Xi et al., 1999; Hallbeck, 2000; Hallbeck et al., 2001). Others possess met-endorphin (up to 20%) and dopamine, met-enkephalin (up to 10 %), leu-enkephalin, somatostatin, ANG II and ANP (Sawchenko and Swanson, 1982a; Cechetto and Saper, 1988; Strack et al., 1989b; Jansen et al., 1995b). Functional in vivo studies show that spinal levels of VP and OT increase with PVN stimulation (Pittman et al., 1984; Malpas and Coote, 1994). The presence of V1aR and OTR has been confirmed in the gray matter of the spinal cord (Desaulles et al., 1995; Sermasi et al., 1998; Pyner, 2009). Intrathecal pretreatment with V1aR antagonist in the lower thoracic region prevents the increase in renal sympathetic nerve activity and mean arterial pressure normally triggered by the stimulation of the PVN (Malpas and Coote, 1994). It cannot be excluded that some of the OT effects are conveyed via spinal V1aRs as well (Sermasi and Coote, 1994). Similar effects are also observed with OTR selective antagonists which abolish the effects of increased heart rate following the PVN stimulation (Yang et al., 2009). These experimental data highlight VP and OT as PVN SPAN transmitters. VP and OT induce cardioacceleratory and pressor effects in lower and upper thoracic spinal cord respectively (Pyner, 2009). The majority of 5 March 2022 \| Volume 13 \| Article 858941 Savić et al. PVN in BP Control vasopressinergic projections implicated in cardiovascular control originate in the lateral and ventral medial subdivision of parvocellular PVN and some portion is found in dorsal parvocellular part (Riphagen and Pittman, 1989; Hallbeck and Blomqvist, 1999). Oxytocinergic SPANs are located in the lateral parvocellular region and dorsal cap (Sawchenko and Swanson, 1982a; Pyner, 2009). The effects of dopamine as a putative SPAN neurotransmitter are controversial (Strack et al., 1989b; Nunn et al., 2011). Some studies on rats suggest its excitatory influence on SPNs, while others report an inhibitory activity (Gladwell et al., 1999; Gladwell and Coote, 1999a,b; Yang et al., 2002). Pharmacological experiments with a glutamate antagonist, following chemical stimulation of PVN, imply glutamate as an additional excitatory neurotransmitter of SPANs (Yang et al., 2002). Although SPANs are positive for enkephalins and ANG II, their influence on sympathetic outflow has not yet been recorded (Sawchenko and Swanson, 1982a; Cechetto and Saper, 1988; Jansen et al., 1995b; Hallbeck and Blomqvist, 1999; Hallbeck et al., 2001). Despite its abundant expression in the nervous system, the presence of inhibitory γ-aminobutyric acid (GABA) as a SPAN output neurotransmitter has not been confirmed (Macdonald and Olsen, 1994; Watkins et al., 2009). increase in BP and heart rate (Martin et al., 1991; Martin and Haywood, 1993). Interfering with GABA orchestrated SPAN activity is an appealing therapeutic opportunity, since increasing GABA inhibitory influence would lead to reduction of sympathetic tone and consequently BP decrease (Nunn et al., 2011). Although injections of ATII in the PVN change blood pressure via angiotensin II receptor type 1 (AT1R; Bains et al., 1992), and this involves the activity of SPANs, it seems that this connection is indirect (Bains and Ferguson, 1995; Li et al., 2003). AT1Rs are expressed in parvocellular division of the PVN on neurons projecting to medulla, not IML (Oldfield et al., 2001; Cato and Toney, 2005). Spinally projecting pre-autonomic neurons, as well as PCNs which project to RVLM are barosensitive. Under basal physiological conditions, these neurons exhibit spontaneous activity, but they are inhibited by rising pressure (Dampney et al., 2018). Axons arising from caudal NTS neurons terminate on PCNs in the dorsal cap of the PVN. Putative targets of these projections are pre-sympathetic PCNs or GABA interneurons (Pyner, 2009; Dampney, 2017). Other studies do not impose an important role of SPANs in the baroreflex response (Haselton et al., 1994). Volume load is another feedback mechanism that can modify the activity of SPANs, with PVN being a command center of low-pressure blood volume receptors located in the veno-atrial junction (Gupta et al., 1966; Lovick and Coote, 1988; Lovick et al., 1993; Deng and Kaufman, 1995). Additionally, SPANs can be modulated by some types of stressors, such as psychological stress. It has been shown that conditional fear engages around 10 % of SPANs (Carrive and Gorissen, 2008; Dampney et al., 2018). Also SPANs are affected by temperature, different humoral factors, and inputs from higher brain areas (Dampney et al., 2018). Modulation of SPAN Activity Spinally projecting pre-autonomic neuron activity can be modulated in various ways. A lot has been discovered about the neurotransmitter content of SPANs, but the data regarding receptors expressed on SPANs and molecules their receptors bind is lacking (Nunn et al., 2011). Learning about the neurotransmitters which can modulate the activity of SPANs will open venues to novel therapeutic agents. It is well established that the PVN is involved in increased sympathetic activity driven by osmotic stimulation, but the mechanisms behind it are poorly understood. Some studies suggest involvement of intranuclear VP released from soma and dendrites of MCNs (Son et al., 2013; Ribeiro et al., 2015). Intranuclear VP stimulates V1aRs expressed on pre-sympathetic parvocellular subdivisions (including SPANs) of the PVN. This leads to increase in sympathetic outflow toward heart, blood vessels and kidneys, followed by BP increase, suggesting this pathway as potential pathophysiological mechanism in neurogenic hypertension (Son et al., 2013; Ribeiro et al., 2015). Under basal physiological conditions, PVN neurons are tonically inhibited by surrounding GABA neurons, keeping the spontaneously generated nerve impulses at low rate, despite the excitatory influence of glutamate (Kannan et al., 1989; Martin et al., 1991; Badoer et al., 2002; Li et al., 2006). SPANs are confirmed to be silenced by GABA (Cui et al., 2001; Li et al., 2002). This inhibition could be dependent on extrasynaptic GABA (“volume” GABA transmission), but it can also be affected by the rate that glial cells take up GABA from the extracellular space (Brickley et al., 1996; Farrant and Nusser, 2005; Park et al., 2009). GABAA α2-subunit is abundantly expressed in the PVN (Fritschy and Mohler, 1995). Blockage of GABAA receptors by the selective antagonist bicuculline leads to an Frontiers in Physiology \| www.frontiersin.org PARAVENTRICULAR NUCLEUS AND BLOOD PRESSURE SHORT-TERM VARIABILITY The peripheral sympathetic nervous system controlling the cardiovascular system has rhythmic activity that creates distinct patterns of sympathetic nerve discharge (SND) in response to physiological demands and pathophysiological conditions. Using frequency analysis of SND, components of SND were identified including fast cardiac and respiratory rhythms, and slow vasomotor rhythm. It is generally believed that the brain is the source of SND. Thus, which parts of the brain are involved, and how they generate peripheral SND rhythms is still unanswered. Two main theories have been postulated, both of which raised considerable criticism. A theory of a central oscillator/pacemaker suggests that the RVLM is the main structure responsible for generating peripheral sympathetic discharge patterns via axonal projections to preganglionic neurons in IML. Recordings of intracellular neuronal activity from medullary slices, uncovered ramp like depolarization following each action potential, leading to subsequent action potential suggestive of pacemaker activity 6 March 2022 \| Volume 13 \| Article 858941 Savić et al. PVN in BP Control (Li and Guyenet, 1996). This observation was seriously challenged by the in vivo study in rats by Lipski et al. (1996) who observed that RVLM neurons fire irregularly, at much higher discharge rates than the ones in SND, with no evidence of gradual depolarization between individual action potentials (Lipski et al., 1996). They suggested that the regular pattern of firing of RVLM neurons seen in medullary slices, was produced by deafferentation. The work of Lipski and colleagues imposes the network hypothesis, where the activity of pre-sympathetic neurons depends on their antecedent excitatory inputs opposed by tonic inhibitory inputs (setting the level of their excitability). For detailed review refer to Malpas (1998). Geber and Barman postulated a theory of a network of brainstem neurons, whose combined action creates inherent rhythmicity entrained by the baroreflex. These include sympatho-excitatory and sympatho-inhibitory neurons distributed over a wide portion of the lower brainstem that do not have necessarily spontaneous activity (Barman and Gebber, 2000). In addition to RVLM neurons, hypothalamic PVN SPANs provide monosynaptic inputs to preganglionic neurons in IML. Malpas and Coote (1994) were first to provide evidence in anesthetized rats that chemical stimulation of the PVN by microinjections of homocysteic acid increases the amplitude of renal SND, by recruiting more active fibers, and that this can be blocked by intrathecal injection of V1R antagonist. At the same time the frequency of renal SND, which is modulated by periodic baroreflex inputs (Ninomiya et al., 1990; Malpas and Ninomiya, 1992), remained unaffected. The first experimental evidence that PVN SPANs can incite BPV at the same stimulation frequency was demonstrated in rats by Stauss and associates (Stauss and Kregel, 1996; Stauss et al., 1997). PVN electrically stimulated at frequencies between 0.1 Hz - 0.5 Hz were found to create the same frequencies in the SND pattern and generate BPV in the low frequency (LF) band, which is abolished by α-adrenergic blockade (Japundzic et al., 1990). Stimulation frequencies above 0.5 Hz did not induce BP variations as the blood vessels behaved like cut off filters to high frequencies. Studies on isolated rat vascular smooth muscle cells showed that transmission of fast SND rhythms to BPV is limited by the sluggish, metabotropic α-adrenoceptor signaling, and not by an intrinsic inability of the cells to contract and relax at higher rates (Julien et al., 2001). Thus, SND frequencies higher than 0.5 Hz in rats induce vasoconstriction, increase peripheral resistance and the mean value of BP. It follows that the faster components of BPV, the cardiac component and the respiratory component, are non-neural, created by the perturbations of the circulation induced by the contracting heart and inspiratory movements (Japundzic et al., 1990; Japundzic-Zigon, 1998). The non-neural origin also stands for the slowest, and dominant component of BPV, the very low frequency (VLF) component, which was found to be created by inherent myogenic activity of mesenteric and renal vasculature (VanBavel et al., 1991; Janssen et al., 1995). Although non-neuronal in nature, all the components of BPV can be modulated by the activity of the nervous system and neurohormones (Japundžić-Žigon et al., 2020). Frontiers in Physiology \| www.frontiersin.org We have investigated the neurochemical contribution of the PVN to short-term BPV, and found that both VP and OT modulate short-term BPV. Using pharmacological and genetic tools in conscious rats we have shown that VP modulates BPV in a complex manner: peripherally as a hormone and centrally as neurotransmitter/modulator (Japundzic-Zigon, 2001, 2013; Japundzić-Zigon et al., 2004, 2018, 2020; Milutinović et al., 2006a,b; Stojicić et al., 2008; Milutinović-Smiljanić et al., 2013; Lozić et al., 2016; Savić et al., 2020). Using spectral analysis of BPV, we found that peripheral administration of non-peptide and selective V1aR or V2R antagonists to conscious normotensive rats under baseline physiological conditions increases BPV, suggesting a buffering role for VP in the VLF domain (Japundzic-Zigon, 2001). We postulated that the decrease of VLF-BPV by VP could be mediated either by the enhancement of the baroreflex sensitivity which normally opposes VLF-BPV, or by the modulation of vasomotion in mesenteric and renal vascular beds (Janssen et al., 1995). In SHR, the buffering capacity of VP on BPV under baseline condition is not preserved (Japundzić-Zigon et al., 2004). This could be due to pathological remodeling of the vasculature in SHR (Head, 1991; Vågnes et al., 2000) making it more sensitive to vasoconstrictors, including VP. Another possibility, even more likely is that impaired baroreflex in SHRs (Dampney, 2017) reduces its capacity to buffer VLF-BPV (Dampney, 2017). However, during hemorrhage, when VP is released in excess in blood, in support of circulation, it acts similarly in normotensive and hypertensive rat strains, and prevents the respiratory related high frequency (HF-BPV) increase, possibly as a consequence of V1aR-mediated vasoconstriction which prevents the unloading of thoracic vessels underlying HF-BPV increase. Intracerebroventricular injection of selective V1aR, V1bR and V2R antagonists to conscious normotensive rats uncovered that VP acts also centrally to buffer VLF-BPV under baseline physiological conditions by the stimulation of V1aRs possibly in AP, accessible from both sides of the blood brain barrier. However, when VP release is stimulated by stress (Stojicić et al., 2006, 2008; Milutinović et al., 2006b), or by drugs (Milutinović et al., 2006a) and when VP is injected centrally (Milutinović et al., 2006b), an increase of the sympathetically mediated LF-BPV and of the respiration mediated HF-BPV was observed (Milutinović et al., 2006a,b). These effects of VP could involve central V1aR found in abundance in the RVLM where integration of the sympathetic outflow to vasculature occurs; and in the pre-Bötzinger area, where the breathing pattern is set. These central effects of VP could be beneficial and contribute to the lifesaving effect of VP in hemorrhagic, septic and cardiogenic shock as a results of increased tissue oxygenation and additional sympathetic activation, acting synergistically with powerful V1aR mediated peripheral vasoconstriction (Levy et al., 2018). VP can also modulate respiration indirectly (Stojicić et al., 2008) by an anxiogenic action characterized by hyperventilation that occurs possibly by the stimulation of V1bR in bed nucleus stria terminalis (Griebel et al., 2002). Paraventricular nucleus is a recognized key integrative site of the behavioral, autonomic and endocrine response to stress, expressing V1aRs and V1bRs on somata and dendrites of MCNs 7 March 2022 \| Volume 13 \| Article 858941 Savić et al. PVN in BP Control and surrounding glia. Stress has been shown to induce VP and OT release in the PVN too (Nishioka et al., 1998; Landgraf and Neumann, 2004), and we have shown that VP and OT act locally, in an autocrine and paracrine manner, to modulate the neuro-cardiogenic stress response. Using adenoviral gene transfer technology we have increased the gene expression and the number of V1aRs in the PVN of Wistar rats. The V1aR rat phenotype had decreased sensitivity of the baroreflex under baseline physiological conditions which was further decreased by stress along with a marked increase of the sympathetically mediated LF-BPV and LF heart rate variability (LF-HRV). These effects could be abolished by intranuclear application of V1aR antagonist. This suggests that V1aR in the PVN can increase the sympathetic outflow to the periphery and modulate BPV and HRV during stress (Lozić et al., 2014; Japundžić-Žigon et al., 2020). In clinical practice, the increase in LF-HRV has been found to predict the occurrence of life threatening arrhythmias in susceptible populations (Huikuri and Stein, 2013). In contrast to V1aR, OTR over-expression in the PVN of Wistar rats had no effect on BPV under baseline conditions but this rat phenotype exhibited reduced baroreflex desensitization by stress and reduced LF-BP increase suggesting that OTR over-expressing rat phenotype is resilient to stress (Lozić et al., 2014; Japundžić-Žigon et al., 2020). In this context, it is important to stress that the modulation of the baroreceptor desensitization during stress by VP seems to be complex and involves more than one central structure and type of VRs (Milutinović-Smiljanić et al., 2013). A number of clinical studies in hypertensive patients unequivocally show that enhanced BPV increases the risk for developing cardiovascular complications (Mancia and Grassi, 2000). Thus, BPV, and especially sympathetically derived LF-BPV, emerged as an independent predictor of stroke, coronary artery disease, heart and renal failure, as well as all-cause mortality (Mancia et al., 1994; Mancia and Parati, 2003; Messerli et al., 2019; Parati et al., 2020). Our group investigated expression of PVN VP and VRs in the genesis of hypertension. Borderline hypertensive rats (BHR) have a genetic predisposition for hypertension and will develop it when exposed to environmental challenges. Under baseline physiological conditions BHRs have increased expression of VP and V1bR in the PVN and consequently increased plasma VP concentrations, as a constitutive trait (Savić et al., 2020). Spectral markers of sympathetic activity toward blood vessels, LF-BP, and the heart, LF/HF-HR, are comparable to normotensive rats under baseline physiological conditions, suggesting that increased expression of V1bR and VP in BHRs is confined to magnocellular (endocrine) portion of the PVN affecting plasma VP only. However, when exposed to repeated stress and prolonged isotonic saline load, BHRs exhibited LF-BPV increase depicting sympathetic overload, and overt hypertension. In these rats no changes in VP and VR gene transcription in the PVN was noted. Moreover, systemic VP release was decreased, refuting involvement of VP in stressinduced hypertension (Savić et al., 2020). CONCLUSION It is well established that PVN has a paramount role in cardiovascular regulation and contributes to the severity of cardiovascular diseases. Both neuroendocrine and autonomic PVN, have a dynamic part in adjusting the circulation to physiological demands and in the modulation of short-term BPV. Thus, elucidating the tightly intertwined mechanisms underlying complexity of the PVN network in health and disease may open up new therapeutic venues. AUTHOR CONTRIBUTIONS BS, DM, and NJ-Ž: outlining paper draft, writing and refining the manuscript, and critical reading of the manuscript. All of the authors have read and approved the manuscript. FUNDING This work was supported by Serbian Ministry of Education, Science and Technological Development (no. 200110, BS, NJ-Ž), British Heart Foundation (RG/11/28714, DM; FS/12/5/29339, DM), and BBSRC (BB/J005452/1, DM). REFERENCES Altemus, M., Redwine, L. S., Leong, Y. M., Frye, C. A., Porges, S. W., and Carter, C. S. (2001). Responses to laboratory psychosocial stress in postpartum women. Psychosom. Med. 63, 814–821. doi: 10.1097/00006842200109000-00015 Altura, B. M., and Altura, B. T. (1984). Actions of vasopressin, oxytocin, and synthetic analogs on vascular smooth muscle. Fed. Proc. 43, 80–86. 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The vasopressin 1b receptor is prominent in the hippocampal area CA2 where it is unaffected by restraint stress or adrenalectomy. Neuroscience 143, 1031–1039. doi: 10.1016/j.neuroscience.2006.08.040 Conflict of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Publisher’s Note: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may 13 March 2022 \| Volume 13 \| Article 858941 Savić et al. PVN in BP Control (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. be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Copyright © 2022 Savić, Murphy and Japundžić-Žigon. This is an open-access article distributed under the terms of the Creative Commons Attribution License Frontiers in Physiology \| www.frontiersin.org 14 March 2022 \| Volume 13 \| Article 858941
https://openalex.org/W4213275237	https://www.researchsquare.com/article/rs-50198/v1.pdf?c=1597087608000	English	null	Multinomial Propensity Score for Ternary Exposure for Genetic Study	Research Square (Research Square)	2,020	cc-by	5,908	Multinomial Propensity Score for Ternary Exposure for Genetic Study Cathy S. J. Fann Academia Sinica Cathy S. J. Fann Academia Sinica Thai Son Dinh National Changhua University of Educati Yu-Hsien Chang National Changhua University of Educati Jia Jyun Sie National Changhua University of Educati Ie-Bin Lian (  maiblian@cc.ncue.edu.tw National Changhua University of Educati Research article Keywords: propensity adjustment, confounding effect, SNP, contrast coding Posted Date: August 10th, 2020 DOI: https://doi.org/10.21203/rs.3.rs-50198/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Posted Date: August 10th, 2020 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 Multinomial propensity score for ternary exposure for genetic study Cathy S. J. Fann1, Thai Son Dinh2, Yu-Hsien Chang3, Jia Jyun Sie2, Ie-Bin Lian3* 1. Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan 2. Department of Mathematics, National Changhua University of Education, Changhua, Taiwan 3. Institute of Statistics and Information Science, National Changhua University of Education, Changhua, Taiwan. 1 ABSTRACT Background: Propensity score (PS) is a popular method for reducing multiple confounding effects in observational studies. It is applicable mainly for situations wherein the exposure/treatment of interest is dichotomous and the PS can be estimated through logistic regression. However, multinomial exposures with 3 or more levels are not rare, e.g., when considering genetic variants, such as single nucleotide polymorphisms (SNPs), which have 3 levels (aa/aA/AA), as an exposure. Conventional PS is inapplicable for this situation unless the 3 levels are collapsed into 2 classes first. Methods: A simulation study was conducted to compare the performance of the proposed multinomial propensity score (MPS) method under various contrast codings and approaches, including regression adjustment and matching. Results: MPS methods had more reasonable type I error rate than the non-MPS methods, of which the latter could be as high as 30~50%. Compared with MPS-direct adjusted methods, MPS-matched cohort methods have better power but larger type I error rate. Performance of contrast codings depend on the selection of MPS models. Conclusions: In general, two combinations had relatively better performance in our simulation of ternary exposure: MPS-matched cohort method with Helmert contrast and MPS-direct adjusted regression with treatment contrasts. Compared with the latter, the former had better power but larger type I error rate as a trade-off. 2 2 Keywords: propensity adjustment, confounding effect, SNP, contrast coding. Keywords: propensity adjustment, confounding effect, SNP, contrast coding. 3 Background Propensity score (PS) approach is a useful and popular method in epidemiology for mitigating confounding effects in observational studies [1]. It was first proposed by Rosenbaum and Rubin to infer cause and effect from observational studies [2]. A PS is estimated through a conditional probability wherein a particular treatment is assigned given a vector of observed covariates. Adjustment for the PS is sufficient to remove bias due to all observed covariates. 4 An observational study attempts to estimate the effects of a treatment or exposure (X) on the outcomes (Y) of subjects who are not assigned at random to the treatment or control group [3]. In such a study, the adjustment for confounders (Zs) that are associated with X and Y is crucial. Otherwise, inference can be severely biased. Common ways for adjustment include matching, stratification, and covariate-adjusted regression [4]. However, when potential confounders are numerous, the above approaches may be implausible. After stratifying on many covariates, some strata will have insufficient cases for analysis even for datasets with large samples. For covariate-adjusted regression, adjustment with excessive covariates or by applying a variable selection scheme can result in severe over-dispersion on the inference [5]. In such cases, PS is considered to be an efficient way to reduce the dimensions of 4 covariates for valid inference. It adjusts for the effects of multiple confounders to estimate treatment effects and balances covariates (such as age, gender, or population principal components) such that the treatment and control groups are comparable [1]. For a binary exposure X, the logistic regression model is often used to estimate the true PS by regressing X on observed covariates. Several ways to adjust for covariates when estimating the effects of treatment on outcomes by using PS include matching, stratification, direct adjustment in regression, and inverse probability treatment weighing [6]. Ali et al. reported that in studies using PS to control for confounding, matching on the PS is the most common approach (68.9%), followed by PS direct adjustment (20.9%); together, such studies comprise more than 90% of the applications involving PS [7]. Therefore, in this work, we focused on the categories of the MPS-direct adjusted and MPS-matched methods. Most of the applications of PS have been in binary exposure. However, multinomial exposures with 3 or more levels are not rare, e.g., smoking can have 3 statuses, namely, “never”, “current”, and “ever-smoking”. Background In genetic studies, specific genetic variants, such as single nucleotide polymorphisms (SNPs), which have 3 levels (aa/aA/AA), can also be considered as an exposure. Conventional PS is inapplicable in this situation unless some levels are collapsed first. his situation unless some levels are collapsed first. 5 5 Yoshida et. al. (2019) considered an extended propensity score for exposure X with J+1 levels [8]. They suggested using PS vector 𝒆′ = (𝑒0 … 𝑒𝐽) with 1 probability of assignment for each level where 𝑒𝑗= P(X = j\|covariate Z′s) for j ∊{0,1, …, J} and used simulation to study the influence of the proposed ternary PS trimming methods for the inverse probability weighting approach. Using the same PS vectors, Wang et al. [9] proposed an application of stratification on the multiple propensity score to dose-response relationships in drug safety studies, and Wang et al, [10] generalized the pair-patching scheme to a trio-matching via defining a new distance among subjects from 3 treatment groups and discuss the choice of optimal caliper. Another alternative for multiple treatments is the generalized boosted model, a machine learning approach that uses inverse probability weighting regression to capture complex relationships between a treatment assignment and pretreatment covariates [11]. 6 For the application of PS in genetic association studies, Jiang and Zhang (2011) suggested using nonparametric techniques to obtain PS while adjusting for covariates, such as population stratification or environmental factors for SNPs of interest, to identify disease associations [12]. Instead of directly testing epistatic effects from numerous combinations of SNPs, Sengupta Chattopadhyaya et al. (2016) proposed using PS as a dimension-reduction tool to improve the marginal single-point 6 association result for each SNP by accounting for the loss of heritability [13]. However, to be able to apply conventional PS, which assumes binary X for logistic regression, the 3-level SNP variable is first collapsed to 2 levels for dominant or recessive traits. In this study, we compare 3 contrast codings for multinomial propensity scores (MPSs) to cope with genetic marker such as SNP, when it is considered as an exposure of interest. Simulation was used to assess the performances of the codings. association result for each SNP by accounting for the loss of heritability [13]. However, to be able to apply conventional PS, which assumes binary X for logistic regression, the 3-level SNP variable is first collapsed to 2 levels for dominant or recessive traits. Methods Let y be the outcome variable, x be the risk factor of interest, and Z = (z1…zp) be numerous covariates. Among these covariates, some are confounders of x that are associated with x and y. The purpose of studies is to investigate the association between x and y. We consider the following generalized linear model system among x, y, and Z: 𝑓(𝑦) = 𝑥+ 𝑍𝛾+ , 𝑔(𝑥) = 𝑍𝛽+ . 𝑓(𝑦) = 𝑥+ 𝑍𝛾+ , 𝑔(𝑥) = 𝑍𝛽+ . For binary y and binary x, the conventional procedure of PS is as follows: (i) Fitting the logistic regression of x on the zs to obtain the prediction of x as PS: logit (𝑥) = log (𝑃(𝑥= 1)/𝑃(𝑥= 0) = 𝑍𝛽. logit (𝑥) = log (𝑃(𝑥= 1)/𝑃(𝑥= 0) = 𝑍𝛽. 7 (ii) Stratifying or matching x and y on the basis of PS, or, for direct adjustment, fitting the logistic regression of y on x and PS. logit (𝑦) = log (𝑃(𝑦= 1)/𝑃(𝑦= 0) = 𝑥+ 𝑍𝛾. logit (𝑦) = log (𝑃(𝑦= 1)/𝑃(𝑦= 0) = 𝑥+ 𝑍𝛾. In the case of binary y and ternary x with numerous Zs, we considered the following 3 In the case of binary y and ternary x with numerous Zs, we considered the following 3 contrast codings to convert x into dummy variables before estimating PS. contrast codings to convert x into dummy variables before estimating PS. In treatment contrast, 1 group needs to be assigned as the baseline reference, and x is decomposed into dummy variables T1 and T2 as follows: x [ 0 1 2 ] → T1 T2 [ 1 0 0 1 0 0 ]. x [ 0 1 2 ] → T1 T2 [ 1 0 0 1 0 0 ]. In the following example, AA (x = 2) was used as the reference group, as follows: In the following example, AA (x = 2) was used as the reference group, as follows: 𝑥= ( 𝑎𝑎 𝐴𝑎 𝐴𝐴 ) = ( 0 1 2 ) = ( 1 1 0 1 0 1 1 0 0 ) ( 𝛼0 𝛼1 𝛼2 )=( 𝛼0 + 𝛼1 𝛼0 + 𝛼2 𝛼0 ). Therefore, 𝛼1 = (𝑎𝑎−𝐴𝐴) and 𝛼2 = (𝐴𝑎−𝐴𝐴). If A is the disease allele, 1 is Therefore, 𝛼1 = (𝑎𝑎−𝐴𝐴) and 𝛼2 = (𝐴𝑎−𝐴𝐴). If A is the disease allele, 1 is Therefore, 𝛼1 = (𝑎𝑎−𝐴𝐴) and 𝛼2 = (𝐴𝑎−𝐴𝐴). Methods If A is the disease allele, 1 is expected to be significant in all 3 (dominant, recessive, and additive) traits, whereas 2 expected to be significant in all 3 (dominant, recessive, and additive) traits, whereas 2 is expected to be significant in the additive and recessive traits. is expected to be significant in the additive and recessive traits. In Helmert contrasts, In Helmert contrasts, In Helmert contrasts, 8 8 Therefore, Therefore, Therefore, 𝑥= ( 𝑎𝑎 𝐴𝑎 𝐴𝐴 ) = ( 0 1 2 ) = ( 1 −1 −1 1 1 −1 1 0 2 ) ( 𝛼0 𝛼1 𝛼2 )=( 𝛼0 −𝛼1 −𝛼2 𝛼0 + 𝛼1 −𝛼2 𝛼0 + 𝛼2 ). 𝛼1 = (𝐴𝑎−𝑎𝑎)/2, 𝛼2 = [𝐴𝐴−(𝐴𝑎+ 𝑎𝑎)/2]/2. i.e., the significance of 𝛼1, 𝛼1 = (𝐴𝑎−𝑎𝑎)/2, 𝛼2 = [𝐴𝐴−(𝐴𝑎+ 𝑎𝑎)/2]/2. i.e., the significance of 𝛼1, suggests the difference between (aa, 𝐴𝑎), and that of 𝛼2 suggests the difference is expected to be significant in the dominant and additive traits, whereas 2 is expecte is expected to be significant in the dominant and additive traits, whereas 2 is expected to be significant for the recessive and additive traits and has weak power for the dominant trait due to the effect size being partially offset by the cancel-out effect between AA and Aa. to be significant for the recessive and additive traits and has weak power for the Finally, we also consider a custom contrast, which we refer to as SNP contrasts, as shown below: Under the contrasts, 9 𝑥= ( 𝑎𝑎 𝐴𝑎 𝐴𝐴 ) = ( 0 1 2 ) = ( 1 0 0 1 0 1 1 1 1 ) ( 𝛼0 𝛼1 𝛼2 )=( 𝛼0 𝛼0 + 𝛼2 𝛼0 + 𝛼1 + 𝛼2 ). Consequently, 𝛼1 = (𝐴𝐴−𝐴𝑎) and 𝛼2 = (𝐴𝑎−𝑎𝑎). Given A as disease allele, a Consequently, 𝛼1 = (𝐴𝐴−𝐴𝑎) and 𝛼2 = (𝐴𝑎−𝑎𝑎). Given A as disease allele, a dominant trait is expected to have significant 𝛼2 but nonsignificant 𝛼1, whereas an additive trait is expected to have significant 𝛼1 and 𝛼2. If 𝛼1 is significant but 𝛼2 is not, this situation indicates that (𝐴𝐴) is different from (aa, 𝐴𝑎). This situation is referred to as the recessive trait. If 𝛼2is significant but 𝛼1is not, this indicates that (𝐴𝐴, 𝐴𝑎) is different from (aa). This situation is referred to as the dominant trait. On the other hand, if 𝛼1and 𝛼2 are significant and have the same sign, this situation can be referred to as the additive trait. sign, this situation can be referred to as the additive trait. Therefore, 10 For each contrast variable x1 and x2, we estimate the corresponding PSs via the stepwise logistic regression of xi on Zs as PSi = P( xi = 1 \|selected 𝑍s), i = 1, 2, and then fit y on (x1, x2, PS1, and PS2) along with some Zs selected through the variable selection procedure. We refer to the above procedure as the MPS method. In direct-adjustment approach, y is regressed on x1, x2, PS1, and PS2 directly. In case-control matching, since only 2 groups (case and control) needs to be balanced, the conventional matching scheme can be applied. In cohort matching with 3 exposure case-control matching, since only 2 groups (case and control) needs to be balanced, the conventional matching scheme can be applied. In cohort matching with 3 exposure 10 levels, instead of the trio-matching used by Wang et al. [10], we adopt a simpler double-pair-matching scheme which can be easily implemented in R package. Regarding the exposure SNP marker =0/1/2 as group 1/2/3 respectively, when treatment contrast is used, a subject from group 3 was matched with one from group 1 based on PS1, and one from group2 based on PS2 separately, that together form a matched trio. Analogously when Helmert contrast is used, after a matched pair between group 1 and 2 based on PS1 being formed, their average PS2 was calculated, and then a subject from group 3 with the closest PS2 to the average was selected to form a trio. Simulation study was conducted using R package (version 4.0.1) to assess the performance among the combination of 8 models (2 non-PS models, 3 MPS-direct adjustment models and 3 MPS-matching models as shown in Table 1) and 3 types of contrast codings (treatment, SNP and Helmert) via simulation. 11 Comparing the simulation setups of Yoshida et al. (2019) for the log-linear model and Lian (2003) for the logit model [5,8] reveals that Yoshida’s simulation is mainly used for assessing marginal estimands. However, in our case wherein SNPs were considered as exposures, we focused on assessing its conditional effect with other SNPs and phenotypes being adjusted as covariates. Therefore, in our simulation, the exposure– outcome functions were specified, and the later setup by Lian (2003) was adopted. 11 Simulation setup The data of sample size m = 100, such that the m by 1 vectors binary outcome y; The data of sample size m = 100, such that the m by 1 vectors binary outcome y; ternary exposure of interest x; and 20 covariates 𝑧1– 𝑧20, were generated by using the f ll i li ti t ternary exposure of interest x; and 20 covariates 𝑧1– 𝑧20, were generated by using the ternary exposure of interest x; and 20 covariates 𝑧1– 𝑧20, were generated by using the following linear equation system: logit (w𝑖𝑗)= 𝛽∙(z𝑖1 + ⋯+ z𝑖 10), 𝑗= 1, 2, x𝑖= 𝑓(w𝑖1, w𝑖2), logit (y𝑖) = · x𝑖+ 𝛾∙(z𝑖6 + ⋯+ z𝑖 15), where w𝑖𝑗 ~ Bernoulli(pij), with pij = e β × ∑ Zij 10 i=1 1 + e β × ∑ Zij 10 i=1 , i = 1~m, j −1,2. where w𝑖𝑗 ~ Bernoulli(pij), with Function f specifies the type of the genetic trait of x: 𝑥= w1 + w2 for an additive trait, 𝑥= w1 ∗w2 for a recessive trait, and 𝑥= w1 + w2 −w1w2 for a dominant trait. If a SNP takes a value as the number of A, i.e., SNP = 0 for (a, a), = 1 for (A, a) or (a, A), and = 2 for (A, A), then x = SNP for an additive trait. For a recessive trait, x = 1 if SNP = 2, and =0 otherwise. For a dominant trait, x = 1 if SNP > 0 and =0 otherwise. 12 According to the above linear system, z1– z5 are associated x only; z6– z10 are confounders that are associated with x and y; z11– z15 are associated with y only; and z16– z20 are unassociated with neither x nor y. The effect sizes re denoted by () as 12 shown in Fig 1. The parameter setup used in this study is given by Table 1. The zis were generated from N(0,1), whereas w and y were generated from Bernoulli distribution as shown in Fig 2. The following models are the methods compared in this work. The corresponding fitting procedures are listed in Table 2. --- Insert Tables 1–2 and Figs 1–2 here --- 13 Note that Model 1 is an unadjusted regression model, and Model 2 is a commonly used covariate-adjusted regression model. Simulation setup Models 3-5 are MPS- and covariate-direct adjusted logistic regression (LR) models, with PS being forced-in and covariates being selected by using a stepwise procedure with levels 0, 0.05, and 0.15. In terms of results, Model 5 adjusts for the most covariates, whereas Model 3 adjusts for PS only. Models 6-8 are MPS-matched methods, wherein Model 6 is a matched case–control condition logistic regression (CLR), and Model 7 is a matched cohort CLR. For Model 8, the Cochran–Armitage Trend (CAT) test with 3 weighting schemes, namely, additive, recessive, and dominant, were used [14]. CAT, which uses the Chi-square statistics of 1 degree of freedom, is considered to be more powerful than the regular Peason’s Chi-square of 2 degrees of freedom [15]. Here, Model 6 was used as a reference for comparison with Models 7 and 8 because matched case–control is inefficient or may actually introduce additional cofounders and bias and is not recommended [16-17]. 13 Note that Model 1 is an unadjusted regression model, and Model 2 is a commonly used covariate-adjusted regression model. Models 3-5 are MPS- and covariate-direct adjusted logistic regression (LR) models, with PS being forced-in and covariates being selected by using a stepwise procedure with levels 0, 0.05, and 0.15. In terms of results, Model 5 adjusts for the most covariates, whereas Model 3 adjusts for PS only. Models 6-8 are MPS-matched methods, wherein Model 6 is a matched case–control condition logistic regression (CLR), and Model 7 is a matched cohort CLR. For Model 8, the Cochran–Armitage Trend (CAT) test with 3 weighting schemes, namely, additive, recessive, and dominant, were used [14]. CAT, which uses the Chi-square statistics of 1 degree of freedom, is considered to be more powerful than the regular Peason’s Chi-square of 2 degrees of freedom [15]. Here, Model 6 was used as a reference for comparison with Models 7 and 8 because matched case–control is inefficient or may actually introduce additional cofounders and bias and is not recommended [16-17]. 13 13 Assessment of performance A total of 12 parameter setups () and 3 genetic traits (additive, recessive, and dominant) on exposure X were assumed in the simulation. For each of the 12 × 3 scenarios, n = 1000 replicates were generated. For the regression method (Models 1–7), a test on ternary exposure comprised 2 nominal T-statistics ∝̂1/SE(∝̂1) and ∝̂2/ SE(∝̂2), where the estimate of ∝̂𝑖 and its nominal standard error SE(∝̂𝑖) corresponded to each contrast. Here, exposure was considered as significant if at least 1 of the contrasts tested significant at the level of 0.05. In this case, the expected type I error that at least 1 of the 2 contrast variables is significant is 1 − (0.95)2 = 9.75%. Tables 3– 10 list the proportions of significance and the empirical standard deviations of nominal T-statistics (denoted as STD_T) for each of the 8 models and 3 contrast codings listed in Table 1 based on 1000 replicated samples corresponding to 1 of the parameter/trait scenario listed in Table 2. Notably, the proportion of significance is regarded as the type I error rate when the true  is 0 (no x–y association) and is regarded as power when ≠0. As a nominal Wald’s statistics on , the T-statistic is supposed to follow an asymptotic standard normal distribution with variance 1; however, the variable selection procedure and the confounding effect can distort the assumption [5,18]. With STD_T > 1, the T-statistic has a heavier tail than the standard normal and therefore the A total of 12 parameter setups () and 3 genetic traits (additive, recessive, and dominant) on exposure X were assumed in the simulation. For each of the 12 × 3 scenarios, n = 1000 replicates were generated. For the regression method (Models 1–7), a test on ternary exposure comprised 2 nominal T-statistics ∝̂1/SE(∝̂1) and ∝̂2/ SE(∝̂2), where the estimate of ∝̂𝑖 and its nominal standard error SE(∝̂𝑖) corresponded to each contrast. Here, exposure was considered as significant if at least 1 of the contrasts tested significant at the level of 0.05. In this case, the expected type I error that at least 1 of the 2 contrast variables is significant is 1 − (0.95)2 = 9.75%. Assessment of performance Tables 3– 10 list the proportions of significance and the empirical standard deviations of nominal T-statistics (denoted as STD_T) for each of the 8 models and 3 contrast codings listed in Table 1 based on 1000 replicated samples corresponding to 1 of the parameter/trait scenario listed in Table 2. Notably, the proportion of significance is regarded as the type I error rate when the true  is 0 (no x–y association) and is regarded as power when ≠0. As a nominal Wald’s statistics on , the T-statistic is supposed to follow an asymptotic standard normal distribution with variance 1; however, the variable selection procedure and the confounding effect can distort the assumption [5,18]. With STD_T > 1, the T-statistic has a heavier tail than the standard normal and therefore the 14 null hypothesis is likely to be rejected; this situation results in a high type I error rate. This scenario is known as over-dispersion. By contrast, STD_T < 1 is known as under-dispersion, which is likely to produce a conservative result. Results As mentioned in the previous session, a ternary exposure is decomposed into contrast variables T1, T2 for treatment contrast; S1, S2 for SNP contrast; and H1, H2 for Helmets contrast. Therefore, 2 estimated coefficients were obtained. Table 2 depicts the assessment of the performance of different contrasts and regression procedures under the null hypothesis (unassociated x–y with= 0) and strong confounding (= 0.5 = 0.5). Here, type I error is referred to as the case when at least 1 contrast variable is significant at the level of 0.05. The results are shown in Tables 3–10. --- Insert Table 3–10 here --- --- Insert Table 3–10 here --- Type I error comparison Type I error comparison 15 Tables 3–4 list the type I error and std of T-statistics for Models 1–8 and 3 contrast codings across 12 simulated scenarios: 2 confounding (high and moderate), 2 x–y association (high and moderate), and 3 genetic traits (additive, dominant, and recessive). For each model, the contrast with the smallest type I error rate among the 3 15 15 genetic traits is bolded. genetic traits is bolded. As expected, Model 1 had unacceptably large type I error rates that ranged from 30%–50% for the high-confounding scenario and from 15%–17% for the low-confounding scenario. After adjusting for the selected covariates, Model 2 reduced the type I error from Model 1. However, the type I error of Model 2 remained considerably larger than that of other MPS-adjusted/matched methods. The nominal standard deviation of the test statistics (STD_T) for Model 2 was also severely inflated and ranged from 1.25–1.5-fold in the high-confounding scenario and from 1.15–1.2 in the low-confounding scenario. The averaged type I error rates among the MPS-adjusted models followed the order of Model 6 < 0.0975 ≒ Model 3 ≒ Model 4 < Model 7 < Model 5 < Model 8 < Model 2 < Model 1, where 0.0975 is the expected error rate. Among the MPS-direct adjusted models, Model 5 had the largest type I error possibly due to over-adjusting by selecting too many covariates. By neglecting Model 6, the MPS-matched method provided larger type I error than MPS-direct adjusted methods. 16 Among the 3 contrast codings, averaged SNP contrast had the smallest type I error, followed by Helmert contrast, and treatment contrast had the largest type I error. This situation was roughly true across all 3 genetic traits and 8 methods. The tendency shown in Table 4 for the low-confounding scenario was similar to that shown in Table 3 but with a smaller size of type I error 3, but with a smaller size of type I error. Given that Models 1 and 2 had considerably larger type I error rates than other models, only MPS-Models 3–8 were compared for power. Given that Models 1 and 2 had considerably larger type I error rates than other models, only MPS-Models 3–8 were compared for power. Power comparison Power comparison Tables 5–8 list the power and std of T-statistics for Models 3–8 and 3 contrast codings across 12 simulated scenarios: 2 confounding (high and moderate), 2 x–y associations (high and moderate), and 3 genetic traits. The contrast with the largest power among the 3 genetic traits is bolded. Model 6 (MPS-matched case–control) had the worst power, which coincided with the conclusion that matched case–control is inefficient or may actually introduce additional cofounding and bias [16-17] and is therefore not recommended. As expected, the powers of all models increased as the x–y association increased. In general, the MPS-matched cohort (Models 7 and 8) had better power than the 17 MPS-direct adjusted cohort (Models 3–5). In general, the power followed the order of MPS-direct adjusted cohort (Models 3–5). In general, the power followed the order of Model 8 > Model 7 > Model 3≒Model 4≒Model 5 > Model 6. Model 8 > Model 7 > Model 3≒Model 4≒Model 5 > Model 6. Among the MPS-direct adjusted models, treatment contrast had better power than other contrast codings for additive and recessive genetic traits. However, when the trait was dominant, Helmert contrast had better power than the other codings. On the other hand, among the MPS-matched cohort models, CAT (Model 8) outperformed CLR (Model 7) for dominant traits, but both models were equally good for additive and recessive traits. Helmert contrast had the best power among the 3 contrasts across all 3 traits. The power of Model 8 with Helmert contrast for additive traits, for example, was as high as 93.9% under the scenario of high x–y association and high confounding and as low as 54.1% under the scenario of low x–y association and low confounding. The power of Model 3 with treatment contrast for an additive trait was as high as 79.2% in the scenario of high x–y association and low confounding and as low as 36.4% in the scenario of low x–y association and high confounding. When confounding is absent As a reference, Tables 9 and 10 show the results obtained with MPS when 18 18 confounding variables were absent. As shown in Table 9, some zs were associated with y, but none were associated with x. As illustrated in Table 10, some zs were associated with x, but none were associated with y. In these cases, the MPS-matched cohort had lower power than the MPS-direct adjusted regression because matching might be unnecessary to reduce the sample size due to the drop in unmatched observations. unnecessary to reduce the sample size due to the reduction in unmatched observations unnecessary to reduce the sample size due to the reduction in unmatched observations. unnecessary to reduce the sample size due to the reduction in unmatched observations. As for the contrast codings, when MPS-direct adjusted models are used, treatment contrast has better power for additive and recessive genetic traits, whereas Helmert contrast has better power for dominant traits. When the MPS-matched cohort method is used, Helmert Contrast had better power for all 3 genetic traits. used, Helmert Contrast had better power for all 3 genetic traits. Discussion 19 Our results showed that in the presence of high confounding effect from multiple confounders, MPS methods had much more reasonable type I error rate than the non-MPS methods. The conventional covariate-adjusted method could have high type I error (~30%) due to the sever over-dispersion of the nominal T-statistics. This was caused by the covariate selection procedure, which normally results the under-estimating the standard deviation for the target coefficient [5]. Compared with MPS-direct adjusted methods, MPS-matched methods have better power but larger type I error rate. Among the MPS-matched methods, MPS-matched cohort CAT has slightly better power than MPS-matched cohort CLR, again, with larger type I error as a trade off. When MPS-direct adjusted regression is used, the selection of excessive other covariates into the model is not recommended. MPS-matched cohort may have lower power than MPS-direct adjusted regression because matching may be 19 Our results showed that in the presence of high confounding effect from multiple confounders, MPS methods had much more reasonable type I error rate than the non-MPS methods. The conventional covariate-adjusted method could have high type I error (~30%) due to the sever over-dispersion of the nominal T-statistics. This was caused by the covariate selection procedure, which normally results the under-estimating the standard deviation for the target coefficient [5]. Compared with MPS-direct adjusted methods, MPS-matched methods have better power but larger type I error rate. Among the MPS-matched methods, MPS-matched cohort CAT has slightly better power than MPS-matched cohort CLR, again, with larger type I error as a trade off. When MPS-direct adjusted regression is used, the selection of excessive other covariates into the model is not recommended. MPS-matched cohort may have lower power than MPS-direct adjusted regression because matching may be Our results showed that in the presence of high confounding effect from multiple confounders, MPS methods had much more reasonable type I error rate than the 19 Ethics approval and consent to participate This study involved only computer simulation data, no IRB approval or consent to participate is needed. Conclusions To summarize, for ternary exposure, when a strong confounding effect due to observed covariates is believed to exist, then the MPS-matched cohort method with Helmert contrast is recommended. Otherwise, MPS-direct adjusted regression with treatment and Helmert contrasts are worth trying. This work limited on discussing MPS-direct adjustment and MPS-matching separately. Nguyen et al. (2017) had proposed an approach of combining double-adjustment and matching in propensity score analysis [19]. Our results may suggest a future study on such combination in MPS. 20 20 Consent for publication The manuscript contains no individual person’s data in any form. Availability of data and materials R code for simulation in this study is available upon request. Competing interests Declarations Ethics approval and consent to participate Funding The research is self-funded, except that publication fee will be supported by the affiliated institute of corresponding as an encouragement of research. 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Double-adjustment in propensity score matching analysis: choosing a threshold for considering residual imbalance. BMC Med Res Methodol. 2017;17(1):78. Published 2017 Apr 28. doi:10.1186/s12874-017-0338-0 Reference Matching, an appealing method to avoid confounding?. Nephron Clin Pract. 2011;118(4):c315-c318. doi:10.1159/000323136 18. Lian, I. (1995), Asymptotics of Forced-In Variables From Screening Processes", Commun. in Statistics - Theory and Method, 24(1) 131-151. 24 Figure 2 Flow chart of generalizing x, y, and zs. 19. Nguyen TL, Collins GS, Spence J, et al. Double-adjustment in propensity score matching analysis: choosing a threshold for considering residual imbalance. BMC Med Res Methodol. 2017;17(1):78. Published 2017 Apr 28. doi:10.1186/s12874-017-0338-0 19. Nguyen TL, Collins GS, Spence J, et al. Double-adjustment in propensity score matching analysis: choosing a threshold for considering residual imbalance. BMC Med Res Methodol. 2017;17(1):78. Published 2017 Apr 28. doi:10.1186/s12874-017-0338-0 25 25 Figures Figures Figures Figure 1 z1– z5 are associated with x only; z6–z10 are confounders that are associated with x and y; z11–z15 are associated with y only; and z16– z20 are not associated with either x or y. Effect sizes are denoted by (α, β, γ) Figure 1 z1– z5 are associated with x only; z6–z10 are confounders that are associated with x and y; z11–z15 are associated with y only; and z16– z20 are not associated with either x or y. Effect sizes are denoted by (α, β, γ) Supplementary Files list of supplementary ¦les associated with this preprint. Click to download. This is a list of supplementary ¦les associated with this preprint. Click to d TABLE2000727.pdf TABLE2000727.pdf TABLE2000727.pdf
https://openalex.org/W2331791140	https://www.scielo.br/j/csc/a/jRG35pnf3N753r7R7XrJCct/?lang=pt&format=pdf	Portuguese	null	Estilo de vida em pacientes portadores de diabetes mellitus tipo 1: uma revisão sistemática	Ciência & Saúde Coletiva	2,016	cc-by	5,296	1 Departamento de Odontopediatria, Ortodontia e Saúde Coletiva, Faculdade de Odontologia de Bauru, Universidade de São Paulo. Av. Octávio Pinheiro Brisolla 9-75. 17012- 901 Bauru SP Brasil. shcperes@usp.br 2 Universidade Estadual Paulista Júlio de Mesquita Filho. São Paulo SP Brasil. Estilo de vida em pacientes portadores de diabetes mellitus tipo 1: uma revisão sistemática revisão review revisão review Lifestyle of patients with diabetes mellitus type 1: a systematic review Silvia Helena de Carvalho Sales-Peres 1 Maria de Fatima Santos Guedes 1 Letícia Marques Sá 1 Carlos Antonio Negrato 2 José Roberto Pereira Lauris 1 Silvia Helena de Carvalho Sales-Peres 1 Maria de Fatima Santos Guedes 1 Letícia Marques Sá 1 Carlos Antonio Negrato 2 José Roberto Pereira Lauris 1 Abstract The aim of this review was to veri- fy data concerning the relationship between the existent lifestyle and glycemic control in patients with Diabetes Mellitus Type 1 (DM1). The meth- ods applied included the literature search strategy, selection of studies by means of inclusion and ex- clusion strategies, according to the characteristics of the studies. The search was conducted in the Lilacs, Medline, PubMed, Cochrame, SciELO and IBECS databases between in the period between 2005 and 2014. The articles selected were studies in humans, investing lifestyle, physical activities and glycemic levels. Of the 1798 studies initially identified, 11 met the eligibility criteria. Among the studies analyzed, 1 cohort; 1 longitudinal pro- spective, 1 case control and 8 transversal studies that approached the proposed theme were re- lated. Regular physical activity was the variable that presented greatest relationship with the im- provement in glycemic levels. Healthy active life, balanced diet, physical activities and education in diabetes improved the glycemic control of the DM1 patient. The results allowed the authors to conclude that a lifestyle based on physical activ- ities interfered directly in the health of patients with DM1, in addition to contributing the glyce- mic control. Resumo O objetivo desta revisão foi verificar dados concernentes sobre a relação existente en- tre estilo de vida e controle glicêmico em pacientes com Diabetes Mellitus tipo 1 (DM1). Os métodos aplicados incluíram estratégia de busca na litera- tura, seleção dos estudos por meio dos critérios de inclusão e exclusão de acordo com as característi- cas dos estudos. A busca foi realizada nas bases de dados Lilacs, Medline, PubMed, Cochrame, SciE- LO e IBECS entre 2005 e 2014. Os artigos selecio- nados foram estudos em humanos, investigando estilo de vida, atividades físicas e níveis glicêmi- cos. Dos 1798 estudos identificados inicialmente, 11 atendiam aos critérios de elegibilidade. Den- tre os estudos analisados foram relacionados 1 de coorte, 1 longitudinal prospectivo, 1 caso controle e 8 transversais que abordavam o tema proposto. DOI: 10.1590/1413-81232015214.20242015 DOI: 10.1590/1413-81232015214.20242015 1197 Estilo de vida em pacientes portadores de diabetes mellitus tipo 1: uma revisão sistemática A atividade física regular foi a variável que apre- sentou maior relação com a melhora nos níveis glicêmicos. Vida ativa saudável, dieta balanceada, atividades físicas e educação em diabetes melho- raram o controle glicêmico do paciente DM1. Os resultados permitem concluir que o estilo de vida pautado em atividades físicas interfere direta- mente na saúde do paciente com DM1, inclusive contribuindo para o controle glicêmico. Palavras-chave Diabetes Mellitus Tipo 1, Estilo de vida, Atividade física, Aspectos psicológicos, Condições socioeconômicas Key words Diabetes Mellitus Type 1, Lifestyle, Physical activity, Psychological aspects, Socioeco- nomic conditions Key words Diabetes Mellitus Type 1, Lifestyle, Physical activity, Psychological aspects, Socioeco- nomic conditions 1198 Sales-Peres SHC et al. 1198 119 Sales-Peres SHC et al. Introdução plicações, pois a adesão ao tratamento é a chave para alcançar os objetivos desejados8. O Diabetes Mellitus Tipo 1 (DM1), presente em 5 a 10% dos casos dessa doença, é o resultado da destruição de células betapancreáticas com con- sequente deficiência de insulina. Os principais marcadores imunológicos do comprometimento pancreático são os anticorpos anti-ilhota, anti -insulina e antidecarboxilase do ácido glutâmi- co e estão presentes em 90% dos pacientes por ocasião do diagnóstico1. O diabetes tipo 1 ocorre habitualmente em crianças e adolescentes, entre- tanto, pode manifestar-se também em adultos, geralmente de forma mais insidiosa. Pacientes com esse tipo de diabetes necessariamente de- pendem da administração de insulina2. Visto que o diabetes exige um controle in- tenso para evitar complicações, o emocional in- fluenciará de forma significativa nesse controle, tendo em vista que tal patologia pode provocar vários sentimentos negativos. Assim, muitas ve- zes, torna-se necessário o acompanhamento psi- cológico, em grupo ou individual, para melhorar a qualidade de vida9. Esta revisão sistemática tem como objetivo primário relacionar os desfechos estilo de vida, atividade física, aspectos psicológicos e condições socioeconômicas em indivíduos com DM1. O se- gundo objetivo foi relacionar o estilo de vida no controle glicêmico. O principal objetivo do tratamento é preve- nir o aparecimento ou a progressão das compli- cações crônicas, como as microvasculares (reti- nopatia, nefropatia e neuropatia diabética) e as macrovasculares (acidente vascular cerebral e doença arterial periférica), ao mesmo tempo mi- nimizando os riscos das agudas como a hipogli- cemia severa3. Metodologia A pergunta elaborada para a realização desta revisão sistemática foi determinar se o paciente DM1 sofre influencias dos desfechos estilo de vida, atividade física, aspectos psicológicos e con- dições socioeconômicas. O estilo de vida é um importante determi- nante do controle glicêmico em pacientes diabé- ticos tipo 1 e 2. O tratamento do DM1 interfere no estilo de vida, é complicado, doloroso, depen- de de autodisciplina e é essencial à sobrevida do paciente4. A abordagem terapêutica envolve vá- rios níveis de atuação, como a insulinoterapia, a orientação alimentar, a aquisição de conheci- mentos sobre a doença, a habilidade de autoapli- cação da insulina e o automonitorização da glice- mia, a manutenção da atividade física regular e o apoio psicossocial5. Estratégia de busca A revisão sistemática daliteraturafoi realizada no segundo semestre de 2014 abrangendo uma grande gama de achados de uma pesquisa con- duzida na Lilacs, Medline, PubMed, Cochrame, SciELO e IBECS . A seleção dos descritores uti- lizados no processo de revisão foi efetuada me- diante consulta ao DECs (descritores de assunto em ciências da saúde da Bireme).Recorreu-se aos operadores lógicos “AND”, “OR” e “AND NOT” para combinação dos descritores e termos utili- zados para rastreamento das publicações. Devido a muitos efeitos benéficos, a atividade física regular é indicada para pacientes com DM1, uma vez que melhora o controle metabólico e di- minui o risco cardiovascular, também agrega um efeito importante na prevenção das complicações crônicas desta patologia6. Contudo, muitos indi- víduos com DM1 não seguem a recomendação de praticar atividade física por um período míni- mo de 30 min, durante cinco dias por semana ou de intensidade vigorosa atividade física aeróbia para um mínimo de 20 min em três dias a cada semana7. Fato este, que favoreceria a continuida- de de um estilo de vida ativo ao longo da vida. Artigos publicados foram procurados base- ados nos descritores Diabetes Mellitus Tipo 1, Estilo de Vida, Atividade Física, Aspectos Psicoló- gicos e Condições Socioeconômicas escritos nos idiomas inglês, português e espanhol, abrangen- do artigos publicados nos últimos cinco anos, de janeiro de 2010 a outubro de 2014, uma vez que a literatura recente abarca o tema de forma mais sólida. Esta revisão objetivou incluir evidências recentes de pesquisas psicossocial e econômica em indivíduos com DM1. Pesquisas recentes têm abordado estilo de vida moderno na saúde geral10. No DM1 é importanterealizar uma dieta ba- lanceada, adotando conhecimentos quanto ao consumo correto de carboidratos, proteínas e gorduras. A observação das quantidades e quali- dades necessárias de cada grupo alimentar possi- bilita o controle glicêmico e a prevenção de com- As bases de dados foram pesquisadas com as seguintes palavras chaves de busca: “DM1” [MESH], and “lifestyle” [MESH] and “physica- lactivity” [MESH] na base de dados PUBMED; e 1199 “DM1” [DESC], “estilo de vida”[DESC], “ativida- de física” [DESC],“aspectos psicológicos” [DESC] e “condições socioeconômicas” [DESC] nas bases de dados Lilacs e SciELO. Critérios de inclusão e exclusão Através deste procedimento de busca, foram identificadas, inicialmente, 1798 publicações po- tencialmente elegíveis para inclusão nesta revi- são.Em seguida, identificaram-se os artigos que atenderam aos seguintes critérios de inclusão: a) artigos publicados entre 2010 a 2014, b) artigos de pesquisa com seres humanos, c) idiomas In- glês, Espanhol e Português; d) todos os estudos publicados envolvendo algum dos desfechos, es- tilo de vida, atividade física, aspectos psicológicos e condições socioeconômicas, específicos para pacientes com DM1. Foram excluídos artigos du- plicados e de revisão. Resultados No Quadro 1 são apresentadas informações ge- rais sobre os 11 estudos incluídos. Um artigo utilizou delineamento de caso controle11, um o estudo de coorte12um o Pros- pectivo13todos os demais foram transversais14-20. Estratégia de busca Na base de dados Pub- med foi realizada a seguinte estratégia de busca: foram inseridos no campo da pesquisa os termos de acordo com o [MESH] “DM1” AND “estilo de vida” AND “atividade física”, com filtro de busca considerando artigos publicados nos últimos 5 anos, além do filtro para idiomas considerando artigos em inglês, espanhol e português. Cochrane = 6) foram considerados elegíveis para a segunda fase desta revisão, que consistiu da lei- tura dos resumos. Após avaliação dos resumos, os estudos que pareciam preencher os critérios de inclusão foram lidos na íntegra.Um revisor ex- traiu os dados e o outro verificou os resultados. Dois revisores avaliaram a qualidade e a força de evidência. Ao final da avaliação, 11 artigos aten- deram a todos os critérios de inclusão (Figura 1). Na categoria características da amostra foram inseridos os participantes do estudo e a demo- grafia, bem como os instrumentos de avaliação utilizados para mensurar os desfechos analisa- dos. Na categoria detalhe dos métodos foram inseridos os métodos utilizados em cada estudo e a análise estatística escolhida. E na última cate- goria, detalhes dos resultados foram descritos os principais resultados e conclusões de cada estudo (Quadro 1). Extração dos dados A composição das amostras variou quanto à faixa etária, sendo que quatro artigos incluíram crianças11,16-18, dois os responsáveis dos meno- res18,20 e outros também adolescentes e adultos. Após a primeira análise, com avaliação dos títulos, 66 artigos (PubMed = 29 Medline= 31 Figura 1. Fluxograma da seleção dos artigos e das bases de dados. 5 anos seres humanos artigos duplicados Total - 11 artigos completos incluídos na revisão PUBMED 775 192 29 5 lilacs 1 1 0 0 COCHRANE 85 47 6 1 MEDLINE 934 427 31 5 SCIELO 2 1 0 0 IBECS 1 1 0 0 Leitura dos títulos Leitura dos resumos (n = 66) Não atenderam critérios de inclusão/exclusão (n = 55) Figura 1. Fluxograma da seleção dos artigos e das bases de dados. 1200 Sales-Peres SHC et al. 120 Sales-Peres SHC et al. continua Quadro 1. Descrição dos estudos selecionados para a revisão. Fator de Impacto A2 B4 A2 Design do estudo Estudo transversal Estudo coorte Estudo transversal País Tamanho da amostra Canadá GDM1 = 75 GC = 75 E.U.A Homens GDM1 = 211 GC = 67 Suécia DM1 = 292 (masc e fem) Faixa etária > 18 anos De 12 a 19 18 a 59 Fatores de Inclusão e de Exclusão Inclusão: DM1 + que 6 meses de diagnóstico > 18 anos e com capacidade de utilizar bicicleta ergométrica Exclusão: complicações crônicas do diabetes Inclusão: DM1com 5 anos de diagnóstico; pacientes tratados no Barbara Davis Center for Childhood Diabetes Exclusão: irmão de DM1 para controle; pacientes com diabetes que não DM1. Inclusão: idade 18 a 59 anos e diagnóstico de diabetes mais que 1 ano Exclusão: comorbidades somáticas graves, transtorno mental grave, pouco conhecimento da língua sueca Métodos Utilização de sensor de movimento Teste de aptidão cardiorrespiratória Avaliação (IMC e RCQ, massa magra e gorda medidas) Avaliação da presença diabetes, HbAc1, perfil lipídico e PA * Questionários- dieta diária e tempo de permanência na frente da televisão; Atividade física e dados antropométricos. Orientações sobre dieta e exercício físico. * Seguimento- 4 anos. * Escala de Depressão e Ansiedade e Escala de Alexetimia de Toronto. * Dados antropométricos e clínicos. Resultados - Composição corporal não foi diferente entre os grupos estudados. Extração dos dados - Estilo de vida ativo, leva a uma melhor composição corporal em pacientes com ou sem diabetes Aumento no consumo de vegetais, frutas e atividades físicas; aumento no consumo de alimentos fritos e de café da manhã. - A depressão, principalmente em mulheres, é de importância para o controle do nível glicêmico, como a obesidade e o tabagismo. - A alexitimia está associada à depressão. Conclusões A composição corporal obteve um melhor nível com um perfil de estilo de vida ativo nos dois grupos. Algumas melhorias na dieta e na atividade física em ambos os grupos. Adolescência é um período de modificações no comportamento. Os fatores psicológicos foram relacionados com controle glicêmico. Maior cuidado em pacientes com depressão e alexitimia, para que possam obter um melhor controle glicêmico. Referência: Brazeau et al.14, 2012 Referência: Bishop et al.12, 2014 Referência: Melin et al.15, 2013 adro 1. Descrição dos estudos selecionados para a revisão 1201 continua Quadro 1. continuação Fator de Impacto B4 A1 Design do estudo Estudo prospectivo Estudo transversal Estudo transversal País Tamanho da amostra Índia DM1 = 56 (31 masc e 25 fem) GC = 50 (30 masc e 20 fem) Itália DM1 = 129 GC = 214 Alemanha DM1 = 296 (crianças, adolescentes e jovens adultos) Faixa etária 15 a 17 Idade escolar < 22 anos Fatores de Inclusão e de Exclusão NT NT NT Métodos Intervenções: Palestras sobre saúde; Questionários; Orientação individual, para pais e professores; Promoção de atividade física; Mudança no cardápio da cantina; programa de sustentabilidade na escola. Questionários (via telefone) sobre atividade física, dados clínicos e estilo de vida sedentária. Dados antropométricos (IMC) Dados clínicos HbA1c * Questionários de auto relato (German Health Interview e Examination Survey). * Índice de Winkler para avaliar o nível socioeconômico. Resultados - Após seis meses houve melhora na função da célula beta; - Diminuição dos níveis de proteína c-reativa ultrassensível; - Redução significante da circunferência abdominal. - Crianças diabéticas: tiveram mesma intensidade de atividade física; mais envolvidas em esportes de grupo; consumiram menos alimentos ao assistir TV ou imediatamente após atividade física. - A regressão múltipla identificou que tempo de diabetes, nível socioeconômico e horas gastas na mídia por dia têm relação linear com os níveis de HBa1c. São fatores de risco para controle metabólico. Extração dos dados Conclusões Utilizar o mesmo tipo de intervenção para prevenir o DM1, melhorando a resistência à insulina e prevenção do DM2 A maior parte das crianças com DM1 praticam atividade física como o GC e a consideram divertimento e não obrigação médica. O nível socioeconômico e o tempo diário gasto em atividade de mídia são importantes para o aumento da HbA1c. Não foi encontrada relação com atividade física. Referência: Singhal et al.13, 2011 Referência: Fainardi et al.16, 2011 Referência: Galler et al.10, 2011 120 Sales-Peres SHC et al. 1202 continua Quadro 1. continuação Fator de Impacto B4 B1 B1 Design do estudo Estudo transversal Estudo transversal Estudo transversal País Tamanho da amostra E.U.A. DM1 = 349 E.U.A 430.912 de 50 Estados e todos os territórios dos Estados Unidos. E.U.A. DM1 = 89 Faixa etária 9 a 17 anos > 18 anos 13 a 19 anos Fatores de Inclusão e de Exclusão Inclusão: diagnóstico de DM1 pelo menos 6 meses Exclusão: com outra doença crônica NT Exclusão: não poderiam estar grávida e não falar Inglês. Apresentar retardo mental ou DM1 menos de 1 ano. Métodos Duas etapas: 1- 200 indivíduos foram avaliados quanto memória, habilidadede aprendizado e manuseio da doença. 2- 149 indivíduos foram orientados para prevenir a piora dos cuidados com diabetes. Questionário para autoavaliação de saúde (SRH) na área de atividade física, saúde e histórico médico. * Questionários para avaliação sobre sexualidade, preconcepção, contracepção e gravidez; avaliar a relação com seus médicos e liberdade para solicitar controle de nascimento. Resultados Diferenças étnicas nos níveis de HbA1 e nos cuidados com a doença. A análise multivariada identificou dentre os fatores sociodemográficos, condição socioeconômica explicar melhor as diferenças. - Foi ótima em DM1 (53,3%),DM2 (52,2%) e 86,2% sem DM. - Quanto mais ativos eram os indivíduos, mais alta era a taxa de SRH. - 50% tiveram relações sexuais sem contracepção. -Metade das pacientes não tinham discutido controle de natalidade. - Dois terços delas se sentiam confortáveis em conversar esse tema com profissionais da saúde. Conclusões A educação para o diabetes deve também abranger os pais dos diabéticos, orientando sobre: estilo de vida saudável, controle de nível glicêmico e classe socioeconômica. Orientação para incorporar atividade física no estilo de vida; Promover meios para que haja melhora de saúde e bem estar da população. Adolescentes com diabetes sentem-se envergonhadas em solicitar orientações a profissionais sobre controle de natalidade. Extração dos dados 1204 Quatro estudos foram realizados nos Estados Unidos12,18,19, dois na Suécia11,15 e os demais na Ín- dia13, Canadá14,Itália16, Alemanha17 e Brasil20. dades físicas realizadas, permanência em frente a TV e dieta diária. Os adolescentes receberam instruções referentes à dieta e à prática de exer- cícios físicos. Os adolescentes apresentaram algu- mas melhorias, tais como aumento no consumo de vegetais, frutas e atividades físicas. Entretanto, relataram aumento no consumo de frituras. Fato que evidencia que a adolescência é a fase da vida na qual ocorre modificações no comportamento e nos hábitos alimentares12. Seis estudos mencionaram que o trabalho foi aprovado por um Comitê de Ética, três fizeram menção a, pelo menos, um aspecto ético (que os participantes deram o consentimento). Vale sa- lientar que foi avaliado apenas se o artigo fazia menção aos aspectos, podendo os mesmos terem sido atendidos independente da menção no cor- po do texto. Em um estudo prospectivo envolvendo jo- vens, entre 15 e 17 anos, foram aplicadas estra- tégias com orientação individual, promoção de atividade física, mudança do cardápio da cantina, orientação para pais e professores e formação de grupos de alunos voluntários para sustentabili- dade do programa na escola. Após seis meses de estudo foram identificadas melhoras na função da célula beta, melhorando a resistência à insu- lina e a prevenção do DM2. Além da diminuição nos níveis de proteína c reativa e na redução sig- nificativa da circunferência abdominal13. Em 07 manuscritos, os fatores de inclusão e exclusão estão detalhados11,12,14,15,17,19,20. Quanto aos instrumentos utilizados para coletas de da- dos, verificou-se que apenas dois11,14 utilizaram o instrumento sensor de movimento, oito usaram questionários11-13,16-20, dois relataram orientações sobre dieta e exercícios12,13 e três empregaram da- dos antropométricos12,15,17. Todos os estudos apresentaram risco de viés. Entretanto, a evidência científica é escassa para muitos dos desfechos examinados. Ao se considerar a importância do estilo de vida ao longo da vida do indivíduo,uma das grandes estratégias a ser aplicada é a educação em saúde sobre o DM1, a qual deve abranger não somente os pacientes envolvidos, mas também os pais dos diabéticos, orientando-os para um estilo de vida saudável e como melhorar o controle do nível glicêmico18. Já que as mães são as princi- pais cuidadoras das crianças e adolescentes com DM1, elas devem tomar conhecimento sobre os riscos do mau controle glicêmico, para que não haja dificuldade em definir limites para as crian- ças e adolescentes com DM1. Extração dos dados Educadores em diabetes devem orientá-las também neste aspecto. Referência: Haskell et al.7, 2007 Referência: Tsai et al.18, 2010 Referência: Schwarz et al.19, 2010 continua 1203 Quadro 1. continuação Fator de Impacto B1 B1 Design do estudo Estudo transversal Estudo caso controle País Tamanho da amostra Brasil DM1 = 1.079 (pais de pacientes) Suécia GDM1 = 24 GC = 26 Faixa etária < 18 anos < 7anos Fatores de Inclusão e de Exclusão Inclusão: ter diabetes a mais de 1 ano e realizar o seguimento por pelo menos 1 ano. Inclusão: < 7 anos e duração do DM1 mais de três meses a entrada do estudo. Exclusão: Outra doença que afeta a capacidade de estar fisicamente ativo, diagnóstico de diabetes conhecidas por não serem tipo 1, conhecimento insuficiente da língua sueca e privação social. Métodos * Estudo Multicêntrico; * 28 clínicas públicas em 20 cidades. * Questionários com questões psicossociais foram dirigidas aos pais, dados clínicos de prontuários, a partir da última consulta médica. * O perfil demográfico, educacional e socioeconômico. * Os níveis de HbA1c registrados. Avaliação de altura e peso (IMC) e HbA1c. Atividade física foi medida usando um sensor de movimento e um sensor e taxa de coração combinado,em dois períodos de tempo durante 1 ano. * Questionário autoaplicável aos pais sobre educação, emprego e estado civil. Resultados - Queixas: desconforto e ansiedade; depressão mais em mães; a maioria relatou mudanças na família depois do diagnóstico; complicações e cuidados sobrecarregavam os pais; as crianças mais rebeldes apresentavam HbA1c mais altos; pais cujos filhos tinham sido hospitalizados por DM1 apresentavam mais ansiedade e depressão. - As crianças com diabetes foram consideradas menos ativas. - O tempo sedentário, não foi significativo nos dois grupos. - PA e tempo gasto em atividade física moderada e vigorosa foram mais prevalentes em meninos. Foi observado efeito significativo da idade. Conclusões O estilo de vida de todos familiares mudou, após o diagnóstico de diabetes, interferindo na qualidade de vida. As mães são as principais cuidadoras das crianças e adolescentes com DM1. Dificuldade em definir limites o que dificultou ocontrole glicêmico. Importância da família no engajamento do paciente DM1, junto ao seu tratamento. A atividade física é reduzida em crianças com DM1. Referência: Malerbi et al.20, 2012 Referência: Sundberg et al.11, 2012 1204 Sales-Peres SHC et al. 120 Sales-Peres SHC et al. Extração dos dados Também devem atuar no estilo de vida de todos familiares, espe- cialmente após o diagnóstico de diabetes em um de seus membros, interferindo favoravelmente na qualidade de vida20. Discussão Os estudos incluídos nesta revisão serão discuti- dos de acordo com as faixas etárias e as variáveis investigadas. O estudo de caso-controle inserido nesta revisão demonstrou que as crianças meno- res de 7 anos de idade portadoras de DM1 são me- nos ativas fisicamente do que as saudáveis. As me- ninas com DM1 correm maior risco de não serem fisicamente ativas. A inatividade física aumenta o risco de doenças cardiovasculares, complicações e baixa aptidão física, portanto devem ser propostas atividades para aumentar o gasto energético em crianças com DM111. Resultados diferentes foram apresentados em pesquisa realizada na Itália, na qual crianças diabéticas, em idade escolar, foram mais envolvidas em esportes e fizeram da ativi- dade física um bom passatempo e não uma obri- gação médica16. Foi possível verificar que o nível socioeconômico e o tempo gasto em atividade de mídia dificultam o controle glicêmico, aumentan- do o risco metabólico17. A importância do grau de atividade física no tratamento de DM1 pode au- mentar a sensibilização para este problema. Dessa forma, estratégias que visem estimular pais, cui- dadores e creches para incentivar essas crianças a aumentar a sua necessária atividade física devem ser elaboradas em programas de saúde. Outro aspecto importante encontrado nesta revisão foram estudos que identificaram fatores psicológicos relacionados com o mau controle glicêmico, como a depressão e a alexitimia, prin- cipalmente em mulheres diabéticas15. Estas se sentem envergonhadas em solicitar orientações aos profissionais sobre controle da natalidade,u- ma vez que acreditam, erroneamente, que tais métodos são menos efetivos naquelas com esta condição19. A equipe multidisciplinar, através de uma educação para os pacientes diabéticos, pode proporcionar a estes uma melhor qualidade de vida, orientando-os quanto à composição de die- ta saudável eà incorporação da atividade física como estilo de vida adequado18-20. Assim como as Adolescentes com DM1, entre 12 e 19 anos, foram acompanhados em um estudo de coorte, com seguimento de 4 anos, para verificar ativi- 1205 gravidez indesejada e não se sentem confortáveis para perguntar ao profissional de saúde sobre o controle da natalidade19. Os educadores que tra- balham junto aos grupos de pacientes portado- res de diabetes devem iniciar o aconselhamento da preconcepção na puberdade, pois discutir as opções com essas adolescentes pode melhorar os resultados da gravidez. Discussão A inserção de mulheres jovens com DM1 em redes de atenção à saúde de doenças crônicas não transmissíveis o mais cedo possível poderá melhorar seu desempenho atual e futuro, reduzindo muitas vezes a necessidade da utilização desses serviços em maiores níveis de complexidade da atenção. cidades devem promover meios para tal, melho- randoa saúde e o bem estar da população19. A investigação conduzida por Haskellet al.7 teve por objetivo identificar o manuseio e a pre- venção de DM1, sendo dividida em duas etapas. Na primeira foi identificada a memória, a habili- dade de aprendizado e o manuseio da DM1. Já na segunda, foram relacionados métodos para pre- venir a piora dos cuidados com DM1. Na análise multivariada foram confirmadas as diferenças étnicas nos níveis de HbA1 e nos cuidados com a doença. Entretanto, os fatores sociodemográficos mostraram que seriam melhor explicados pelo status socioeconômico.Os cuidados com o dia- betes deve também abranger os pais dos diabé- ticos, salientando a importância do estilo de vida saudável e como melhorar ocontrole de nível gli- cêmico, levando em consideração principalmen- te seu status socioeconômico. Reforçando esses achados e os complementando, outra pesquisai- dentificouque o tempo de diabetes, o nível so- cioeconômico e as horas gastas na mídia por dia têm relação linear com osníveis de HBa1c7.Ou seja, são fatores de risco para controle metabó- lico inadequado. Por outro lado, discordando de Haskellet al.7, não foi encontrada associação com atividade física. As crianças diabéticas quando comparadas aos controles apresentam a mesma intensidade de atividade física, porém são mais envolvidas em esportes de grupo, gostam de pra- ticar tais atividades como um bom passatempo e uma oportunidade de fazer novas amizades. As crianças com diabetes parecemingerir menos ali- mentos, enquanto assistem televisão ou imedia- tamente após atividade física.A maior parte das crianças com DM1 podem praticar atividade fí- sica como os nãodiabéticos e a consideram como um divertimento e não uma obrigação médica16. A presente revisão sistemática ressalta a im- portância da elaboração de programas de saú- de,com enfoque em atividades físicas para indi- víduos DM1,que podem oferecer benefício para o controle glicêmico. Programas de Intervenção no estilo de vida, baseado na Web (Web-base- dlifestyleintervention) ou em Telessaúde, devem ser elaborados para sua utilização por pacientes DM1. Estudos futuros de longa duração deverão ser conduzidos para elucidar melhor a relação entre estilo de vida e diabetes tipo 1. Conclusão A presente revisão sistemática permite concluir que o estilo de vida pautado em atividades físicas interfere diretamente na saúde do paciente com DM1, inclusive contribuindo para o controle glicêmico. A promoção da saúde do DM1 deve se pautar em atividades físicas regulares, orien- tações específicas quanto aos aspectos da sexua- lidade humana e práticas para reduzir o estresse diário. Além de orientar sobre os riscos dos dis- túrbios de ansiedade e depressão, para melhorar o estilo de vida e controlar o nível glicêmico. g ç A pesquisa de Schwarz et al.19, com adoles- centes americanas maiores de 18 anos portadoras de DM1, demonstrou que metade das adolescen- tes sexualmente ativas tiveram relações sexuais sem cuidados na contracepção. Entre os relatos encontrados destacaram existir poucas opções de controle da concepção para diabéticas e ou- tras acreditam, erroneamente, que os métodos de controles de natalidade são menos efetivos em mulheres com DM1.Cerca da metade das pacien- tes não tinha discutido controle de natalidade com seus médicos e um terço delas não recebeu qualquer instrução sobre o assunto. O achado mais preocupante é que somente dois terços delas se sentiam confortáveis em conversar sobre esse tema com profissionais da saúde. Muitas mulhe- res adolescentes com DM1 apresentam risco de Colaboradores SHC Sales-Peres trabalhou na concepção (pes- quisa bibliográfica) e redação final do artigo; MFS Guedesna concepção (pesquisa bibliográ- fica) e redação do artigo; LM Sá na concepção (pesquisa bibliográfica) e redação do artigo; CA Negratona concepção (pesquisa bibliográfica); JRP Lauris na revisão crítica do artigo e aprova- ção da versão a ser publicada. 1206 Sales-Peres SHC et al. 1206 120 Sales-Peres SHC et al. Referências Brazeau AS, Leroux C, Mircescu H, Rabasa-Lho- ret R. Physical activity level and body composition among adults with type 1 diabetes. Diabet Med 2012; 29(11):e402-e408. 14. Fraguas R, Soares SMS, Bronstein MD.Depressão e dia- betes mellitus. Rev Psiquiatr Clin 2009; 36(Supl. 3):93- 99. 1. Oliveira JEP, Vencio S. Diretrizes da Sociedade Brasi- leira de Diabetes: 2013-2014. São Paulo: AC Farmacêu- tica; 2014. 2. Melin EO, Thunander M, Svensson R, Landin-Olsson M, Thulesius HO. Depression, obesity, and smoking were independently associated with inadequate glyce- mic control in patients with type 1 diabetes. Eur J En- docrinol 2013; 168(6):861-869. 15. Canadian Diabetes Association Clinical Practice Guidelines Expert Committee. Canadian Diabetes As- sociation 2013 Clinical Practice Guidelines for the Pre- vention and Management of Diabetes in Canada. Can J Diabetes 2013; 37(Supl. 1):S1-S212. 3. Fainardi V, Scarabello C, Cangelosi A, Fanciullo L, Mas- trorilli C, Giannini C, Mohn A, Iafusco D, La Loggia A, Lombardo F, Toni S, Valerio G, Franzese A, Prisco F, Chiarelli F, Vanelli M. Physical activity and sedentary lifestyle in children with type 1 diabetes: a multicentre Italian study. Acta Biomed 2011; 82(2):124-131. 16. Goes APP, Vieira MRR, Liberatore-Junior RR. Diabetes mellitus tipo 1 no contexto familiar e social. Rev Paul Pediatr 2007; 25(2):124-128. 4. Setian N, Damiani D, Dichtchekenian V, Manna TD. Diabetes mellito. In: Marcondes E, Vaz FAC, Ramos JLA, Okay Y, editores. Pediatria básica. 9ª ed. São Paulo: Sarvier; 2003. p. 382-392 5. Erkkola M, Salmenhaara M, Nwaru BI, Uusitalo L, Kronberg-Kippilä C, Ahonen S, Veijola R, Knip M, Virtanen SM. Sociodemographic determinants of early weaning: a Finnish birth cohort study in infants with human leucocyte antigen-conferred susceptibility to type 1 diabetes. Public Health Nutr 2013; 16(2):296- 304. 17. De Angelis K, da Pureza DY, Flores LJ, Rodrigues B, Melo KF, Schaan BD, Irigoyen MC. Physiologicaleffect- sofexercise training in patientswithtype 1 diabetes. Arq Bras Endocrinol Metabol 2006; 50(6):1005-1013. 6. Tsai J, Ford ES, Li C, Zhao G, Balluz LS. Artigo apresentado em 22/06/2015 Aprovado em 16/11/2015 Versão final apresentada em 18/11/2015 Colaboradores Physical activity and optimal self-rated health of adults with and with- out diabetes. BMC Public Health 2010; 10:365. 18. Haskell WL, Lee IM, Pate RR, Powell KE, Blair SN, Franklin BA, Macera CA, Heath GW, Thompson PD, Bauman A. Physical activity and public health: updated recommendation for adults from the American College of Sports Medicine and the American Heart Associa- tion. Med Sci Sports Exerc 2007; 39(8):1423‑1434. 7. Schwarz EB, Sobota M, Charron-Prochownik D. Per- ceived access to contraception among adolescents with diabetes: barriers to preventing pregnancy complica- tions. Diabetes Educ 2010; 36(3):489-494. 19. Lottenberg AM. Diet composition along the evolution of type 1 diabetes mellitus. Arq Bras Endocrinol Metabol 2008; 52(2):250-259. 8. Malerbi FE, Negrato CA, Gomes MB; Brazilian Type 1 Diabetes Study Group (BrazDiab1SG). Assessment of psychosocial variables by parents of youth with type 1 diabetes mellitus. Diabetol Metab Syndr 2012; 4(1):48. 20. Marcelino DB, Carvalho MDB. Reflexões sobre o dia- betes tipo 1 e sua relação com o emocional. Psicol Reflex Crit 2005; 18(1):72-77. 9. Galler A, Lindau M, Ernert A, Thalemann R, Raile K. Associations between media consumption habits, physical activity, socioeconomic status, and glycemic control in children, adolescents, and young adults with type 1 diabetes. Diabetes Care 2011; 34(11):2356-2359. 10. Sundberg F, Forsander G, Fasth A, Ekelund U. Children younger than 7 years with type 1 diabetes are less physi- cally active than healthy controls. Acta Pædiatrica 2012; 101(11):1164-1169. 11. Bishop FK, Wadwa RP, Snell-Bergeon J, Nguyen N, Maahs DM. Changes in diet and physical activity in ad- olescents with and without type 1 diabetes over time. Int J Pediatr Endocrinol 2014; 2014(1):17. 12. Singhal N, Misra A, Shah P, Gulati S, Bhatt S, Sharma S, Pandey RM. Impact of intensive school-based nutri- tion education and lifestyle interventions on insulin resistance, β-cell function, disposition index, and sub- clinical inflammation among Asian Indian adolescents: A controlled intervention study. 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https://openalex.org/W4206792669	https://www.matec-conferences.org/articles/matecconf/pdf/2022/02/matecconf_icpcm2022_02019.pdf	English	null	Simulation on adjacent line mutual interference of high-speed railway track circuit	MATEC web of conferences	2,022	cc-by	3,335	* Corresponding author: ysw@bjtu.edu.cn Simulation on adjacent line mutual interference of high-speed railway track circuit Qihui Xiong, Shiwu Yang, and Chang Liu 1 School of Electronic and Information Engineering, Beijing Jiaotong University, No.3, Shangyuan Village, Haidian District, 100044 Beijing, China Qihui Xiong, Shiwu Yang, and Chang Liu 1 School of Electronic and Information Engineering, Beijing Jiaotong University, No.3, Shangyuan Village, Haidian District, 100044 Beijing, China Qihui o g, Shiwu a g , a d Chang u 1 School of Electronic and Information Engineering, Beijing Jiaotong University, No.3, Shangyu Village, Haidian District, 100044 Beijing, China Abstract. As the density of the high-speed railway network continues to increase, the problem of electromagnetic interference on adjacent lines has become increasingly prominent. This paper focuses on the electromagnetic interference of adjacent lines caused by rail and line in the signal transmission process of the high-speed rail track circuit. Firstly, complete the establishment of the four-terminal network model of the ZPW-2000A track circuit system and the cab signal entry current crosstalk model, calculation of interference voltage under different parallel length of signal frequency. Then the interference factors and coupling mechanism of adjacent lines are analysed to realize calculation of interference amount. Finally, according to the sensitivity index of the cab signal, the maximum parallel length of adjacent sections is given respect, and the interference protection suggestions of adjacent lines are put forward. The research work of this paper provides a theoretical basis for suppressing the interference of adjacent lines and guarantees the safe and efficient operation of high-speed trains. Keywords: High-speed railway, ZPW-2000A track circuit, Adjacent line interference, Rail mutual inductance, Inductive coupling. © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). https://doi.org/10.1051/matecconf/202235502019 https://doi.org/10.1051/matecconf/202235502019 MATEC Web of Conferences 355, 02019 (2022) ICPCM2021 1 Introduction The track circuit is an important device for detecting train occupancy, rail integrity, and train-ground information transmission in the railway signal system. Its reliability is directly related to the safety and efficiency of trains. In the high-speed and heavy-haul electrified railway, the track circuit is affected by the surrounding strong electromagnetic interference sources (such as the traction power supply system) [1]. Due to the increase in the density of the road network, the interference between adjacent lines in the railway site shows the characteristics of irregular interference waveforms, uncertain sources of interference, and difficulty incomplete elimination, which is easy to cause missing or upgrading cab signal code [2]. Therefore, it is significance to establish the simulation model of adjacent line interference of track circuit to realize the protection of electromagnetic interference of railway signal system to ensure the safe and efficient operation of trains. * Corresponding author: ysw@bjtu.edu.cn MATEC Web of Conferences 355, 02019 (2022) ICPCM2021 https://doi.org/10.1051/matecconf/202235502019 https://doi.org/10.1051/matecconf/202235502019 As a traditional and inherent problem of railway signals, adjacent line interference has always attracted the attention of domestic and foreign researchers. Lucca[3] et al. studied the influence of inductive interference caused by the electrified double track on the track circuit, and proposed an algorithm for sensitivity analysis of the electromagnetic interference level generated; E. M. Tarasov[4] et al. proposed a method to determine the parameter matrix coefficients of railway multiple lines and analyzed the adjacent line interference between asymmetrical track circuits; Manakov AD[5] et al. proposed a method to determine the interference current of the double-track circuit; Cuiqin Zhao[6] et al. established a multi-line parallel ballasted track coupling interference equivalent circuit model; Yuequan Li[7] et al. studied the coupling interference at the transverse connecting line at the entrance of electrified railway; Jialiang Liu[8] and others realized the numerical calculation of interference current in the adjacent segment. Most of the above-mentioned research is based on general-speed railways, while high-speed railways have different configurations. And in the face of more stringent safety requirements and a more complex electromagnetic environment, the mechanism of adjacent line interference is still worthy of further study. 2 Overview of adjacent track circuit interference factors The track circuit of this line can receive not only the signal of this line but also the track circuit signal of adjacent lines. When analyzing the interference problem of adjacent lines, the track circuit of this line is called the interfered loop, and the track circuit of adjacent lines is the interfering loop. China's high-speed railway mainly uses ZPW-2000 series track circuits, with a carrier frequency ranging from 1700 to 2600 Hz. Because the signal current flowing through the track circuit is a high carrier frequency, and the inductive characteristics of the rail, the varying signal current in the interfering loop forms an interference signal in the interfered loop through mutual inductance between rails, which is called mutual inductance coupling. The existence of capacitance between parallel lines makes the energy of other lines couple to interfered loop, which is called capacitive coupling. The current in the rail will leak to another rail through the track bed, forming many parallel leakage channels between the interfering loop and the interfered loop, so that the signal current of the interfering loop is conducted to the interfered loop via the ballast leakage resistance, which is called Ballast resistance leakage conduction. Under actual conditions on-site, due to factors such as humidity, rainfall, snowfall, etc., the resistance of the ballast is reduced, and the leakage of the ballast bed is more serious, which not only causes the red band phenomenon in this track section but also makes the adjacent line interference problem more serious. In the shunt state, the shunt current is relatively large, and the problem of adjacent line interference is particularly complicated. To sum up, mutual inductance coupling, capacitance coupling, ballast resistance leakage conduction and other electromagnetic interference work together, resulting in adjacent line interference problem. When the interference is serious, it may cause distortion of cab signal receiving current waveform, affecting the normal operation of the track circuit. 2 2 MATEC Web of Conferences 355, 02019 (2022) ICPCM2021 https://doi.org/10.1051/matecconf/202235502019 3.1 Modelling and verification of ZPW-2000A track circuit The track circuit can be regarded as an electrical circuit with uniformly distributed parameters. Generally, the uniform transmission line theory is used to establish a four-port network model for analysis and calculation. This paper has completed the modelling and simulation of ZPW-2000A type non-insulated track circuit, and considers the mutual crosstalk system model of adjacent jointless track circuits in the same direction. And analyze the influence of electromagnetic interference caused by mutual inductance coupling, capacitive coupling, and track bed leakage conduction on cab signals. According to the structural characteristics of the ZPW-2000A track circuit, a hierarchical four-port network model of component level, module level, and system level is established as shown in Figure 1. In shunt state, because the transient component of the signal attenuates quickly when the train slides on the track circuit, it is only necessary to f R (taking 0.15Ω) at different positions. Trans- mitter Nt(x) Nd(n-x) Rf Zsr · Us· Is· IRf· IT· UT· Zr · Ur· Ir· Fig. 2. Cab signal voltage model. (taking 0.15Ω) at different positions. consider the cascaded shunt resistance f R Tuning NT N(x1) N(x2) N(xn) Matching Transformer NMatch Transmitter cable NCable Transmitter N(xm) Tuning NT Matching Transformer NMatch Transmitter cable NCable Receiver Attenuator NAtten Fig. 1. ZPW-2000A four-port network model. Fig. 2. Cab signal voltage model. Fig. 1. ZPW-2000A four-port network model. The equivalent model of cab signal voltage in shunting state is shown in Figure 2. 3.1 Modelling and verification of ZPW-2000A track circuit value, inductance value and compensation capacitor spacing of the track circuit in different carrier frequency sections, the current received by the cab is lower in the section with higher carrier frequency. interfering loop interfered loop a1 a2 p1 p2 Ra1 Ra2 Rp1 Rp2 L a1 L a2 L p1 L p2 Map12 Map11 Map21 Map22 Cap11 Cap12 Cap21 Cap22 Rda11 Rda12 Rdp11 Rdp12 ·Ia1 Ua · Ia2 · Up · (CI) earth Fig. 3. Simulation results of cab signal receiving voltage. Fig. 4. the calculation model of adjacent line coupling interference. 40 120 200 280 360 440 520 600 680 760 840 920 1000 1080 1160 Cab shunting position/m 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 Receiving voltage of cab signal/V 1700 2000 2300 2600 interfering loop interfered loop a1 a2 p1 p2 Ra1 Ra2 Rp1 Rp2 L a1 L a2 L p1 L p2 Map12 Map11 Map21 Map22 Cap11 Cap12 Cap21 Cap22 Rda11 Rda12 Rdp11 Rdp12 ·Ia1 Ua · Ia2 · Up · (CI) earth Fig. 4. the calculation model of adjacent line coupling interference. interfering loop interfered loop a1 a2 p1 p2 Ra1 Ra2 Rp1 Rp2 L a1 L a2 L p1 L p2 Map12 Map11 Map21 Map22 Cap11 Cap12 Cap21 Cap22 Rda11 Rda12 Rdp11 Rdp12 ·Ia1 Ua · Ia2 · Up · (CI) earth Fig. 4. the calculation model of adjacent line coupling interference. Fig. 3. Simulation results of cab signal receiving voltage. Fig. 3. Simulation results of cab signal Fig. 4. the calculation model of adjacent line coupling interference. 3.1 Modelling and verification of ZPW-2000A track circuit sr Z is the apparent impedance of the receiving end, which can be expressed as:  r is 11 12 21 22 ( ) ( ) ( ) ( ) d r d sr d r d N n x Z N n x Z N n x Z N n x − + − = − + −    (1) The cab short circuit current ( ) Rf I x  is: 11 12 ( ) ( )( ( )) / ( ) s t f t f sr sr U N x R N x R Z x Z x = ⋅ + +   (2) (2) 11 12 ( ) ( )( ( )) / ( ) s t f t f sr sr N x R N x R Z x Z x = ⋅ + +   (2) f f The following quantitative relationship between the cab signal receiving voltage and current is[9]: The following quantitative relationship between the cab signal receiving voltage and current is[9]: 200 ( ) ( ) 255 Rf Rf V x I x =   (3) (3) Without loss of generality, take the track circuit length xL as 1200m, the compensation capacitor C as 25μF, the cable transmission length d L as 10km. The transmission voltage is three levels (140V) and the ballast resistance d R as 2 km Ω⋅ . Through MATLAB software simulation, the cab signal receiving voltage under different carrier frequencies is obtained, as shown in Figure 3. It can be seen that due to the influence of the compensation capacitor, the received voltage value of the cab signal fluctuates and rises from the receiving end, and a turning point appears at each compensation capacitor. Its function is to reduce line loss, increase transmission distance, and improve the reliability of information exchange between vehicles and ground. In addition, due to the difference in resistance 3 3 3 MATEC Web of Conferences 355, 02019 (2022) ICPCM2021 MATEC Web of Conferences 355, 02019 (2022) MATEC Web of Conferences 355, 02019 (2022) ICPCM2021 https://doi.org/10.1051/matecconf/202235502019 value, inductance value and compensation capacitor spacing of the track circuit in different carrier frequency sections, the current received by the cab is lower in the section with higher carrier frequency. 4.1 Index evaluation of adjacent line interference The interference of adjacent lines is easy to cause the wrong action of on-board equipment, so it is necessary to evaluate the interference of adjacent lines according to the safety index of cab. When the interfered loop is in the adjustment state, the interference of adjacent lines shall not exceed the sensitivity of cab signal to prevent the "red light band" phenomenon; when the interfered loop is in shunting state, the interference from adjacent lines shall not exceed the reliable falling value of cab signal to prevent signal upgrading accident. Under the above calculation conditions, the maximum parallel length is shown in Table 1. If it exceeds the recommended value, it may cause equipment misoperation and increase the hidden danger of driving safety. Table 1. Maximum parallel length under different carrier frequencies. Table 1. Maximum parallel length under different carrier frequencies. carrier frequency (Hz) adjustment state shunting state Maximum interference(mV) Maximum parallel length(m) Maximum interference(mV) Maximum parallel length(m) 1700 92.5 712 69.4 446 2000 92.5 623 69.4 383 2300 92.5 551 69.4 363 2600 92.5 533 69.4 314 3.2 Theoretical calculation of adjacent line interference modelling 5. Equivalent circuit of capacitance coupling and ballast resistance conduction. Fig. 6. Interference voltage with parallel length under different carrier frequencies. In order to study the influence of carrier frequency on adjacent line coupling interference voltage, the interference voltage of the interfered loop is simulated under the conditions of 1700Hz, 2000Hz, 2300hz and 2600Hz carrier frequency respectively of the interfering loop. The simulation results are shown in Figure 6. It can be seen that when the carrier frequency is 2600Hz, the interference voltage to the interfered loop is the largest. 100 200 300 400 500 600 700 800 900 1000 1100 1200 Parallel length/m 0 20 40 60 80 100 120 140 Inerference voltage/mV 1700Hz 2000Hz 2300Hz 2600Hz Cap11 Cap21 Rdp11 Rda11 Rda12 Rdp11 Zcr1 · Zcr2 · Zcr3 · Zcr4 · R a1 R a2 Ua · Cap12 Rda11 Rdp12 Cap22 Rda12 Rdp12 (CR) Up · Iz2 · Iz1 · Fig. 5. Equivalent circuit of capacitance coupling and ballast resistance conduction. Fig. 6. Interference voltage with parallel length under different carrier frequencies. Fig. 5. Equivalent circuit of capacitance coupling and ballast resistance conduction. In order to study the influence of carrier frequency on adjacent line coupling interference voltage, the interference voltage of the interfered loop is simulated under the conditions of 1700Hz, 2000Hz, 2300hz and 2600Hz carrier frequency respectively of the interfering loop. The simulation results are shown in Figure 6. It can be seen that when the carrier frequency is 2600Hz, the interference voltage to the interfered loop is the largest. 3.2 Theoretical calculation of adjacent line interference modelling Figure 4 shows the calculation model of adjacent line coupling interference. The calculation formula of mutual inductance coefficient ap M of parallel running railway lines a and p is as follows: 12 21 6 11 22 ( 200 ln ) 10 ap ap ap ap ap D D M k D D − × = × × × × (4) (4) In the formula, k is the correction factor, which is related to the carrier frequency. Since the carrier frequency of the signal flowing through the rail is much less than 4 MHz, the mutual impedance between two parallel track circuits a and p can be calculated according to Carson's theory [10] as: 2 4 10 0.00289 ln g ap ap ap D Z jwM f j f D π − = = × + (5) (5) Among them, f is the carrier frequency of the signal flowing through the rail; =660 / g d D f σ is the equivalent depth of the line, d σ is the earth conductivity (take the average, -2 =10 m d σ Ω⋅ ). Then the mutual inductance voltage generated can be obtained as follows: (MI) p p ap a a U Z I D =   (6) (6) Capacitance coupling is also called electric field coupling. For the convenience of modelling, capacitance coupling and ballast resistance conduction are combined for analysis in Fig. 5. The interference voltage (CR) p U generated by capacitance coupling and ballast resistance leakage conduction interference in the interfered loop is as follows: (CR) 1 2 2 4 p z cr z cr U I Z I Z = −     (7) (7) Adjacent line interference will directly affect the value of cab signal entry current in the series circuit. G U is the interference voltage of the interfering loop to the interfered loop. MATEC Web of Conferences 355, 02019 (2022) ICPCM2021 MATEC Web of Conferences 355, 02019 (2022) https://doi.org/10.1051/matecconf/202235502019 ICPCM2021 Fig. 6. Interference voltage with parallel length under different carrier frequencies. 100 200 300 400 500 600 700 800 900 1000 1100 1200 Parallel length/m 0 20 40 60 80 100 120 140 Inerference voltage/mV 1700Hz 2000Hz 2300Hz 2600Hz Cap11 Cap21 Rdp11 Rda11 Rda12 Rdp11 Zcr1 · Zcr2 · Zcr3 · Zcr4 · R a1 R a2 Ua · Cap12 Rda11 Rdp12 Cap22 Rda12 Rdp12 (CR) Up · Iz2 · Iz1 · Fig. 4.2 Suggestions on adjacent line interference protection The existence of adjacent line interference greatly improves the probability of locomotive signal upgrading or code dropping, and increases the hidden danger of train operation. Based on the above theoretical analysis, the following measures can be taken to protect the engineering design. g g g (1) Try to avoid the configuration of the same carrier frequency between adjacent line 5 5 MATEC Web of Conferences 355, 02019 (2022) ICPCM2021 https://doi.org/10.1051/matecconf/202235502019 MATEC Web of Conferences 355, 02019 (2022) ICPCM2021 https://doi.org/10.1051/matecconf/202235502019 (2) Appropriately increase the line spacing of lines with the same carrier frequency; (3) The tuning area is staggered to reduce the parallel length; (3) The tuning area is staggered to reduce the parallel length; (4) The spacing of compensating capacitors is different to reduce the area of equivalent loop. (4) The spacing of compensating capacitors is different to reduce the area of equivalent loop. 5 Conclusion This paper focuses on the problem of electromagnetic interference between adjacent lines in multi-line parallel section of high-speed railway. The specific conclusions are as follows: (1) In the context of high-speed railway, the main factors causing adjacent line interference is rail mutual inductance coupling, which can reach 100mV under the most unfavorable conditions. (1) In the context of high-speed railway, the main factors causing adjacent line interference is rail mutual inductance coupling, which can reach 100mV under the most unfavorable conditions. (2) From the perspective of normal operation of cab equipment, it is suggested to increase line spacing and reduce the parallel length between lines to reduce the interference of adjacent lines. (2) From the perspective of normal operation of cab equipment, it is suggested to increase line spacing and reduce the parallel length between lines to reduce the interference of adjacent lines. References 1. Liu C , Yang S , Cui Y , et al. Optimization method of switch jumper setting based on strategies for reducing conductive interference in railway[J]. Proceedings of the Institution of Mechanical Engineers Part F Journal of Rail and Rapid Transit, 2020:095440972095130. 2. Liu C , Yang S , Cui Y , et al. Quantitative analysis on coupling of traction current into cab signaling in electrified railways[J]. Railway Engineering Science, 2020, 28(3):275- 289. 3. Lucca, Giovanni. Influence of railway line characteristics in inductive interference on railway track circuits[J]. IET Sci-ence, Measurement Technology, 2019. 4. E. M. Tarasov et al. A method for determination of the matrix coefficients of the A - parameters of a multipole that simulates the impact of the adjacent track circuit[J]. Russian Electrical Engineering, 2017, 88(3) : 99-104. 5. Manakov A D, Kudryavtsev V A, Os'Minin A T. Methods of determining the interference current in rails of double sub-way track circuits[J]. Russian Electrical Engineering, 2016, 87(5):282-285. 6. ZHAO Cuiqin, WU Xiaochun. Analysis of the Interference Effect of Rail Mutual Impedance on the Information Trans-mission of Adjacent Lines[J]. Journal of Lanzhou Jiaotong University, 2020, 039(001):73-79. (in Chinese) 7. LI Yuequan. Analysis and Solution of Same Frequency Interference Between Multi- line Parallel Sections[J]. Value Engineering, 2018,37(028):189-191. (in Chinese) 8. LIU Jialiang, BI Hongjun, YANG Shiwu etc. Simulation Research on Interference of Adjacent Section of ZPW-2000 Non-insulated Track Circuit[J]. Railway computer application, 2014,23(05):45-48. (in Chinese) 9. Ministry of Railways of the People's Republic of China. Technical Standards for Railway Signal Maintenance Rules I[M]. 3rd edition. Beijing: China Railway Press, 2014: 54-59. (in Chinese) 10. DU Xuelong, WANG Zhixin, ZOU Jun. Simplified formula for fast calculation of mutual impedance of high-speed rail-way track circuit considering the influence of the earth[J]. Journal of Electrotechnical Technology, 2016,31(04):1-6. (in Chinese) 6 6
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https://openalex.org/W2952606444	https://escholarship.org/content/qt880951pv/qt880951pv.pdf?t=pgsixu	English	null	Network Formation by Reinforcement Learning: The Long and Medium Run	Oxford University Press eBooks	2,014	cc-by	8,085	UC Irvine Copyright Information This work is made available under the terms of a Creative Commons Attribution License, availalbe at https://creativecommons.org/licenses/by/4.0/ Peer reviewed UC Irvine UC Irvine Previously Published Works Title Network formation by reinforcement learning: the long and medium run Permalink https://escholarship.org/uc/item/880951pv Journal Mathematical Social Sciences, 48(3) ISSN 0165-4896 Authors Pemantle, Robin Skyrms, Brian Publication Date 2004-11-01 DOI 10.1016/j.mathsocsci.2004.03.007 Copyright Information This work is made available under the terms of a Creative Commons Attr availalbe at https://creativecommons.org/licenses/by/4.0/ Peer reviewed UC Irvine UC Irvine Previously Published Works Title Network formation by reinforcement learning: the long and medium run Permalink https://escholarship.org/uc/item/880951pv Journal Mathematical Social Sciences, 48(3) ISSN 0165-4896 Authors Pemantle, Robin Skyrms, Brian Publication Date 2004-11-01 DOI 10.1016/j.mathsocsci.2004.03.007 Copyright Information This work is made available under the terms of a Creative Commons Attr availalbe at https://creativecommons.org/licenses/by/4.0/ Peer reviewed 1Research supported in part by National Science Foundation grant # DMS 0103635 2Department of Mathematics, The Ohio State University, 231 W. 18 Ave., Columbus, OH 43210 3School of Social Sciences, University of California at Irvine, Irvine, CA 92607 1Research supported in part by National Science Foundation grant # DMS 0103635 2D f M h i Th Ohi S U i i 231 W 18 A C l b 2Department of Mathematics, The Ohio State University, 231 W. 18 Ave., Columbus, OH 43210 3School of Social Sciences, University of California at Irvine, Irvine, CA 92607 Powered by the California Digital Library University of California eScholarship.org Network formation by reinforcement learning: the long and medium run Network formation by reinforcement learning: the long and medium run ork formation by reinforcement learning: the long and medium run ath/0404106v1 [math.PR] 5 Apr 2004 arXiv:math/0404106v1 [math.PR] 5 Apr 2004 Robin Pemantle 1,2 Brian Skyrms 3 Robin Pemantle 1,2 Brian Skyrms 3 arXiv:math/0404106v1 [math.PR] 5 arXiv:math/0404106v1 [math. 3School of Social Sciences, University of California at Irvine, Irvine, CA 92607 2Department of Mathematics, The Ohio State University, 231 W. 18 Ave., Colum 3 ABSTRACT: We investigate a simple stochastic model of social network formation by the process of reinforcement learning with discounting of the past. In the limit, for any value of the discounting parameter, small, stable cliques are formed. However, the time it takes to reach the limiting state in which cliques have formed is very sensitive to the discounting parameter. Depending on this value, the limiting result may or may not be a good predictor for realistic observation times. Keywords: absorption, discount, urn model, Friedman urn, Stag Hunt, urn, stochastic approxima- tion, meta-stable, trap, three-player game, potential well, exponential time, quasi-stationary. Subject classiﬁcation: Primary: 60J20 1 Introduction Each day, each member of a small group of individuals selects two others with whom to interact. The individuals are of various types, and their types determine the payoﬀto each from the interaction. That is to say that the interaction is modeled as a symmetric 3-person game. Probabilities of selecting individuals evolve by reinforcement learning, where the reinforcements are the payoﬀof the interaction. We consider two games. The ﬁrst is a degenerate game, “Three’s Company”. Here there is only one type and everyone gets equal reinforcement for every interaction. The analysis of “Three’s company” is then used in the analysis of a second game, a three-person Stag Hunt. Here there are two types, Stag Hunters and Hare Hunters. Hare Hunters always get a payoﬀof 3, while a Stag Hunter gets a payoﬀof 4 if he interacts with two other Stag Hunters, otherwise he gets nothing. The point of this modeling exercise is threefold. First, there is a substantial literature, reviewed in the next section, that compares learning models to laboratory data in order to make inferences about underlying psychological mechanisms for learning and strategy formation. Our model explores subgroup formation in a way that is informed by previous research on plausible mechanisms and parameter values. Secondly, the notion of modeling the co-evolution of interaction networks and strategies was set out in [SP00]. This begs the formulation and analysis of basic stochastic network models. To this end, we provide an analysis of two such models. Our analysis is suﬃciently robust so as to shed light on similar models, many of which have been studied in less depth. Finally, many of the models previously studied share a common mathematical description. We aim to provide a collection of rigorous results on this class of models, which will allow scientists quickly to understand the long- and medium-term behavior of these stochastic models. 2 Reinforcement learning and reinforcement processes Mathematically, a reinforcement process as deﬁned in the literature on reinforced random walks [CD97, Dav90, Dav99, Pem88, Pem92] need not have this Markov property. The current probability can depend on the history of choices and payoﬀs, via summary statistics or propensities associated to the possible actions. The probability vector is a function of all the propensities (though in general not a one-to-one function if the process is not Markovian). The possibility of using such processes to model reinforcement learning was introduced by Luce [Luc59]. Luce considered a range of models for the evolution of the propensities. The payoﬀs for an action taken might modify its propensity multiplicatively, additively, or in some combination. In Luce’s gamma model, the new propensity, v′(i), for an action i, is the sum of γi and the product of the old propensity and a factor, βi, v′(i) = βiv(i) + γi . where both βi and γi are functions of the payoﬀfor action i. Luce investigated his models using the linear response rule, ( ) p(i) = v(i) P j v(j) which simply normalizes the propensities. However the separation of the questions of propensity evolution and response rule opens the possibility of other alternatives such as the logistic response rule: (b (i)) which simply normalizes the propensities. However the separation of the questions of propensity evolution and response rule opens the possibility of other alternatives such as the logistic response rule: (b ( )) p(i) = exp(bv(i)) P j exp(bv(j)) p(i) = exp(bv(i)) P j exp(bv(j)) with b a learning parameter. This response rule is used in the learning models of Busemeyer and Townsend [BT93] and Camerer and Ho [CH99]. One advantage to the logistic response rule is that it allows deterrent reinforcement to be modeled, since probabilities are proportional to exponentials of prepoensities, thus remain positive when propensities are allowed to become negative. with b a learning parameter. This response rule is used in the learning models of Busemeyer and Townsend [BT93] and Camerer and Ho [CH99]. One advantage to the logistic response rule is that it allows deterrent reinforcement to be modeled, since probabilities are proportional to exponentials of prepoensities, thus remain positive when propensities are allowed to become negative. The ﬁrst expermental corroboration of these models, of which we are aware, was by Herrn- stein [Her70]. 2 Reinforcement learning and reinforcement processes Reinforcement learning, as the term is used in psychology, and reinforcement models, as used in applied mathematics, are not coextensive. We will model reinforcement learning by a certain kind of reinforcement process, following [Her70] and [RE95]. Reinforcement learning is to be contrasted to cognitive or belief-based learning. In the latter, each agent is assumed to have some kind of internal model for the other agents and any unknown parameters. Observations alter an agent’s beliefs, which then govern the agent’s future actions. In a reinforcement learning model, observations aﬀect future actions through a similar kind of change of the agent’s state, but the translation of observations into propensities for action are not assumed to be mediated by anything capable of high-level processing. Instead, it is assumed that the agent’s actions are governed by previous observations in a simple and explicit way. One may view this as an exreme form of bounded rationality. This kind of model is natural when modeling agents with less advanced brains than humans have (e.g., slime molds [OS97]), but has also been proposed for humans in situations of low information. Stochastic models of reinforcement learning were introduced by Estes [Est50] and by Bush and Mosteller [BM55]. On each of a series of trials the learning agent chooses between a ﬁnite set of alternative acts, and gets a payoﬀ. The choice is assumed to be governed by a probability vector that evolves according to prescribed learning dynamics. In the learning dynamics investigated by Estes and by Bush and Mosteller, the new probability vector after a trial is a weighted average of the previous probability vector and the vector putting unit weight on the act just chosen. The weight on the unit vector is the product of the payoﬀand a learning rate parameter. This kind of learning 1 satisﬁes what Bush and Mosteller call independence of path: the probaiblity vector at time n + 1 depends only on the probability vector at time n, the act chosen, and the magnitude of the payoﬀ. As a consequence, if the payoﬀs to the acts are ﬁxed, the learning process is a Markov chain with the probability vector as its state. Some Markov models for learning have been ﬁrmly established in the psychology literature, appearing in survey texts by the early 1970’s [IT69, Nor72]. 2 Reinforcement learning and reinforcement processes Thorndike had previously proposed the “Law of Eﬀect”, which Herrnstein quantiﬁes as the “Matching Law”: the probability of choosing an action is proportional to the accumulated rewards. Let propensities evolve by adding payoﬀs, that is, βi = 1 and γi is zero if the action i was not taken, and is otherwise equal to the payoﬀ. If we follow the linear response rule, we obtain Herrnstein’s matching law. Herrnstein reports data from laboratory experiments with humans as well as with animals, from which one may conclude the broad applicability of the model. There is a special case whose limiting behavior is well known. If each action is equally reinforced, the process is mathematically equivalent to P´olya’s urn process [EP23], with each action represented by a diﬀerent color of ball initially in the urn. The process converges to a random limit, whose support is the whole probability simplex. In other words, any limiting state of propensities or probabilities is possible. In 1960, Suppes and Atkinson [SA60] introduced interactive reinforcement to model learning behavior in games. A number of players choose between alternatives as before, but the payoﬀs to 2 each player now depend on the acts chosen by all players. Players modify their choice probabilities by reinforcement learning dynamics of the Bush-Mosteller type. If joint actions ﬁx the payoﬀs and we take the state of the system to be the vector, indexed by players, of vectors of choice probabilities, then the dynamics will be Markovian. Insofar as multi-agent reinforcement learning has been studied, it has been largely in the frame- work of Suppes and Atkinson. Macy [Mac90, Mac91] applies multi-player reinforcement to study collective action problems from a bounded rationality viewpoint. Borgers and Sarin [BS97] draw a connection between multi-agent Bush-Mosteller dynamics and the replicator dynamics of evolu- tionary game theory [MS82], showing that the two coincide in a certain limit. Perhaps the greatest impulse to this direction of study was the widely cited 1995 paper of Roth and Erev [RE95]. They proposed a multi-agent reinforcement model based on Herrnstein’s linear reinforement and response. Here and in subsequent publications [ER98, BE98], they show a good ﬁt with a wide range of em- pirical data. Limiting behavior in the basic model has recently been studied by Beggs [Beg02] and by Ianni [Ian02]. In [SP00], both basic and discounted versions of Roth-Erev learning are applied to social network formation. 2 Reinforcement learning and reinforcement processes Individuals begin with prior propensities to interact with each other, and interactions are modeled as two-person games. Individuals have given strategies, and interactions between indi- viduals evolve by reinforcement learning. The analysis begins with a series of results on “Making Friends”, a network formation model in the special case where the game interaction is trivial. Non- trivial strategic interaction is then introduced, and it is shown that the co-evolution of network and strategy depends on relative rates of evolution as well as on other features of the model. The present work is a natural outgrowth of the investigations begun in [SP00]. In the richer context of multi-agent interactions, more phenomena arise, namely clique formation and a meta- stable state of high network connectivity for an initial epoch whose length depends dramatically on the discounting parameter. In Section 5.3 we discuss the implications of these features for a wide class of models. 3 Mathematical background Our ultimate goal is to understand qualitative phenomena such as clique formation, or tendency of the interaction frequencies toward some limiting values. The mathematical literature on reinforce- ment processes contains results in these directions. It will be instructive to review these, and to examine the mathematical classiﬁcation of such processes, although we will need to go beyond this level of analysis to explain the behavior of network models such as Three’s Company on timescales we can observe. Reinforcement processes fall into two main types, trapping and non-trapping. A process is said to be trapping if there are proper subsets of actions for each player such that there is a positive probability that all players always play from this subset of actions. For example, if the repetition of any single vector (i) of actions (action ij for player j) is suﬃciently self-reinforcing that it might cause action i to be perpetuated forever, then the process is trapping. The speciﬁc dynamics investigated by Bush and Mosteller in 1955 are trapping, as are most logistic response models. By contrast, models that give all times in the past an equal eﬀect on the present, such as Herrnstein’s dynamics and Roth-Erev dynamics, tend not to be trapping. 3 One of several modiﬁcations suggested by Roth and Erev to maximize agreement of their model with the data is to introduce a discounting parameter x ∈(0, 1). The past is discounted via multiplication by a factor of (1 −x) at each step. Formally, this is a version of Luce’s gamma model with βi = 1 −x for all i. It is known from the theory of urn processes that discounting may cause trapping. For example, it follows from a theorem of H. Rubin reported in [Dav90] that if P´olya’s urn is altered by discounting the past, there will be a point in time beyond which only one color is ever chosen. This holds as well with Roth-Erev type models: the discounted Roth-Erev model is trapping, while the undiscounted model is not. In [SP00], discounted and nondiscounted versions of several games are studied, and equilibria examined for stability. Again, discounting causes trapping, and we investigate the robustness of the trapping when the discounting parameter becomes negligible. In a related paper, Bonacich and Liggett [BL02] investigate Bush-Mosteller dynamics in a two-person interaction representing gift giving. Their model has discounting, and they ﬁnd a set of trapping states. 3 Mathematical background It is in general an outstanding problem in the theoretical study of reinforcement models to show that trapping must occur with probability 1 if it occurs with positive probability. This was only recently proved, for instance, for the reinforced random walk on a graph with three vertices, via a complicated argument [Lim01]. Much of the eﬀort that has gone into the mathematical study of these models has been directed at these diﬃcult limiting questions. In the non-trapping case, even though the choice of action does not ﬁxate, the probaiblities for some of the actions may tend to zero. A series of papers in the 1990’s by Benaim and others [BH95, Ben98, Ben99] establishes some basic tests for whether in undiscounted Roth-Erev type models, probabilties will tend toward determinstic vectors. From the point of view of applications, in a situation where it can be proven or surmised that trapping occurs, we are mainly interested in characterizing the states in which we may become trapped and in determining how long will it be before the process becomes trapped. Recalling our initial discussion of modeling goals, we are particularly interested in results that are robust as parameters and modeling details vary, or when they are not robust, of understanding how these details of the model aﬀect observed qualitative behavior. 4.1 Speciﬁcation of the model The game “Three’s Company” models collaboration of trios of agents from a ﬁxed population. At each time step, each agent selects two others with whom to form a temporary collusion. An agent may be involved in multiple collusions during a single time step: one that she initiates, and zero or more initiated by another agent. Analogously to the basic game “Making Friends”, introduced in [SP00], Three’s Company has a constant reward structure: every collaboration results in an identical positive outcome, so every agent in every temporary collusion increases by an identical amount her propensity to choose each of the other two agents in the trio. The choice probabilities follow what could be called mulitlinear response. The probability of an agent choosing to form a trio with two other agents i and j is taken to be proportional to the product of her propensity for i with her propensity for j. In addition to providing a model for self-organization based on a simple matching law type of response mechanism, this model is meant to provide a basis for the analysis 4 of games such as the three person stag hunting game discussed in the next section. We now give a more formal mathematical deﬁnition of Three’s Company, taken from [PS03a]. Fix a positive integer N ≥4, representing the size of the population. For t ≥0 and 1 ≤i, j ≤N, deﬁne random variables W(i, j, t) and U(i, t) inductively on a common probability space (Ω, F, P) as follows. The W variables are positive numbers, and the U variables are subsets of the population of cardinality 3. One may think of the U variables as random triangles in the complete graph with a vertex representing each agent. The variable U(i, t) is equal to the trio formed by agent i at time t. The W variables represent propensities: W(i, j, t) will be the propensity for player i to choose player j on the time step t. The initialization is W(i, j, 0) = 1 for all i ̸= j, while W(i, i, 0) = 0). We write W(e, t) for W(i, j, t) when e is the edge (unordered set) {i, j} (note that the evolution rules below imply that W(i, j, t) = W(j, i, t) for all i, j and t). 4.1 Speciﬁcation of the model The inductive step, for t ≥0, deﬁnes probabilities (formally, conditional probabilities given the past) for the variables U(i, t) in terms of the variables W(r, s, t), r, s ≤N, and then deﬁnes W(i, j, t + 1) in terms of W(i, j, t) and the variables U(r, t), r ≤N. The equations are: P(U(i, t) = S \| Ft) = 1i∈S Q r,s∈S,r<s W(r, s, t) P S′:i∈S′ Q r,s∈S′,r<s W(r, s, t) ; (4.1) W(i, j, t + 1) = (1 −x)W(i, j, t) + N X r=1 1i,j∈U(r,t) . (4.2) (4.1) (4.2) Here (1 −x) is the factor per unit time by which the past is discounted, and the σ-ﬁeld conditioned on is the process up to time t, Here (1 −x) is the factor per unit time by which the past is discounted, and the σ-ﬁeld conditioned on is the process up to time t, Ft := σ {W(i, j, u) : u ≤t} . The following alternative statement of the evolution equation (4.1) is useful for those familiar with the analytic machinery (c.f [Pem92]) that is typically used to reduce such a process to a stochastic approximation. Think of the normalized matrix Wt := 1 P i,j W(i, j, t) W(·, ·, t) as the state vector. This is then an asymptotically time-homogeneous Markov chain, with an evolu- tion rule E (Wt+1 −Wt \| Ft) = g(t) [µ(Wt) + ξt] (4.3) (4.3) where g(t) = 1/x + O(1/t), the drift vector ﬁeld µ maps the simplex of normalized matrices into its tangent space and may be explicitly computed, and ξt are martingale increments of order 1. In the non-discounted case, g(t) = 1/t+O(1/t), and much information about the long term behavior of this process can be discovered by an analysis of the the ﬂow dX/dt = µ(X) [Ben99]. In the discounted case, g(t) does not go to zero and an alternative analysis is required. Equations (4.1) and (4.2) completely specify the model for the given parameters N and x. Simula- tions for a population of size 6 (N = 6) showed the following behavior. 4.2 Analysis of the model Equations (4.1) and (4.2) completely specify the model for the given parameters N and x. Simula- tions for a population of size 6 (N = 6) showed the following behavior. 5 When x = .5 (a rather steep discount rate, though not unheard of in psychological laboratory experiments [BS02]), all 1,000 trials broke up into two cliques of size 3, with no interactions across clique boundaries. In larger populations, with the same discount rate, again decomposition into cliques occurs, this time of sizes 3, 4 and 5, whose members interact exclusively with other members of the same clique. When N = 6 and x = .4 we found that 994 out of the 1,000 trials had decomposed into two cliques of three (we allowed the process to continue for 1,000,000 time steps). When x was decreased to .3, only 13 of the 1,000 trials showed decomposition into cliques, while in the remainder of the trials all six members of the population remained well connected through the 1,000,000 time steps. Finally, when x = .2, a reasonable discount rate for individuals though still steeper than in most economic models, none out of 1,000 trials had broken into cliques. All six members of the population remained well connected after 1,000,000 time steps. To summarize the simulation data, high discount rates lead to trapping, with each agent re- stricting her choices to members of a clique of size 3 (or, in larger populations, size 4 or 5). Less steep discount rates lead to less trapping or no trapping at all. Interestingly, the simulation data is contradicted by the following theorem, proved in the appendix. Theorem 4.1 In Three’s Company, with any population size N ≥6 and any discount rate x ∈(0, 1), with probability 1 the population may be partitioned into subsets of sizes 3, 4 and 5, such that each member of each subset chooses each other with positive limiting frequency, and chooses members outside the subset only ﬁnitely often. Every partition into sets of sizes 3, 4 and 5 has positive probability of occurring. In other words, despite the simulation data, trapping always occurs. The set of traps is the set of all ways of decomposing into cliques of sizes 3, 4 and 5. The apparent contradiction between the simulation and the theorem is resolved by Theorem 4.2, whose proof is given in the companion paper [PS03a]. 4.2 Analysis of the model The theorem states that the time for the population to break into cliques increases exponentially in 1/x as the discount rate 1 −x increases to 1. Theorem 4.2 For each N ≥6 there is a δ > 0 and numbers cN > 0 such that in Three’s Company with N players and discount rate 1 −x, the probability is at least δ that each player will play with each other player beyond time exp(cNx−1). □ 4In this model, since any number of collusions is permitted for an agent on each time step, the sub-optimality is manifested not through wasted time on the stag hunter’s part. Instead, it is a societal opportunity cost, borne by all the rabbit hunters passed over in favor of the stag hunter. 5.1 Speciﬁcation of the model (5.4) (5.4) The factor in front of the last sum is 2 rather than 4 because the sum counts the trio {q, r, s}, chosen by agent q, exactly twice: as (q, r, s) and as (q, s, r). 5.1 Speciﬁcation of the model We now replace the uniformly positive reward structure by a nontrivial game, which is a three player version of Rousseau’s Stag Hunt. For the purposes of our model, agents are divided into two types, hare hunters and stag hunters. That is, we model strategic choice as unchanging, at least on the time scale where network evolution is taking place. No matter which other two agents a hare hunter goes hunting with, the hare hunter comes back with a hare (hares can be caught by individuals). A 6 stag hunter, on the other hand, comes home empty-handed unless in a trio of three stag hunters, in which case each comes home with one third share of a stag. One third of a stag is better than a whole hare, but evidently riskier because it will not materialize if any member of the hunting party decides to play it safe and focus attention on bagging a hare. In the three player stag hunting game, as in Three’s Company, at each time step each agent chooses two others with whom to form a collusion. The payoﬀs are as follows. Whenever a hare hunter is a member of a trio, his reward is 3. A stag hunter’s reward is 4 if in a trio of three stag hunters and 0 otherwise. A formal model is as follows. Let N = 2n be an even integer representing the size of the population and let x ∈(0, 1) be the discount parameter. The variables {W(i, j, t), U(i, t) : 1 ≤i, j ≤N; t ≥0} are deﬁned again on (Ω, F, P) with the W variables taking positive values and representing propensities and the U variables taking values in the subsets of {1, . . ., N} of cardinality 3 and representing choices of trios. We initialize the W variables by W(i, j, 0) = 1 −δij, just as before, and we invoke a linear response mechanism (4.1) just as before. Now, instead of the trivial reward structure (4.2), the propensities evolve according to the hunting bounties W(i, j, t + 1) = (1 −x)W(i, j, t) + 31i≤n N X r=1 1i,j∈U(r,t) +21i>n N X q,r,s=n+1 1i∈U(q,t)={q,r,s} . 5.2 Analysis of the model The propensities for stag hunters to choose rabbit hunters remain at their initial values, whence stag hunters choose other stag hunters with limiting probability 1. The stag hunters are never aﬀected by the rabbit hunters’ choices, so the stag hunters mimic Three’s Company among themselves precisely except for the times, numbering only O(log t) by time t, when they choose rabbit hunters. We know therefore, that eventually they fall into cliques of size 3, 4 and 5, but that this will take a long time if the discount parameter is small. Rabbit hunters may form cliques of size 3, 4 and 5 as well, but because they are rewarded for choosing stag hunters, they may also attach to stag hunters. The chosen stag hunters have cliques of their own and ignore the rabbit hunters, except during the times that they are purposelessly called to hunt with them. These attachements can be one rabbit continually calling on a particular pair of stags or two rabbits continually calling on a single stag. In either case the one or two rabbits are isolated from all hunters other than their chosen stag hunters. What matters here is not the details of the trapping state but the time scale on which the trap forms and the likelihood of a rabbit hunter ending up in a sub-optimal trap4. This likelihood 4In this model, since any number of collusions is permitted for an agent on each time step, the sub-optimality is manifested not through wasted time on the stag hunter’s part. Instead, it is a societal opportunity cost, borne by all the rabbit hunters passed over in favor of the stag hunter. 7 decreases as the discount rate becomes small for the following reason. Rabbit hunters choosing to hunt with stag hunters are getting no reciprocal invitations, whereas any time they choose to hunt with other rabbit hunters, their mutual success creates a likelihood of future reciprocal invitations. These reciprocal invitations are then successful and increase the original hunter’s propensity for choosing the other rabbit hunter. Thus, on average, propensity for a rabbit hunter to form a hunting party with other rabbit hunters will increase faster than propensity to call on stag hunters, and the relative weights will drift toward the rabbit-rabbit groupings. 5.2 Analysis of the model The smaller the discount parameter, x, the more chance this has to occur before a chance run of similar choices locks an agent into a particular clique. Simulations show that stag hunters ﬁnd each other rapidly. With 6 stag hunters and six rabbit hunters and a discount rate of .5, the probability that a stag hunter will visit a rabbit hunter usually drops below half a percent in 25 iterations. For 50 iterations of the process this always happened in 1000 trials, and this remains true for values of x between .5 and .1. For x=.01, 100 iterations of the process suﬃces for stag hunters to meet stag hunters at this level and for 200 iterations are enough when x=.001. Rabbit hunters ﬁnd each other more slowly, except when they are frozen into interactions with stag hunters. When the past is heavily discounted the latter possibility a serious one. At x=.5, at least one rabbit hunter interacted with a stag hunter (after 10,000 iterations) in 384 of 1,000 trials. This number dropped to 217 for x=.4, 74 for x=.3, 6 for x=.2, and 0 for x=.1. Reliable clique formation among stag hunters is much slower in line with results of the last section, taking about 100,000 iterations for x=.5 and 1,000,000 iterations for x=.4. 6 Conclusion Our analysis reinforces the emphasis of Suppes and Atkinson, and of Roth and Erev, on the medium run for empirical applications. Long run limiting behavior may simply never be seen. It is useful to quantify the time scale on which we can expect medium run behavior to persist, and Theorem 4.2 is meant to serve as a prototypical result in this direction. Indeed, Theorem 4.2 is proved via a stronger result [PS03a, Theorem 4.1], which applies to many trapping models as the discount rate becomes negligible. As to the nature of the medium run behavior, analyses tend to be model-dependent. 5.3 Further discussion The two models discussed in this paper are highly idealized. But from these, we can learn some general principles as to how to analyze a much wider class of models. The ﬁrst principle is that when x is near zero, the process should for a long time behave similarly to the non-discounted process (x = 0). Here, following [Ben99], one must ﬁnd equlibria for the ﬂow dX/dt = µ(X), and classify these as to stability. Unstable equilibria, in general, do not matter (though see [PS03b] for cases in which the eﬀects of unstable equilibria may last quite a while). Stable equilibria may be possible trapping states, or may not be. The interesting case is when a stable equilibrium for the non-discounted process is not a possible trapping state for the discounted process. In this case, the process may get pseudo-trapped there, that is, may remain there for a very long time. Just how long will depend on the model, though Theorem 4.2 extends rather robustly to a broader class of linearly stable states (for the non-discounted process) that are non-trapping for the discounted process. Another mathematical technique relevant to these analyses, which we have not yet tried to apply, is quasi-stationary analysis. Recall that equation (4.3) describes and asymptotically time- homogeneous Markov chain. If there is trapping, this chain is not ergodic. A chain that is not ergodic may be conditioned to stay in a set of transient states. The stationary measure of the conditioned chain is called a quasi-stationary measure for the original chain. The study of these was begun in the 1960’s by Seneta and others (see, e.g., [DS65]) and there is now an extensive literature. In particular, it is sometimes possible to understand the time scale on which the process leaves the transient states. 8 8 7 Appendix: proof of Theorem 4.1 Let G(t) be the graph whose edges are all e such that e ⊆U(i, t) for some i, that is, the set of edges whose weights are increased from time t to time t + 1. The following two easy lemmas capture some helpful estimates. Lemma 7.1 (i) X e W(e, t) →3Nx−1 Lemma 7.1 (i) Lemma 7.1 (i) X e W(e, t) →3Nx−1 exponentially fast as t →∞. exponentially fast as t →∞. (ii) If e ∈G(t) then (ii) If e ∈G(t) then W(e, t + k) ≥(1 −x)k−1 . W(e, t + k) ≥(1 −x)k−1 . Proof: The ﬁrst part is a consequence of the equation for the evolution of the total weight: X e W(i, t + 1) = (1 −x) X e W(e, t) + 3N . The second part follows from the ﬁrst, and from the fact that when e ∈G(t) then W(e, t + 1) ≥1 and hence W(e, t + k) ≥(1 −x)k−1. □ Let G denote the transitive (irreﬂexive) closure of a graph G; thus G is the smallest disjoint union of complete graphs that contains G. Lemma 7.2 There are constant c, depending on N and x, such that with probability at least c, every edge e ∈G(t) satisﬁes W(e, t + N 2) ≥(1 −x)N 2 . (7.1) (7.1) Proof: Let H be a connected component of G(t). Fix a vertex v ∈H and let w be any other vertex of H. There is a path from v to w of length at most N; denote this path (v = v1, v2, . . . , vr = w). If r = 2 then the inequality (7.1) for e ∈G(t) follows from Lemma 7.1. If r ≥3, we let E(H, v, w, 1) be the event that for every 2 ≤j ≤r −1, the edge {vj−1, vj+1} is in G(t + 1). Since this event contains the intersection over r of the events that U(vj, t) = {vj, vj−1, vj+1}, since Lemma 7.1 bounds each of 9 these probabilities from below, and since the events are conditionally independent given Ft, we have a lower bound on the probability of E(H, v, w, 1). In general, for 1 ≤k ≤r−2, let E(H, v, w, k) be the event that for every 2 ≤j ≤r −k, the edge {vj−1, vj+k is in G(t + k). Lemma 7.1 (i) We claim that conditional on E(H, v, w, l) for all l < k, the conditional probability of E(H, v, w, k) given Ft+k−1 can be bounded below: inductively, Lemma 7.1 bounds from below the product of W(vj, vj−1, t)W(vj, vj+k, t), and hence the probability that U(vj, t) = {vj, vj−1, vj+k}; these conditionally independent probabilities may then be multiplied to prove the claim, with the bound depending only on x and N. ¿From this argument, we see that the intersection E(H, v, w) := T 1≤k≤r−2 E(H, v, w, k) has a probability which is bounded from below. Sequentially, we may choose a sequence of values for w running through all vertices of H at some distance r(w) −1 ≥2 from v, measured in the metric on H. For each such w, we can bound from below the probability that in r −2 more time steps the path from v to w will be transitively completed. We denote these events E′(H, v, w), the prime denoting the time shift to allow events analogous to E(H, v, w) to occur sequentially. Summing the time to run over all w ∈H yields at most N 2 time steps. Let E(H, v) denote the intersection of all the events E′(H, v, w). Inductively, we see that the probability of E(H, v) is bounded from below by a positive number depending only on N and x. Finally, we let (H, v) vary with H exhausting components of G(t) and v a choice function on the vertices of H. The events E(H, v) are all conditionally independent given Ft, so the probability of their intersection, E, is bounded from below by a positive constant which we call c. By Lemma 7.1 once more, on E, we know that (7.1) is satisﬁed for each e ∈G(t). □ Proof of Theorem 4.1: For any subset V of agents, let E(V, t) := \ s≥t \ v∈V,w∈V c {{v, w} /∈G(s)} denote the event that from time t onward, V is isolated from its complement. If V is the vertex set of a component of G(t), then the conditional probability given Ft of the event E(V, t) may be bounded from below as follows. Lemma 7.1 (i) For any v ∈V, w ∈V c, and for any s ≥t, if the edge e := {v, w} is not in G(r) for any t ≤r < s, then by part 1 of Lemma 7.1, its weight W(e, s) is at most (1 −x)s−t3Nx−1. Since P z W(v, z, s) ≥2 for all v, z, s, it follows from the evolution equations that denote the event that from time t onward, V is isolated from its complement. If V is the vertex set of a component of G(t), then the conditional probability given Ft of the event E(V, t) may be bounded from below as follows. For any v ∈V, w ∈V c, and for any s ≥t, if the edge e := {v, w} is not in G(r) for any t ≤r < s, then by part 1 of Lemma 7.1, its weight W(e, s) is at most (1 −x)s−t3Nx−1. Since P z W(v, z, s) ≥2 for all v, z, s, it follows from the evolution equations that P(e ∈G(s) \| Fs) ≤ (1 −x)s−t3Nx−1 2 + (1 −x)s−t3Nx−1 . It follows that Then with probability at least (1 −x)N 2 3N + (1 −x)N 2 !\|J\|+\|K\| (1 −x)N 2 3N + (1 −x)N 2 !\|J\|+\|K\| U(i, t + N 2) ⊆J for every i ∈J and U(i, t + N 2) ⊆K for every i ∈K. In this case, G(t + N 2) has components that are proper subsets of H. By the martingale convergence theorem, U(i, t + N 2) ⊆J for every i ∈J and U(i, t + N 2) ⊆K for every i ∈K. In this case, G(t + N 2) has components that are proper subsets of H. By the martingale convergence theorem, P(H is a component of G∞\| Ft) converges with probability 1 to the indicator function of H being a component of G∞. From the above computation, it is not possible for P(H is a component of G∞\| Ft) to converge to 1 when H has cardinality six or more. Therefore, every component of G∞has cardinality 3, 4 or 5. The rest of the proof is easy. Let V1, . . . , Vk be any partition of [N] into sets of cardinalities 3, 4 and 5. The derivation of (7.2) shows that P   k\ j=1 E(Vj, 1)  > 0 , in other words, with positive probability G∞has k components which are precisely the complete graphs on V1, . . . , Vk. It is elementary that a coupling may be produced between the Three’s Company processes on populations of sizes N and K < N (with the same x value), so that if { ˜W(i, j, t), ˜U(i, t)} are the weight and choice variables for the smaller population, then ˜U(i, t) = U(i, t) and ˜ W(i, j, t + 1) = W(i, j, t + 1) for all t < τ where τ is the ﬁrst time, possibly inﬁnite, at which U(i, t) contains an edge between [K] and {K + 1, . . . , N}. In general, coupling methods show that if P(G∞= G0 \| t) > 1 −ǫ then the conditional distribution of the Three’s Company process from time t onward given Ft and G∞= G0, shifted back t time units and restricted to a component H of G∞, is within ǫ in total variation of the distribution of the Three’s Company process on H started with initial weights W ′(i, j, 0) := W(i, j, t). in other words, with positive probability G∞has k components which are precisely the complete graphs on V1, . . . , Vk. It follows that P (∃v ∈V, w ∈V c : {v, w} ∈G(s) \| Fs) = O Nx−1(1 −x)s−t uniformly in N, x and t as s −t →∞(though the uniformity in N and x is not needed). By the conditional Borel-Cantelli Lemma, it follows that uniformly in N, x and t as s −t →∞(though the uniformity in N and x is not needed). By the conditional Borel-Cantelli Lemma, it follows that P(E(V, t) \| Ft) > c(N, x) (7.2) P(E(V, t) \| Ft) > c(N, x) (7.2) t of a component of G(t). (7.2) on the event that V is the vertex set of a component of G(t). on the event that V is the vertex set of a component of G(t). By the reverse direction of the Conditional Borel-Cantelli Lemma, the event E(V, t) occurs for some t with probability 1 on that event that V is a component of G(t) inﬁnitely often. Let e = {v, w} be any edge. If e /∈G(t) inﬁnitely often, then since there are only ﬁnitely many subsets of vertices, 10 it follows that v ∈V and w ∈W for some disjoint V and W that are inﬁnitely often components of G(t). This implies that e ∈G(t) ﬁnitely often. We have shown that, almost surely, the edges come in two types: those in G(t) ﬁnitely often and those in G(t) all but ﬁnitely often. This further implies that G(t) is eventually constant. Denote this almost sure limit by G∞. It remains to characterize G∞. it follows that v ∈V and w ∈W for some disjoint V and W that are inﬁnitely often components of G(t). This implies that e ∈G(t) ﬁnitely often. We have shown that, almost surely, the edges come in two types: those in G(t) ﬁnitely often and those in G(t) all but ﬁnitely often. This further implies that G(t) is eventually constant. Denote this almost sure limit by G∞. It remains to characterize G∞. It is evident that G∞contains no component of size less than three, since G(t) is the union of triangles U(i, t). Suppose that G(t) = H for some H of cardinality at least six. By Lemma 7.2, conditional on Ft and G(t) = H, W(e, t + N 2) ≥(1 −x)N 2 3N for every e ∈H. Write H as the disjoint union of sets J and K, each of cardinality at least three. P(H is a component of G∞\| Ft) It is elementary that a coupling may be produced between the Three’s Company processes on populations of sizes N and K < N (with the same x value), so that if { ˜W(i, j, t), ˜U(i, t)} are the weight and choice variables for the smaller population, then ˜U(i, t) = U(i, t) and ˜ W(i, j, t + 1) = W(i, j, t + 1) for all t < τ where τ is the ﬁrst time, possibly inﬁnite, at which U(i, t) contains an edge between [K] and {K + 1, . . . , N}. In general, coupling methods show that if P(G∞= G0 \| t) > 1 −ǫ then the conditional distribution of the Three’s Company process from time t onward given Ft and G∞= G0, shifted back t time units and restricted to a component H of G∞, is within ǫ in total variation of the distribution of the Three’s Company process on H started with initial weights W ′(i, j, 0) := W(i, j, t). 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https://openalex.org/W2895798859	https://europepmc.org/articles/pmc6308728?pdf=render	English	null	Magnitude of Cryptococcal Antigenemia among HIV Infected Patients at a Referral Hospital, Northwest Ethiopia	Ethiopian journal of health sciences	1,970	cc-by	3,606	KEYWORDS; Cryptococcal antigenemia, IMMY CrAg® LFA, Bahir Dar, Ethiopia Awoke D. et al. 369 Awoke D. et al. 369 Magnitude of Cryptococcal Antigenemia… 369 OPEN ACCESS BACKGROUND: Cryptococcosis is one of the common opportunistic fungal infections among HIV infected patients living in Sub-Saharan Africa, including Ethiopia. The magnitude of the disease at Felege Hiwot Referral Hospital (FHRH) in particular and in Ethiopia at large is not well explored. Citation: Awoke Derbie, Workineh Ayalew, Daniel Mekonne, Megbaru Alemu, Yihun Mulugeta. Magnitude of Cryptococcal Antigenemia among HIV Infected Patients at a Referral Hospital, Northwest Ethiopia. Ethiop J Health Sci.2018; 28(4): 369. Northwest Ethiopia. Ethiop J Health Sci.2018; 28(4): 369. METHODS: A retrospective document review and analysis was done on records of 137 HIV infected patients who visited FHRH ART clinic from 1 Sep to 30 Dec 2016 and had registered data on their sex, age, CD4 count and cryptococcal antigen screening result. The cryptoccocal antigen (CrAg) detection was done by the IMMY CrAg® LFA (Cryptococcal Antigen Lateral Flow Assay) kit from patient serum as per the manufacturer’s instruction. All data were entered, cleared, and analyzed using SPSS v20. Descriptive data analysis and cross tabulation were done to assess factors associated with cryptococcal antigenemia. Statistical significance was set at p-value less than or equal to 0.05. doi: http:// dx. doi. org/10.4314/ejhs.v28i4.2 Received: December 20, 2017 doi: http:// dx. doi. org/10.4314/ejhs.v28i4.2 Received: December 20, 2017 Accepted: December 23, 2017 Published: July 1, 2018 y Copyright: © 2018 Awoke Derbie, 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 Awoke Derbie, et al . This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Nil Funding: Nil INTRODUCTION in sub-Saharan Africa, between 35%-65%. This compares with 10%-20% in most developed countries. The main reason for this is a delay in presentation with diagnosis only when meningitis is advanced and treatment is less effective, mainly as a result of limited access to lumbar puncture (LP) and rapid diagnostic assays (1). Increasing access to antiretroviral therapy (ART) has transformed the prognosis of HIV infected patients in resource-limited settings. However, treatment coverage remains relatively low, and HIV diagnosis occurs at a late stage. As a result, many patients continue to die of HIV-related opportunistic infections (OIs) in the weeks prior to, and months following initiation of ART (1, 2). Cryptococcus neoformans, a causative agent of cryptococcosis remain a common cause of infectious morbidity and mortality, especially among HIV-positive patients living in Sub- Saharan Africa (3). Cryptococcal disease is one of the most important OIs, and a major contributor to this early mortality, accounting for between 13% and 44% of deaths among HIV infected people living in developing nations. In sub-Saharan Africa alone, there are more than 500,000 deaths each year due to cryptococcal meningitis (CM), which may exceed those attributed to tuberculosis (1). There are three categories of methods that can be used to diagnose cryptococcal meningitis: India Ink microscopy, which can be used on cerebrospinal fluid (CSF); culture, which can be used on CSF or blood; and antigen detection. There are several methods to detect cryptococcal antigen in CSF or serum: latex agglutination (LA), enzyme immunoassay (EIA), and lateral flow assay (LFA) (6-8). A patient who tests positive for cryptococcal antigen can take oral fluconazole to help the body fight the early stage of the infection. This could prevent the infection from developing into meningitis (9). In Ethiopia, where there is poor surveillance system, there are few studies conducted on the level of cryptococcal infection and its associated risk factors (2,10-11). However, the data is missing in our study site where there are a number of HIV patients getting ART service in accordance with the national ART program. Some HIV patients in the study area get tested for cryptococcal infection during follow-up when there is suggestive clinical presentations. Thus, the aim of this study was to assess the prevalence of cryptococcal antigenemia and its associated factors among HIV infected patients at Felege Hiwot Referral Hospital (FHRH) setting. Funding: Nil Competing Interests: The authors declare that this manuscript was approved by all authors in its form and that no competing interest exists. RESULTS: More than half of the participants, 54.7% (75/137), included in the study were females. The median age of the participants was 32.0 years (ranged: 8-52 years). The mean CD4 count was 51.8 with SD of 26.3 (range 3-98). All the patients were HIV stage IV. The proportion of positive cryptococal antigen from serum test was at 11.7% (95% CI: 7.3-18.1%). The IMMY CrAg® LFA result was found statically associated with patient sex (p= 0.045). However, it was not associated with patient age group and the CD4 count (P>0.05) Affiliation and Correspondence: 1Department of Medical Microbiology, Immunology and Parasitology, College of Medicine and Health Sciences, Bahir Dar University, Bahir Dar, Ethiopia 2CDT-Africa, Addis Ababa University, Ethiopia 3Felege Hiowot Referral Hospital, Bahir Dar, Ethiopia 4Biotechnology Research Institute, Bahir Dar University, Ethiopia 5Department of Epidemiology and Biostatstics, College of Medicine and Health Sciences, Bahir Dar University, Bahir Dar, Ethiopia Email: awe.love2000@gmail.com Affiliation and Correspondence: 1Department of Medical Microbiology, Immunology and Parasitology, College of Medicine and Health Sciences, Bahir Dar University, Bahir Dar, Ethiopia 3Felege Hiowot Referral Hospital, Bahir Dar, Ethiopia 4 4Biotechnology Research Institute, Bahir Dar University, Ethiopia 5 CONCLUSIONS: This study provided baseline data on the magnitude of cryptococcal antigenemia among HIV positive patients that is not touched before in the studied area. The results of the study showed that this opportunistic fungal infection is an important health concern among HIV patients. Further studies with sound design employing adequate sample size should be considered. 5Department of Epidemiology and Biostatstics, College of Medicine and Health Sciences, Bahir Dar University, Bahir Dar, Ethiopia Email: awe.love2000@gmail.com DOI: http://dx.doi.org/10.4314/ejhs.v28i4.2 Vol. 28, No. 4 July 2018 Ethiop J Health Sci. 370 INTRODUCTION Cryptococcus is a type of fungus that lives in soil, especially soil that is contaminated with large amounts of bird droppings. Some people inhale the spores from the environment and never get sick, but in people with weak immune systems, the fungus can cause an infection. The only way a person can get sick from this fungus is by directly inhaling the agent from the environment, making it no person to person transmission (1,4). Cryptococcal infection is associated with a range of illness. In some people, it causes a lung infection similar to tuberculosis, or it can cause no symptoms at all. The incubation period is not known, but it is thought that the infection can remain dormant in the body for many years. In immunosuppressed people, particularly HIV- infected once with CD4 counts less than 100, the infection can reactivate and spread throughout the body. When this happens, the infection usually presents as meningitis which is a common cause of death among HIV/AIDS patients (5). DOI: http://dx.doi.org/10.4314/ejhs.v28i4.2 MATERIALS AND METHODS Study design, setting and data collection: A retrospective record review was done on records of 137 HIV infected patients who were attending ART monitoring at FHRH ART clinic. The hospital is located in Bahir Dar town, which is the capital of Amhara National Regional State located 565 km away from the capital Addis Ababa. The FHRH is a tertiary health care level hospital serving the population of Bahir Dar and remote areas of Northwest Ethiopia. The total population served by the hospital is about 12 million. In the The case fatality rate in patients with cryptococcal meningitis, the commonest presentation of HIV-related cryptococcal disease in adults, remains unacceptably high, particularly DOI: http://dx.doi.org/10.4314/ejhs.v28i4.2 Magnitude of Cryptococcal Antigenemia… Awoke D. et al. 371 its one drop dilute were added into a test tube and the lateral flow device that is pre-coated with anti- CrAg monoclonal antibodies and gold conjugated control antibodies was submerged with its white end. After ten minutes of incubation, the result was read and recorded. The antigen antibody complex forms a test line causing a visible line to form. The IMMY CrAg® LFA has sensitivity and specificity of 100% using serum (12-13). Patients found positive for CrAg were managed using fluconzole based antifungal treatment based on the recommended approach. hospital, the ART clinic is operating under the National HIV Program of Ethiopia, under which patients are diagnosed for HIV and get ART treatment and follow-up services (CD4 count, liver and kidney function tests and hematologic evaluation) for free. HIV positive patients, regardless of their ART status, whose CD4 count was less than 100 and had headache with signs suggestive of meningitis were screened for CrAg in the hospital (1). Taking this background, those HIV patients who visited the hospital’s ART clinic from 1 Sep to 30 Dec 2016 and had registered data on their sex, age, CD4 count and cryptococcal antigen screening result were included for analysis. Patient records that missed one of these variables were excluded from the study. Data were retrieved directly from laboratory registration logbook using data extraction sheet on 1to 10 January 2017. Statistical analysis: All data were entered, cleared, and analyzed using SPSS statistical software package, Version 22.0. Descriptive data analysis was used to visualize differences within data, and cross tabulation was done to assess factors associated with cryptococcal antigen. Differences were considered significant when p- value was less than or equal to 0.05. MATERIALS AND METHODS Cryptococcal antigen test: Although culture is the standard method for definitive diagnosis, detection of cryptococcal antigen in serum or cerebrospinal fluid is used for presumptive diagnosis. Cryptococcal antigen screening in peripheral blood is also recommended for HIV- infected persons with CD4 cell counts <100/µl to reduce early deaths while receiving ART (6, 12). Ethical issues: Permission and ethical clearance were obtained from Amhara Regional Health Bureau Institutional Review Board (IRB) to utilize the data. As the data was collected retrospectively, no patient details were linked to the patient’s identity and confidentiality was maintained. RESULTS In the studied area, the cryptoccocal antigen (CrAg) detection was done by the IMMY CrAg® LFA (Cryptococcal Antigen Lateral Flow Assay) kit as per the manufacturer’s instruction. The IMMY CrAg® LFA is an immunochromatographic dipstick assay for the qualitative and semi-quantitative detection of cryptococcal antigen in serum, plasma, whole blood and cerebrospinal fluid (CSF). The CrAg® LFA is a prescription use laboratory assay which can aid in the diagnosis of cryptococcosis (6,12). In this study, from those patients who came with evidence of cryptococcal meningitis, 40µl serum was used to detect the CrAg. The serum and In this study, we analyzed the records of 137 HIV infected patients to assess their cryptococcal antigenemia. More than half of the participants, 75(54.7%), were females. The median age of the participants was 32.0 years (ranged: 8-52 years). The mean CD4 count was at 51.8 with standard deviation of 26.3 (range 3-98). The proportion of positive cryptococal antigenemia from serum test was at 11.7% (95% CI: 7.3-18.1%) (Table1). Further, based on the WHO classification system, all of the HIV positive participants in this study were stage IV. In this study, we analyzed the records of 137 HIV infected patients to assess their cryptococcal antigenemia. More than half of the participants, 75(54.7%), were females. The median age of the participants was 32.0 years (ranged: 8-52 years). The mean CD4 count was at 51.8 with standard deviation of 26.3 (range 3-98). The proportion of positive cryptococal antigenemia from serum test was at 11.7% (95% CI: 7.3-18.1%) (Table1). Further, based on the WHO classification system, all of the HIV positive participants in this study were stage IV. DOI: http://dx.doi.org/10.4314/ejhs.v28i4.2 Vol. 28, No. 4 July 2018 372 Ethiop J Health Sci. Table 1: Demographic and related clinical data of HIV patients screened for cryptococcal antigenemia at FHRH, Bahir Dar, 2016. Table 1: Demographic and related clinical data of HIV patients screened for cryptococcal antigenemia at FHRH, Bahir Dar, 2016. FHRH, Bahir Dar, 2016. RESULTS Variable Frequency Percent Sex Male 62 45.3 Female 75 54.7 Total 137 100 Age group in years 0-14 3 2.2 15-24 11 8.0 25-34 69 50.4 35-44 38 27.7 >44 16 11.7 CD4 count/ µl 0-25 25 18.2 26-50 46 33.6 51-75 36 26.3 76-100 30 21.9 CrAg LFA result Positive 16 11.7 Negative 121 88.3 f d h h C A ® A l i d h C 4 ( 0 0 ) patient age group and the CD4 count (P>0.05) (Table). We found that the IMMY CrAg® LFA result was statically associated with patient sex (p= 0.045). However, it was not found associated with the Table 2: CrAg LFA test result of study subjects at FHRH, Bahir Dar, 2016. Table 2: CrAg LFA test result of study subjects at FHRH, Bahir Dar, 2016. Variable No. Tested CrAg LFA result P value Positive n (%) Negative n (%) Patient Sex Male 62 11 (17.7) 51 (82.3) 0.045 Female 75 5 (6.7) 70 (93.3) Age groups in years 0-14 3 1 (33.3) 2 (66.7) 0.276 15-24 11 0 (0) 11 (100) 25-34 69 7 (10.1) 62 (89.9) 35-44 38 7 (18.4) 31 (81.6) >44 16 1 (6.3) 15 (93.7) CD4 count 0-25 25 3 (12) 22 (88) 0.793 26-50 46 7 (15.2) 39 (84.8) 51-75 36 3 (8.3) 33 (91.7) 76-100 30 3 (10) 27 (90) CrAg LFA: Cryptococcal antigen lateral flow assay test *CrAg LFA: Cryptococcal antigen lateral flow assay test DOI: http://dx.doi.org/10.4314/ejhs.v28i4.2 DISCUSSION disease and high fatality rate, and its clinical symptoms are not typical, misdiagnosis is common in early stage (4). Although the widespread availability of antiretroviral therapy (ART) in developed countries has helped reduce cryptococcal infections in these areas, it is still a major problem in developing countries, like Ethiopia, where access to healthcare is limited (9). Most of the patients with cryptococcal meningitis have serious Each year, Cryptococcus is believed to cause more deaths than tuberculosis in Sub-Sahran Africa (14-15). Therefore, screening individuals with AIDS for serum cryptococcal antigen (CrAg), followed by treatment of CrAg positives DOI: http://dx.doi.org/10.4314/ejhs.v28i4.2 Magnitude of Cryptococcal Antigenemia… Awoke D. et al. 373 In this study, we found that the mean CD4 count of the study participants was 51.8 per 1µl of blood (range 3-98). At the same time, all of them were stage IV. Considering the fact that in such patients with CD4 counts < 100, screening them for CrAg and early ART initiation is the most important and cost-effective preventive strategy to reduce the incidence and high mortality associated with cryptococcal meningitis in HIV-infected patients (1). Since Amphotericin B is not available, the management protocol of CrAg positive HIV patients in FHRH was using fluconzole based antifungal treatment based on the WHO recommended approach (1) and an expert’s opinion. The fluconzole based treatment was given in three phases: 1) induction phase (for two weeks, high dose-600mg BID), 2) consolidation phase (for up to eight weeks, 400mg BID) and 3) maintenance phase (200mg daily for 3-6 months until the patients attains CD4 count of >200). Lumber puncture was also given every 24 hours for these patients until they got relief from the headache. with antifungals, may prevent cryptococcal meningitis (2, 6, 15). In this study, most of the participants living with HIV were females (54.7%). Similarly, in terims of their age category, the majority of HIV patients, 50.4% were in the 25-36 years group which implies that HIV still affects those individuals who are sexually active. Moreover, there is a similar report by Kharsany et al’s study in which adolescent girls and young women aged 15-24 years had up to eight fold higher rates of HIV infection compared to their male peers. DISCUSSION There remains a gap in women initiated HIV prevention technologies especially for women who are unable to negotiate the current HIV prevention options of abstinence, behavior change, condoms and medical male circumcision or early treatment initiation in their relationships (16). In this study, we found that the overall prevalence of cryptococcal antigenemia was 11.7% among HIV infected patients with mean CD4 count at 51.8 cells/µl. According to Shiferaw et al’s study, in Addis Ababa-Ethiopia, the magnitude of cryptococcal antigenemia was 11% among HIV infected patients with CD4 count lessthan100 cells/µl (10). Similarly, according to Bitew et al’s. study in Addis Ababa, the prevalence of cryptococcal antigenemia among HIV positive patients was 8.5% (11). An overall prevalence of 9.9% of C. neoformans infection among HIV patients was also documented by Egbe et al. in Nigeria (17) which makes our finding almost comparable with previous studies done in Ethiopia and elsewhere in the world. The results of our study should be applied with caution. Our findings are subject to at least two limitations: selection bias and quite limited sample size. At the same time, due to the retrospective nature of the study, it was not possible to show detailed clinical picture of HIV patients which might have a significant role to indicate the overall profile of the study participants. No data was found on the ART status and antifungal treatment outcome of patients with positive CrAg test. However, our study is the first of its kind in the studied area that provide baseline information about the magnitude of cryptococcosis among HIV positive patients for further large scale study. The results of this study also showed that this opportunistic fungal infection is an important health problem among HIV patients that needs the attention of physicians who are in charge of attending them. p It is indicated that among immunosuppressed persons, particularly HIV-infected people with CD4 counts under 100, cryptococcal infection is important (16) and is one of the AIDS defining infections (3). Cryptococcal antigenemia was not found associated with patient age group and their CD4 count (P>0.05) (Table 2). On a study done in Nigeria, authors demonstrated that CD4 count less than 200/µl was significantly associated with cryptococcal antigenemia but age group and gender did not show association (17). The statistical difference on CD4 might be because of the small sample size we have employed in the present study. DISCUSSION In conclusion, althrough we have employed quite limited study participants’ data, the reported prevalence of cryptococcal antigenemia calls stakeholders to expand CrAg screening service for individuals with HIV/AIDS, especially for those with CD4 count <100/µl. Additional prospective DOI: http://dx.doi.org/10.4314/ejhs.v28i4.2 July 2018 Vol. 28, No. 4 Ethiop J Health Sci. 374 8. Subramanian S, Mathai D. Clinical manifestations and management of cryptococcal infection. J Postgrad Med. 2005; 51: Suppl 1: 21-6. study with adequate sample size is needed to determine the exact magnitude of the disease and to explore its determinants. ACKNOWLEDGEMENTS 9. Centers for Disease Control and Prevention (CDC), National Center for Emerging and Zoonotic Infectious Diseases Division of Foodborne, Waterborne, and Environmental Diseases. Cryptococcal meningitis: a deadly fungal disease among people living with HIV/AIDS. http://www.cdc.gov/fungal/. We express our deep appreciation to FHRH laboratory staffs for their support during data collection. Our great appreciation is also goes to We express our deep appreciation to FHRH laboratory staffs for their support during data collection. Our great appreciation is also goes to Bahir Dar University for provision of facilities to exercise research. Bahir Dar University for provision of facilities to exercise research. 10. Alemu AS, Kempker RR, Tenna A, et al. High Prevalence of Cryptococcal Antigenemia among HIV-infected Patients Receiving Antiretroviral Therapy in Ethiopia. PLoS ONE 2013; 8(3): e58377. doi:10.1371/journal.pone.0058377 REFERENCES 1. World Health Organization (WHO). Rapid advice: diagnosis, prevention and management of cryptococcal disease in HIV-infected adults, adolescents and children. 2011. <http://apps.who.int/iris/bitstream/10665/44786/ 1/9789241502979_eng.pdf> 1. World Health Organization (WHO). Rapid advice: diagnosis, prevention and management of cryptococcal disease in HIV-infected adults, adolescents and children. 2011. <http://apps.who.int/iris/bitstream/10665/44786/ 1/9789241502979_eng.pdf> 11. Bitew A, Hassen M, Getachew T, Fentaw S. Prevalence of Crytpococcal Infection in Patients Clinically Diagnosed to Have Meningitis in Ethiopia. Clinical Medicine Research. 2016;5 (4):73-76. 2. Beyene T, Woldeamauel Y, Asrat D et al. Comparison of Cryptococcal Antigenemia between Antiretroviral Nai¨ve and Antiretroviral Experienced HIV Positive Patients at Two Hospitals in Ethiopia. PLoS ONE 8 (10): e75585. 12. The IMMY CrAg LFA (Cryptococcal Antigen Lateral Flow Assay). Available at: http://www.immy.com/products/lateral-flow- assays/crag-lfa/. Accessed on 30 Oct 2016. 13. Kozel R, Bauman K. CrAg Lateral Flow Assay for Cryptococcosis. Expert Opin Med Diagn. 2012; 6(3): doi:10.1517/17530059.2012.681300. 3. Lungran P, Vijaya D, Shashi S, et al. Cryptococcosis: Its Prevalence and Clinical Presentation among Hiv Positive and Negative Patients in Rims, Manipur. IOSR Journal of Dental and Medical Sciences. 2014;13(7):38. 14. Warkentien T, Crum-Cianflone F. An Update on Cryptococcosis Among HIV-Infected Persons. Int J STD AIDS. 2010; 21(10): 679. 15. CDC: Cryptococcal Screening. A New Strategy for saving lives among people with HIV/AIDS. <https://www.cdc.gov/fungal/pdf/crypto-screen- strategy-508c.pdf>. 4. Wang H, Yuan X, Zhang L. Latex agglutination: Diagnose the early cryptococcus neoformans test of capsular polysaccharide antigen. Pak. J. Pharm. Sci., 2015; 28(1):307-311. 16. Kharsany B and Karim A. HIV Infection and AIDS in Sub-Saharan Africa: Current Status, Challenges and Opportunities. The Open AIDS Journal, 2016; 10:34-48. 5. Aberg A, Powderly G. Cryptococcosis and HIV. Available at <http://hivinsite.ucsf.edu/InSite?page=kb-05-02- 05>. Accessed on 28 July 2017 17. Egbe A, Omoregie R, Alex-Ighodalo O. Cryptococcus neoformans infection among human immunodeficiency virus patients on highly active antiretroviral therapy in Benin City, Nigeria. N Z J Med Lab Sci. 2015;69: 21- 23. 6. Boulware R, Rolfes A, Rajasingham R, et al. Multisite Validation of Cryptococcal Antigen Lateral Flow Assay and Quantification by Laser Thermal Contrast. Emerging Infectious Diseases 2014; 1. <www.cdc.gov/eid > 7. Saha C, Bhowmik M, Xess I, Padma V, Biswas A. Detection of Cryptococcus by conventional, serological and molecular methods. Journal of Medical Microbiology. 2009; 58: 1098–1105. DOI: http://dx.doi.org/10.4314/ejhs.v28i4.2
https://openalex.org/W2123550866	https://europepmc.org/articles/pmc3653772?pdf=render	English	null	Gene therapy for cardiovascular disease mediated by ultrasound and microbubbles	Cardiovascular ultrasound	2,013	cc-by	10,215	Abstract Gene therapy provides an efficient approach for treatment of cardiovascular disease. To realize the therapeutic effect, both efficient delivery to the target cells and sustained expression of transgenes are required. Ultrasound targeted microbubble destruction (UTMD) technique has become a potential strategy for target-specific gene and drug delivery. When gene-loaded microbubble is injected, the ultrasound-mediated microbubble destruction may spew the transported gene to the targeted cells or organ. Meanwhile, high amplitude oscillations of microbubbles increase the permeability of capillary and cell membrane, facilitating uptake of the released gene into tissue and cell. Therefore, efficiency of gene therapy can be significantly improved. To date, UTMD has been successfully investigated in many diseases, and it has achieved outstanding progress in the last two decades. Herein, we discuss the current status of gene therapy of cardiovascular diseases, and reviewed the progress of the delivery of genes to cardiovascular system by UTMD. Keywords: Ultrasound, Microbubble, Gene therapy, Cardiovascular Keywords: Ultrasound, Microbubble, Gene therapy, Cardiovascular Open Access Open Access Gene therapy for cardiovascular disease mediated by ultrasound and microbubbles Zhi-Yi Chen1, Yan Lin1, Feng Yang1, Lan Jiang1 and Shu ping Ge2* Zhi-Yi Chen1, Yan Lin1, Feng Yang1, Lan Jiang1 and Shu ping Ge2* CARDIOVASCULAR ULTRASOUND CARDIOVASCULAR ULTRASOUND Chen et al. Cardiovascular Ultrasound 2013, 11:11 http://www.cardiovascularultrasound.com/content/11/1/11 CARDIOVASCULAR ULTRASOUND © 2013 Chen et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. * Correspondence: shupingge@126.com 2Section of Cardiology, St. Christopher’s Hospital for Children, Drexel University College of Medicine, 3601 A Street, Philadelphia, PA, USA Full list of author information is available at the end of the article Barriers of gene therapy for cardiovascular diseases With the rapid development of economy, urbanization and changing lifestyles, the number of people with cardiovascu- lar diseases is increasing globally [1]. Even if recent progress have been made in diagnosis and remedy, cardiovascular disease remains the leading cause of mortality in many countries [2]. Therefore, there is a strong impetus for more effective treatment and prevention. As increasing insight into the molecular mechanisms of cardiovascular diseases, gene therapy has been proposed as a promising therapeutic tool for the treatment of cardiovascular diseases [3,4]. To realize efficient delivery of therapeutic genes to the cardiovascular system, a series of barriers related to al- most all aspects of cellular biology have to be overcome. Firstly, the gene vectors need to pass through the endo- thelial barriers in capillary walls when systemically injected. Meanwhile, the plasmid faces a threat of being degraded rapidly by the immune system or DNAse in serum before transfection. On the other hand, viral gene vectors need to avoid the immunoreaction in circulation and transduction of non-target organs, mainly liver and spleen. Secondly, as gene vectors and plasma membrane are negatively charged, the gene vectors have to diffuse through myocardial membrane then bind to cell surface but to be repelled from it. Thirdly, the plasmid needs to avoid being entrapped into lysosome or the endosome, where it will be degraded. Fourthly, the gene vector has to penetrate the nuclear membrane to achieve the goal of gene therapy. Nevertheless, appropriate technologies can be used to make the gene vector itself complete tar- get to the interested area, such as injection catheter, sur- gical operation or UTMD [8,9]. Also directly injection of the vector into myocardium will lead to high local con- centration of the vector. Optimized surface of the vector To realize the therapeutic effect, both efficient delivery to the target cells and sustained expression of transgenes are required. In recent years, a large number of proof- of-principle studies have confirmed that ultrasound targeted microbubble destruction (UTMD) could enhance transfection efficiency of naked plasmid DNA by several orders of magnitude [5-7]. Therefore, it is considered as a promising strategy for gene therapy. Herein, we discuss the current status of gene therapy of cardiovascular dis- eases and review the studies of gene therapy of cardiovas- cular diseases mediated by UTMD. * Correspondence: shupingge@126.com 2Section of Cardiology, St. Current status of gene therapy of cardiovascular disease Nabel et al. [10] were the first to demonstrate gene ther- apy in cardiovascular system in 1989. Since then, gene therapeutic trials for cardiovascular diseases have been performed all over the world. However, advances in the area of gene therapy for cardiovascular disease are not well satisfied because of the lack of gene delivery systems to transfer therapeutic gene to specific target to provide an adequate dose of a therapeutic gene [2]. So far, the gene delivery systems are mainly divided into two kinds, namely, viral systems and non-viral systems [11]. Viral systems derived from adeno-associated virus (AAV) [12], retrovirus [13], lentivirus [14] and adenovirus [15] are one of the successful gene delivery systems, which are used in the majority of current gene therapy researches and clin- ical trials due to their benefits of highly efficient delivery into cells with sustained expression. Recently, Prunier et al. [16] showed that delivering adenovirus expressing sarco- plasmic reticulum Ca2+ ATPase (SERCA2a) into coronary arteries could prevent ventricular arrhythmias in a ische- mia - reperfusion model. Suckau et al. [17] also employed adenoviral and AAV vectors to obtain high RNAi activity. They showed that an adenoviral short hairpin RNA vector could silence phospholamban in cardiomyocytes and im- proved hemodynamics in heart-failure. Meanwhile, they designed a dimeric cardiotropic AAV vector to intraven- ously deliver RNA molecule to the heart for simplified long-term therapy. However, viral gene therapy has been subjected to criticism due to their potential for uncontrol- lable and insertional mutagenesis [18]. Moreover, viral vec- tors will evoke undesired immune response by the virtue of systemic administration, which limits repetitive regi- ments [19,20]. Also the transfection efficiency of viral vectors occur with a relatively low efficiency and organ specificity, which restricts its therapeutic efficacy [21,22]. UTMD has been widely proved as a new strategy of improving the delivery of drugs or genes [29,30]. Due to its advantages of high safety profile, repetitive applicabil- ity, cost-effectiveness and the capability to enhance the permeability of plasma membrane to macromolecules by its bioeffects, it is considered as a feasible tool for gene delivery [31-35]. Chen et al. [36] have proved that UTMD significantly increased the transfection rate of short hairpin RNA (shRNA) vectors in vitro and in vivo, which was equal to that of some cancer cell lines deliv- ered by polyethylenimine (PEI). Qiu et al. Barriers of gene therapy for cardiovascular diseases Christopher’s Hospital for Children, Drexel University College of Medicine, 3601 A Street, Philadelphia, PA, USA Full list of author information is available at the end of the article Page 2 of 14 Chen et al. Cardiovascular Ultrasound 2013, 11:11 http://www.cardiovascularultrasound.com/content/11/1/11 transfection efficiency. Recently, Ko et al. [25] conjugated cell-penetrating transactivating transcriptional activator (TAT) peptide (TATp) and/or monoclonal anti-myosin monoclonal antibody 2G4 (mAb 2G4) which target to car- diac myosin to liposomes for targeted gene delivery to ischemic myocardium. The result showed that in vitro transfection was enhanced by the presence of TATp and was further enhanced by the additional modification with mAb 2G4 antibody. And the transfection efficiency of in vivo experiments was significantly enhanced in the ischemic zone. The main disadvantage of nonviral systems is their low transfection because of the inability of vectors to overcome biological barriers to enter cells [26]. In other hand, physical methods recently have been developed as a feasible nonviral method. However, most of them are mainly based upon invasion procedures, their inherent risks that may outweigh their benefits, which makes them inappropriate for cardiac gene transfer. Durieux et al. [27] conducted a study to investigates the factors regarding gene electrotransfer associated muscle damage, which confirmed that gene electrotransfer associated muscle damage was related to the intracellular presence and ex- pression of plasmid DNA. Besides, the gene expression is confined to the injection site. Therefore, it is necessary to develop an effective and specific gene delivery system suit- able for human body [28]. can realize directional transduction of the vector into the cell and karyon. Current status of gene therapy of cardiovascular disease [37] have proved that enhanced green fluorescent protein (EGFP) plasmids could be delivered effectively into rabbit Achil- les tendons by UTMD, causing no obvious injury. Nonviral systems is consist of chemical methods (such as cationic liposome, nanoparticle and polymers) and physical methods (include gene gun, electroporation, par- ticle bombardment, ultrasound utilization, and magne- tofection) [23,24]. The advantages of nonviral system include availability, cost-effectiveness, and less induction of immune system and no limitation in size of transgenic DNA compared with viral system, which have made them an attractive candidate for gene delivery. Among them, the simplest and most widely nonviral gene vector is naked DNA. However, its transfection efficiency is limited due to the rapid degradation by DNAse and the clearance by the mononuclear phagocyte system in the systemic circulation. Increasing target specificity to diseased tissue can reduce off-target bioeffects and enhance the gene Delivery genes to cardiovascular system by microbubble and ultrasound Ultrasound and microbubble have been widely investi- gated in myocardial infarctions, atherosclerosis, and in hind limb ischemia models in rodents for therapeutic angiogenesis. Early in the last decade, commercial and custom microbubbles were tested for pDNA encoding luciferase delivery in the left ventricle [46]. y It has proved that TFPI-2 played an important role in suppressing thrombosis and arterial re-stenosis, which has been considered as a potential gene for gene therapy of atherosclerosis. Studies have confirmed that TFPI-2 gene can be delivered to the target specifically by the virtue of UTMD. For example, Wang et al. [39] showed gene transfection with SonoVue and TFPI-2 gene could suppress thrombosis and arterial re-stenosis, providing a potential gene therapy approach for atherosclerosis. Compared with adenovirus, the in vivo transfection effi- ciency of SonoVue was higher than that of adenovirus and SonoVue was less damaging when transfecting genes into the arterial wall. Studies have confirmed that thymosin beta 4 (TB4)-protein delivery could stimulate differentiation of resident adult WT1-positive (WT1 is a biomarker for development of embryonic heart and is not normally expressed in adult rat heart) cardiac pro- genitor cells, but its application is limited due to low Mechanisms of UTMD in gene therapy Mechanisms of UTMD in gene therapy UTMD is an immense potential target-specific gene de- livery tool. Its ability to elevate the gene transfection effi- ciency in various studies in vitro and in vivo has been confirmed [38-40], thus being consider as a promising gene carrier approach for gene therapy. Microbubbles (MBs) of UTMD, which may consist of lipids, albumin, saccharide, biocompatible polymers and other materials [41-43] are traditionally used as ultrasound contrast agents due to their physical property of reflecting ultra- sound. Microbubble as cavitation nucleus could expand and contract under the effect of ultrasound, and even be disrupted when the acoustic pressure reaches a much Chen et al. Cardiovascular Ultrasound 2013, 11:11 http://www.cardiovascularultrasound.com/content/11/1/11 Page 3 of 14 Chen et al. Cardiovascular Ultrasound 2013, 11:11 http://www.cardiovascularultrasound.com/content/11/1/11 higher level, which could cause a series of biological ef- fects. The mechanism of transferring gene into cells effectively through UTMD is based on the specific re- sponse of the microbubbles upon exposure to ultra- sound, namely sonoporation. Microbubbles may oscillate when exposed to ultrasound, and then these oscillating microbubbles may rupture. So, the gene therapy vector incorporated with microbubbles can be released with high local concentrations at the site of interest. Mean- while, the destruction of MBs may transiently induce transient holes in membranes in consequence to local shear forces exerted on membranes by fluid flow (‘micro streaming’) around oscillating bubbles, local shock waves (which produce large pressure gradients across a cell), or cavitation microjets, therefore, facilitating drug or gene into cells [44,45], and augmenting the transfection effi- ciency (Figure 1). The advantages of UTMD techniques are as follows: (1) low toxicity, (2) low immunogenicity of the vectors, (3) low invasiveness (e.g., the vector and MB can be intravenously injected), (4) great potential for repetitive application, and (5) organs can be targeted with high specificity. Since UTMD can not only improve the efficiency, but also avoid the immunogenicity, it has been regarded as a new choice for gene therapy. therapy. In recent years, many studies in vitro and in vivo have confirmed that ultrasound and microbubble could significantly elevate the gene transfection efficiency. It is emerging as a potential strategy for treatment of cardio- vascular diseases [11]. Gene therapy mediated by UTMD in cardiovascular disease Due to its advantages of high safety, cost effectiveness, re- petitive applicability, and the possibility to increase the permeability of microvessel and plasma membrane to macromolecules by its bioeffects, ultrasound and micro- bubble has been considered as a powerful tool in gene Figure 1 Schematic diagram of gene therapy mediated by UTMD. Microbubbles carrying therapeutic gene are destroyed at the site of the target tissue, resulting in sonoporation and delivery of the drug directly to the target cell. The process of sonoporation induced by US application leads to transiently holes in cell membrane and capillary, which facilitates the uptake of therapeutic gene. Figure 1 Schematic diagram of gene therapy mediated by UTMD. Microbubbles carrying therapeutic gene are destroyed at the site of the target tissue, resulting in sonoporation and delivery of the drug directly to the target cell. The process of sonoporation induced by US application leads to transiently holes in cell membrane and capillary, which facilitates the uptake of therapeutic gene. Chen et al. Cardiovascular Ultrasound 2013, 11:11 http://www.cardiovascularultrasound.com/content/11/1/11 Page 4 of 14 efficiency. Chen et al. [47] used UTMD to enhance the delivery of the human TB4 gene under a piggybac trans- poson plasmid to normal rat heart. The result showed that WT1 started to express from the nucleus of epicar- dium layer cells one week after UTMD-TB4 treatment (Figure 2), and the level of WT1 mRNA of which treated with UTMD-TB4 group was 43.5-fold higher than in normal control or UTMD-DsRed groups in the 4th week, and c-kit mRNA level in UTMD-TB4 group was 52-fold higher after UTMD-TB4 treatment. parameters for this technique, including the US expos- ure parameters, US frequency, mode of US, mechanical index, and amount of the plasmid DNA, should be opti- mized in the future. Delivery viral vectors to cardiovascular system by ultrasound and microbubble Cardiovascular Ultrasound 2013, 11:11 http://www.cardiovascularultrasound.com/content/11/1/11 http://www.cardiovascularultrasound.com/content/11/1/11 Figure 3 The combination of UTMD and PEI in vivo. A, Phosphate-buffered saline group; B, phosphate-buffered saline + US; C, naked plasmid; D, plasmid + US; E, plasmid + liposome microbubbles (LM); F, plasmid + LM + US; G, plasmid + LM + PEI; H, plasmid + LM + PEI + US. There were no fluorescent signals in the negative control groups (A, B). When the mouse hearts exposed, the bases of the heart tissue samples had more transfected cells than the rest of the samples (D-H). Only a few fluorescent signals could be detected in the absence of US (E). A transmural fluorescent signal could be observed in the anterior wall after the microbubble was destroyed (F). EGFP was principally expressed in the subendocardial layer in the absence of US(C, E, and G and arrows in G). Combining with PEI and UTMD, the distribution of EGFP was not significant (H) [5]. Figure 3 The combination of UTMD and PEI in vivo. A, Phosphate-buffered saline group; B, phosphate-buffered saline + US; C, naked plasmid; D, plasmid + US; E, plasmid + liposome microbubbles (LM); F, plasmid + LM + US; G, plasmid + LM + PEI; H, plasmid + LM + PEI + US. There were no fluorescent signals in the negative control groups (A, B). When the mouse hearts exposed, the bases of the heart tissue samples had more transfected cells than the rest of the samples (D-H). Only a few fluorescent signals could be detected in the absence of US (E). A transmural fluorescent signal could be observed in the anterior wall after the microbubble was destroyed (F). EGFP was principally expressed in the subendocardial layer in the absence of US(C, E, and G and arrows in G). Combining with PEI and UTMD, the distribution of EGFP was not significant (H) [5]. microjet by UTMD can enhance the penetrability of plasma membrane and capillary, thus overcoming the endothelial barrier. Moreover, the microbubbles can sim- ultaneously impose restriction on the immune response to the viruses thus allowing intravascular administration and repetitive injections [51]. ultrasonic standing wave fields could offer a potential ap- proach to increase transduction efficiency of retrovirus- based vector in large-scale settings. Since, systemic administration of these viruses had been a challenge as the drawback of adenoviral vectors has innate host antiviral immune responses. Howard et al. Delivery viral vectors to cardiovascular system by ultrasound and microbubble Due to its high transfection efficiency and sustained ex- pression, viral vector is the priority choice of transferring genes to the target cell. However, it is difficult to restrict the specificity of delivery in viral vectors which are usually delivered systemically, while avoid the immunoreaction. Additionally, the endothelial barrier limits systemic deliv- ery of viral vector such as AAV, which leads to unsatisfac- tory transfection efficiency. UTMD techniques have many advantages [48]. Studies have proved that it had synergism to combine with viral vectors, which offered many benefits [49,50]. Firstly, the microbubbles offer the strength of site- specific release through ultrasound irradiation, thus im- proving viral vector specificity. And the production of Chen et al. [5] have proved that the combination of UTMD with PEI could effectively enhance transfection efficiency of two different naked DNA without causing any apparently side effect (Figure 3). Besides, they dem- onstrated that naked plasmids (luciferase reporter, red fluorescent protein reporter, EGFP) could be effectively delivered to myocardium, combining with liposome microbubble (MB), PEI and ultrasound (US). However, though UTMD-mediated naked DNA in gene therapy is effective, this technology has some limitations. The Figure 2 Exogenous TB4 reprogrammed epicardium layer cells into WT1-positive adult cardiac progenitor cells and then formed new cardiac muscle cells. A: Negative control for 12 weeks post-UTMD-DsRed;B-F: The panels are high power images (scale bar = 50 mm) from TB4-treated rats killed at 1, 2, 3, 4 and 12 weeks after UTMD. WT1 signal from nucleus of epicardium layer cells at 1–3 weeks after UTMD-TB4 treatment and then migrated into myocardium layer to form new cardiac muscle cells (WT1 signal from nucleus of cTnT-positive cells, arrows) at 4 or 12 weeks after UTMD-TB4 treatment [47]. Figure 2 Exogenous TB4 reprogrammed epicardium layer cells into WT1-positive adult cardiac progenitor cells and then formed new cardiac muscle cells. A: Negative control for 12 weeks post-UTMD-DsRed;B-F: The panels are high power images (scale bar = 50 mm) from TB4-treated rats killed at 1, 2, 3, 4 and 12 weeks after UTMD. WT1 signal from nucleus of epicardium layer cells at 1–3 weeks after UTMD-TB4 treatment and then migrated into myocardium layer to form new cardiac muscle cells (WT1 signal from nucleus of cTnT-positive cells, arrows) at 4 or 12 weeks after UTMD-TB4 treatment [47]. Page 5 of 14 Chen et al. Cardiovascular Ultrasound 2013, 11:11 Chen et al. Delivery gene to cardiovascular system by ultrasound and novel microbubble incapable of target cell entry to target cells with ultra- sound exposure of pulsed 1 MHz for 5s. By using virus- loaded microbubbles, ultrasound facilitated the delivery of viral vectors in a restricted area of cells exposed to ≥0.4 MPa peak-negative acoustic pressure. It showed that the technology was an ideal mean suited for targeted delivery. Compared with the other groups, the application of viral vector significantly increased transgene expression, but the toxicity and immunogenicity of the viral vectors still plagued people. The non-viral vectors can enhance the gene transfection rate. Noninvasive UTMD enable the successful transfection of vascular endothelial grow factor (VEGF), stem cell factor or other genes into the infracted heart, thus increasing density of blood vessel, myocardial perfusion and ventricular function. UTMD-mediated plas- mid gene delivery takes advantage of myocardial contrast echocardiography and has numerous merits including low toxicity, lack of immunogenicity, and the potential for re- petitive and targeted application [56,57]. However, there were some injured endothelial cells in part of the vessel wall with the ultrasound exposure. Also, Huang et al. [57] developed new liposomes that could be used to protect and transfer a bioactive gas to target in conjugate with noninvasive UTMD. The results showed that it hold po- tential for gas delivery and could be used to control thera- peutic gas release (Figure 5). The mechanism how ultrasound enhances the trans- fection rate of transgene may be that ultrasound imparts a microjet to the cell and facilitates local delivery of the DNA into the cell. It also presumedly that ultrasound af- fected cell regulatory or transcription factors. However, the precise mechanism accounting for why expose ultra- sound to cells together with and also to viral vector en- hance the gene expression level is still blurred. Specific studies should be conducted to explain these different mechanisms and decide how UTMD influences AAV- mediated gene expression. Therefore, Geers et al. [55] have undertaken a study to interpret the mechanisms behind how the combination of UTMD and AAV medi- ated gene therapy work. They made use of “non-active AAV”, which are AAV vectors chemically modified at their surface with a poly (ethylene glycol) (PEG) brush, and found the “non-active AAV” could be delivered in the cytosol of cells directly through sonoporation. Delivery viral vectors to cardiovascular system by ultrasound and microbubble [51] have tested the ability of microbubble to load and protect an adenoviral vector. The result demon- strated that systemic delivery of the vector incorporated into microbubbles led to specific targeting of the GFP transgene. Microbubbles allow intravenous injection as it can reduce the degradation rate of viruses by immune system. This opens up a new therapeutic frontier for pa- tients needs for a less invasive and highly specific gene delivery system. Müller et al. [49] also conducted a study to research the specificity and transfection rate of gene delivery. In their study, adult rats were injected of microbubbles loaded with AAV-6 or AAV-9 expressing luciferase or EGFP. The result demonstrated that the re- porter gene transfection efficiency significantly increased with ultrasound exposure (Figure 4). Recently, a study by Naka et al. [52] demonstrated that gene expression mediated by retrovirus was significantly increased in all four cell types tested in this study without any adverse bioeffecs when they were exposed for 5 s with the ultrasound of 1.0 W/cm2. The transduction efficiencies of ultrasound was enhanced 6.6-fold, 4.8-fold, 2.3-fold, and 3.2-fold in 293T cells, BAECs, RASMCs, and L6 cells, re- spectively. Furthermore, in the presence of ultrasound and the retrovirus, β-Gal activities of these cells were also in- creased. Chen et al. [46] optimized echocardiographic pa- rameters for successfully delivered adenoviral or plasmid DNA to the heart. The results demonstrated that enhance- ment of transgene expression are detected in the heart tis- sue when treated with UTMD associating with adenoviral or plasmid DNA. And the ultrasound parameters were optimized with a low-transmission frequency (1.3 MHz), maximal mechanical index, and ECG triggering to allow completely fitting the myocardial capillary bed for micro- bubbles dispose. In addition, Lee et al. [53] showed that Also, Taylor et al. [54] carried out a study to assess the feasibility of gene delivery system that combined UTMD and retrovirus. In their study, they added an envelope-deficient retroviral vector which was inherently Page 6 of 14 Chen et al. Cardiovascular Ultrasound 2013, 11:11 http://www.cardiovascularultrasound.com/content/11/1/11 http://www.cardiovascularultrasound.com/content/11/1/11 Figure 4 Localization of gene expression after treatment with UTMD. Adult rats were injected with microbubbles loaded with 1011 genomic particles of AAV-9-cytomegalovirus (CMV)-MLC0.26-EGFP vectors. Delivery viral vectors to cardiovascular system by ultrasound and microbubble (A) No fluorescent signals were detectable in the absence of ultrasound; (B) The anterior wall showed a strong fluorescent signal after UTMD; (C) When is amplified, only few fluorescent signals could be observed in the absence of microbubbles; (D) The merged picture of green fluorescent protein (GFP)-fluorescence and phalloidin co-staining; (E) In the presence of UTMD, multiple GFP-postive cells can be observed; (F) co-staining with phalloidin. Bar a, b: 1 mm and c ~ f: 50 μm [49]. Figure 4 Localization of gene expression after treatment with UTMD. Adult rats were injected with microbubbles loaded with 1011 genomic particles of AAV-9-cytomegalovirus (CMV)-MLC0.26-EGFP vectors. (A) No fluorescent signals were detectable in the absence of ultrasound; (B) The anterior wall showed a strong fluorescent signal after UTMD; (C) When is amplified, only few fluorescent signals could be observed in the absence of microbubbles; (D) The merged picture of green fluorescent protein (GFP)-fluorescence and phalloidin co-staining; (E) In the presence of UTMD, multiple GFP-postive cells can be observed; (F) co-staining with phalloidin. Bar a, b: 1 mm and c ~ f: 50 μm [49]. Delivery gene to cardiovascular system by ultrasound and novel microbubble Thus UTMD can specifically and effectively increase rAAV gene delivery system, and it may afford potential for highly effective gene delivery means for gene therapy of cardiovascular diseases. Despite the existence of many in vitro experimental studies on gene therapy with combination of non-viral vectors and UTMD, research on in vivo therapeutic ap- plication is on the beginning. Saliba et al. [58] developed an interesting safe method for local gene transfer by in- jection of plasmid or siRNA mixed with a standard com- mercial liposome, with the presentence of ultrasound Page 7 of 14 Chen et al. Cardiovascular Ultrasound 2013, 11:11 Page 7 of 14 Chen et al. Cardiovascular Ultrasound 2013, 11:11 http://www.cardiovascularultrasound.com/content/11/1/11 Figure 5 Nitric oxide (NO) was delivered to the cultured vascular smooth muscle cells by ELIP with or without NO-scavenging agent, haemoglobin. Vascular smooth muscle cells were transfected with a fluorescent probe, diaminofluorescein-2 diacetate (DFA-2DA), which reacts with NO to produce a fluorescent signal. In the absence of haemoglobin, A: the cultured cells were treated with free NO and in the absence of haemoglobin; B: the cultured cells were treated with NO encapsulated in ELIP. In the presence of haemoglobin, C: the cultured cells were treated with free NO; D: the cultured cells were treated with NO encapsulated in ELIP. NO-loaded ELIP were able to efficiently deliver NO into cultured cells even in the presence of a potent NO-scavenging agent such as haemoglobin [57]. Figure 5 Nitric oxide (NO) was delivered to the cultured vascular smooth muscle cells by ELIP with or without NO-scavenging agent, haemoglobin. Vascular smooth muscle cells were transfected with a fluorescent probe, diaminofluorescein-2 diacetate (DFA-2DA), which reacts with NO to produce a fluorescent signal. In the absence of haemoglobin, A: the cultured cells were treated with free NO and in the absence of haemoglobin; B: the cultured cells were treated with NO encapsulated in ELIP. In the presence of haemoglobin, C: the cultured cells were treated with free NO; D: the cultured cells were treated with NO encapsulated in ELIP. NO-loaded ELIP were able to efficiently deliver NO into cultured cells even in the presence of a potent NO-scavenging agent such as haemoglobin [57]. application (4.9MHz, 1Hz, 1W/cm2, ultra-harmonic mode, 5min, another 5min), resulted in much higher transfection efficiency. Zhao et al. Delivery gene to cardiovascular system by ultrasound and novel microbubble [59] investigated that the cardio pro- tective effect of the acidic fibroblast growth factor (aFGF) combing with heparin modified microbubbles (aFGF- HMB) under UTMD technique. Echocardiography of the heart parenchyma was enhanced after aFGF-HMB injec- tion. From ultrasonography, aFGF-HMB suspension had good capability in heart ultrasonic contrast imaging. As shown in M-mode echocardiography, the group (aFGF-HMB + US) could remarkably stimulate myocardial vessel neogenesis, resulting in a significant improvement of regional as well as global myocardial contractile func- tions (Figure 6). gene transfection of the ischemic heart for cardiac re- generation. Compared its DNA-carrying capacity and reporter gene transfection efficiency with the commer- cially available Definity microbubble, the cationic microbubbles loaded 70% more plasmid DNA than the Definity microbubbles and UTMD was able to deliver the therapeutic gene to the ischemic rat myocardium and evaluated the effects on apoptosis, angiogenesis, and cardiac function, and provided an efficient platform for gene therapy of the ischemic heart and preserve car- diac function (Figure 8). Atherosclerosis is an inflammatory disease of the vas- culature and risks, which can lead to heart attacks and stroke, so there is a great need for both drug delivery and detection of disease state. Phillips et al. [61] devel- oped the novel microbubbles targeted to vascuolar cell adhesion molecule 1 (VCAM-1), which can be used for simultaneous ultrasound molecular imaging and gene delivery. Compared with nontargeted microbubbles, VCAM-1-targeted microbubbles exhibited a 100-fold in- crease in adhesion to inflamed SMCs. Their studies may aid in the detection and treatment of in-stent restenosis Recently, Fujii et al. [41] used UTMD to deliver an- giogenic genes. The result showed that GFP expression was identified in the hearts of mice that were injected with microbubble/GFP expressing plasmid complex five days after UTMD and confirmed that successful in vivo transfection was achieved by using the microbubble technique (Figure 7). Sun et al. [60] prepared a cationic microbubble with DNA-binding to improve targeted Chen et al. Cardiovascular Ultrasound 2013, 11:11 Page 8 of 14 Chen et al. Cardiovascular Ultrasound 2013, 11:11 http://www.cardiovascularultrasound.com/content/11/1/11 http://www.cardiovascularultrasound.com/content/11/1/11 Figure 6 M-mode echocardiography of Group (aFGF-HMB + US) [59]. Figure 6 M-mode echocardiography of Group (aFGF-HMB + US) [59]. activator, t-PA) gene plasmid, was also employed to cure human thrombosis-related diseases. Delivery gene to cardiovascular system by ultrasound and novel microbubble The results showed that the heart ultrasound visualization increased obviously compared to preinjection, and the baseline image follow- ing the ultrasound treatment decreased after the intraven- ous injection of microbubbles loaded with nanopackaged t-PA gene plasmid [63]. Additionally, Ren et al. [64] reported the preparation of three groups of self-made or be used to detect early atherosclerosis, and subse- quently would realize gene or drug therapy to inflamed vasculature. A body of laboratory work has demonstrated that UTMD was a promising tool for the local delivery of genes and drugs [62]. A novel and potential site-specific gene transfer, combination of ultrasound (1 MHz, 1.5 w/cm2, 10 minutes) and nanopackaged t-PA (Tissue plasminogen Figure 7 Confocal microphotograph of myocardial tissue of mice after treatment to show the efficiency of gene transfection in vivo by UTMD. 5 days after the treatment of UTMD in the myocardial tissue of mice, the samples were immunostained with an antibody against green fluorescent protein (GFP, green), nuclear staining (Hoechst, blue). A ~ C: control group, the mice was injected with empty plasmids; D ~ I: GFP group, the mice was injected with plasmids expressing GFP; G ~ I: the merged images indicated co-localization of GFP and Hoechst. Magnification _400 in A to F. Framed areas in D to F are shown enlarged at _600 in G to I [41]. Figure 7 Confocal microphotograph of myocardial tissue of mice after treatment to show the efficiency of gene transfection in vivo by UTMD. 5 days after the treatment of UTMD in the myocardial tissue of mice, the samples were immunostained with an antibody against green fluorescent protein (GFP, green), nuclear staining (Hoechst, blue). A ~ C: control group, the mice was injected with empty plasmids; D ~ I: GFP group, the mice was injected with plasmids expressing GFP; G ~ I: the merged images indicated co-localization of GFP and Hoechst. Magnification _400 in A to F. Framed areas in D to F are shown enlarged at _600 in G to I [41]. Chen et al. Cardiovascular Ultrasound 2013, 11:11 Page 9 of 14 Chen et al. Cardiovascular Ultrasound 2013, 11:11 http://www.cardiovascularultrasound.com/content/11/1/11 Chen et al. Cardiovascular Ultrasound 2013, 11:11 http://www.cardiovascularultrasound.com/content/11/1/11 Figure 8 Images of rats receiving luciferase plasmid delivered by UTMD. Bioluminescence was significantly higher at 3, 7, and 14 days after gene delivery with the CMB compared with the Definity MB [60]. Delivery gene to cardiovascular system by ultrasound and novel microbubble Figure 8 Images of rats receiving luciferase plasmid delivered by UTMD. Bioluminescence was significantly higher at 3, 7, and 14 days after gene delivery with the CMB compared with the Definity MB [60]. microbubble-loading uPA (1 uPA-MBs, 5 uPA-MBs and 10 uPA-MBs) via freeze-drying methods to achieve a more efficacious and safer thrombolytic effect. It can be used for thrombolysis when combined with low-frequency US in vitro. The loaded uPA exhibited bioactivity by agarose fibrin plate when exposed to US and in vitro thrombolysis also showed higher effects with US exposure related to the group who received uPA-MBs alone, the control group or the US group. In conclusion, the physiochemical proper- ties of these self-made uPA-MBs allow for intravenous in- jection but 1 uPA-MB and 5 uPA-MBs are better than 10 uPA-MBs. The combination of uPA-MBs and US can minish the in vitro dosage of uPA for thrombolysis (Figure 9). Mannell et al. [65] loaded the magnetic (MNP) perfluorocarbon-filled lipid microbubbles with lentiviral particles and associated magnetic targeting of these com- plexes with UTMD. The combination eventually led to a transduction efficiency increase by 30-fold over the appli- cation of naked virus alone. Application of ultrasound and microbubble in RNA interference based gene therapy for cardiovascular desease Application of ultrasound and microbubble in RNA interference based gene therapy for cardiovascular desease microbubble-loading uPA (1 uPA-MBs, 5 uPA-MBs and 10 uPA-MBs) via freeze-drying methods to achieve a more efficacious and safer thrombolytic effect. It can be used for thrombolysis when combined with low-frequency US in vitro. The loaded uPA exhibited bioactivity by agarose fibrin plate when exposed to US and in vitro thrombolysis also showed higher effects with US exposure related to the group who received uPA-MBs alone, the control group or the US group. In conclusion, the physiochemical proper- ties of these self-made uPA-MBs allow for intravenous in- jection but 1 uPA-MB and 5 uPA-MBs are better than 10 uPA-MBs. The combination of uPA-MBs and US can minish the in vitro dosage of uPA for thrombolysis (Figure 9). Mannell et al. [65] loaded the magnetic (MNP) perfluorocarbon-filled lipid microbubbles with lentiviral particles and associated magnetic targeting of these com- plexes with UTMD. The combination eventually led to a transduction efficiency increase by 30-fold over the appli- cation of naked virus alone. RNA interference (RNAi) is a technique that could in- hibit target gene expression based on sequence-specific gene silencing using small interfering RNA (siRNA) [66]. The technique has attracted much attention for clinical use in various diseases, and has potential to treat cardio- vascular diseases. However, transfection of the endothe- lium and myocardial cell with siRNAs in vivo still poses a distinct hurdle [67]. There still need a noninvasive and ef- fective method to transfer siRNA into target cells. Kinoshita et al. [68] demonstrated that delivery siRNA intracellularly via microbubble-enhanced focused ultrasound was viable, and represented a powerful tool for using siRNA in vivo and possibly in the clinical setting. Among the more common approaches employed for siRNA delivery are the use of ultrasound-microbubble which can effectively delivery siRNAs into target areas Page 10 of 14 Chen et al. Cardiovascular Ultrasound 2013, 11:11 Chen et al. Cardiovascular Ultrasound 2013, 11:11 http://www.cardiovascularultrasound.com/content/11/1/11 , http://www.cardiovascularultrasound.com/content/11/1/11 Figure 9 After treatment, the thrombi were observed under the electron microscope. A1, A2: control group; B1, B2: uPA group; C1, C2, C3, C4: uPA + US group; A1, B1, C1, C3 were amplified by 1000 times; A2, B2, C2, C4 were amplified by 4000 times [64]. Figure 9 After treatment, the thrombi were observed under the electron microscope. Application of ultrasound and microbubble in RNA interference based gene therapy for cardiovascular desease A1, A2: control group; B1, B2: uPA group; C1, C2, C3, C4: uPA + US group; A1, B1, C1, C3 were amplified by 1000 times; A2, B2, C2, C4 were amplified by 4000 times [64]. in vivo. Suzuki et al. [33] prepared three kind of siRNA/ microbubble complexes (a fluorescein-labeled siRNA, green fluorescent protein (GFP) siRNA, and intercellular adhesion molecule (ICAM)-1 siRNA), and confirmed that ICAM-1 siRNA/microbubble has the potential to suppress arter- ial neointimal formation using ultrasound-microbubble method (Figure 10). With ultrasound irradiation, siRNA/ microbubble is potent for clinical treatment of cardiovas- cular diseases and other inflammatory disease. After acute myocardial infarction, migration of bone marrow-derived mesenchymal stem cells (MSCs) to vital myocardium is a promising therapeutic method. Ultra- sound irradiation inducing stimulation of microbubbles al- lows locoregional pre-treatment of target tissue, and combination of ultrasound irradiation and stem cell tech- nology may improve transplantation efficacy and targeting of MSCs, and enhance the efficacy of a sustained myocar- dial cell delivery. Ghanem et al. [69] demonstrated that fo- cused ultrasound with stimulated microbubbles improved transplantation efficacy and allowed targeted engraftment of MSCs. Compared to nontargeted areas, significantly more MSCs adhered to the endothelium of targeted tis- sues was observed, and they did not observe any apoptosis After acute myocardial infarction, migration of bone marrow-derived mesenchymal stem cells (MSCs) to vital myocardium is a promising therapeutic method. Ultra- sound irradiation inducing stimulation of microbubbles al- lows locoregional pre-treatment of target tissue, and combination of ultrasound irradiation and stem cell tech- nology may improve transplantation efficacy and targeting of MSCs, and enhance the efficacy of a sustained myocar- dial cell delivery. Ghanem et al. [69] demonstrated that fo- cused ultrasound with stimulated microbubbles improved transplantation efficacy and allowed targeted engraftment of MSCs. Compared to nontargeted areas, significantly more MSCs adhered to the endothelium of targeted tis- sues was observed, and they did not observe any apoptosis Stem Cell Transplant for Treatment of Cardiovascular d b l d d b bbl Figure 12 The MSCs adherent to the aortic endoluminal surface after ultrasound mediated delivery survive and undergo morphological changes (Scale bar = 100 mm), between 20min (two examples in A) 3h (B), and 24 h (C) post delivery [71]. delivery. A variety of experiments have demonstrated that the combination of UTMD and viral and non-viral vectors in gene delivery could not only enhance the effi- ciency of the viral vector, but also avoid its immunogen- icity. Thus it may become a feasible, novel candidate for gene therapy, providing support to gene therapy trial for patients with cardiovascular diseases. phenomenon and/or myocardial necrosis. Otani et al. [70] proved the feasibility of combination of ultrasound and microbubbles for delivery of siRNA into MSC, and the re- sults indicated that ultrasound and microbubble could serve as a nonviral delivery method of siRNA into MSC, which would be a useful appoach for regenerative medi- cine in the future. Moreover, MSCs conld be surface-coated electrostati- cally with gas-filled lipid microbubbles (MB-MSCs) and seeded to targeted areas or a specific vascular segment, which is an emerging therapeutic option. Xu et al. [56] launched experimental research to figure out whether combining lipid-coated microbubbles with diagnostic US could enable the site directed delivery of MSCs into the myocardium even myocardial infarcted rabbits. After the intravenous injection of lipid-coated microbubble ac- company with BM-MSCs into the rabbits, the anterior chest was treated with diagnostic ultrasound for 10 min to induce infusion of BM-MSCs which was labeled with DAPI in the nucleus. The results showed that DAPI positive cells in myocardial infarction area were much more than that of the MSCs infusion group (Figure 11). Toma et al. [71] proposed a novel method by using ultrasound-generated acoustic radiation force combined with MB-MSCs to delivery of therapeutic cells to a spe- cific endovascular treatment site. This approach may be used for endoluminal cellular paving, providing a powerful tool for cell-based gene delivery of injured ar- terial segments (Figure 12). UTMD is a promising technique for gene delivery, but most of its studies are in the preclinical stage. UTMD still remains limited by its safety and efficiency. Authors’ contributions ZYC and SPG are responsible for designing the framework of this article. YL and FY participated in collecting material and drafting the manuscript. LJ participated in picture processing. All authors read and approved the final manuscript. Competing interests Competing interests The authors declare no competing interests. Stem Cell Transplant for Treatment of Cardiovascular d b l d d b bbl Cell-based therapy presents an attractive approach to res- toration of a functional endothelium and myocardium. Page 11 of 14 Chen et al. Cardiovascular Ultrasound 2013, 11:11 Chen et al. Cardiovascular Ultrasound 2013, 11:11 http://www.cardiovascularultrasound.com/content/11/1/11 Figure 10 Arteries harvested on day 28 stained with Elastica van Gieson (EvG). A: Significantly thickened intima in the arteries is seen with ICAM-1 siRNA without ultrasound irradiation; B: B shows significant suppression of neointimal formation in the artery with ICAM-1 siRNA with microbubble administration and ultrasound irradiation. C: VACM-1 siRNA had an effect statistically comparable to that of ICAM-1 siRNA in the prevention of neointimal formation. D: The scrambled siRNA did not suppress neointimal formation [33]. Figure 10 Arteries harvested on day 28 stained with Elastica van Gieson (EvG). A: Significantly thickened intima in the arteries is seen with ICAM-1 siRNA without ultrasound irradiation; B: B shows significant suppression of neointimal formation in the artery with ICAM-1 siRNA with microbubble administration and ultrasound irradiation. C: VACM-1 siRNA had an effect statistically comparable to that of ICAM-1 siRNA in the prevention of neointimal formation. D: The scrambled siRNA did not suppress neointimal formation [33]. Figure 11 DAPI positive cells were detected by fluorescent microscope. Compare with MSC infusion group, the US + Microbubble + MSC group has much more DAPI-positive cells localized in the ischemic myocardium (A-B). MSCs were labeled with DAPI, and all the cells were dyed in bright blue, and observed under light microscope (×200) and fluorescent microscope. (C) MSCs of US + Microbubble + MSC group (×100), (D) MSC infusion group (×100) [56]. Figure 11 DAPI positive cells were detected by fluorescent microscope. Compare with MSC infusion group, the US + Microbubble + MSC group has much more DAPI-positive cells localized in the ischemic myocardium (A-B). MSCs were labeled with DAPI, and all the cells were dyed in bright blue, and observed under light microscope (×200) and fluorescent microscope. (C) MSCs of US + Microbubble + MSC group (×100), (D) MSC infusion group (×100) [56]. Chen et al. Cardiovascular Ultrasound 2013, 11:11 Page 12 of 14 Chen et al. Cardiovascular Ultrasound 2013, 11:11 http://www.cardiovascularultrasound.com/content/11/1/11 http://www.cardiovascularultrasound.com/content/11/1/11 Figure 12 The MSCs adherent to the aortic endoluminal surface after ultrasound mediated delivery survive and undergo morphological changes (Scale bar = 100 mm), between 20min (two examples in A) 3h (B), and 24 h (C) post delivery [71]. Stem Cell Transplant for Treatment of Cardiovascular d b l d d b bbl Future work needs to be done before its clinical appli- cation, including optimization of microbubble prepar- ation technology to efficiently carry gene payloads while maintaining acoustic activity, prolonging circula- tion time to prevent clearance by the mononuclear cell, improving targeting techniques to enhance tissue bind- ing force in areas of high shear stress, and illustration of optimal ultrasound parameters for each microbubble and its intended application. What is more important, as UTMD mediated gene therapy involve the multiple interacting modalities, there needs to be close collabor- ation between chemists, ultrasound engineers, and bi- ologists to move this strategy to fruition. Competing interests The authors declare no competing interests. Competing interests The authors declare no competing interests. References 23. Park HJ, Yang F, Cho SW: Nonviral delivery of genetic med 23. 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Raake PW, Hinkel R, Müller S, Delker S, Kreuzpointner R, Kupatt C, Katus HA, Kleinschmidt JA, Boekstegers P, Müller OJ: Cardio-specific long-term gene expression in a porcine model after selective pressure-regulated retroinfusion of adeno-associated viral (AAV) vectors. Gene Ther 2008, 15:12–17. 34. Zhou S, Li S, Liu Z, Tang Y, Wang Z, Gong J, Liu C: Ultrasound-targeted microbubble destruction mediated herpes simplex virus-thymidine kinase gene treats hepatoma in mice. J Exp Clin Cancer Res 2010, 29:170. 13. Peng CA: Analysis of gene transfer rate with immobilized retroviral vectors. Ann N Y Acad Sci 2009, 1161:26–33. 35. Received: 23 March 2013 Accepted: 9 April 2013 Published: 17 April 2013 Received: 23 March 2013 Accepted: 9 April 2013 Published: 17 April 2013 22. Wu J, Zhang S, Wu X, Dong X, Xu P, Liu X, Li C, Huang Q: Enhanced transduction and improved photoreceptor survival of retinal degeneration by the combinatorial use of rAAV2 with a lower dose of adenovirus. Vision Res 2008, 48:1648–1654. Acknowledgements Increasing evidences prove that UTMD is a promising strategy to improve delivery efficiency, thus emerges as a method with great promise for target-specific gene This work was supported by Research Fund of Doctoral Program of Universities (20124401120002), China Postdoctoral Science Foundation (No. 2011M501375), Research Projects of Guangzhou Education Bureau Page 13 of 14 Page 13 of 14 Chen et al. Cardiovascular Ultrasound 2013, 11:11 http://www.cardiovascularultrasound.com/content/11/1/11 (No. 10A242), Research Projects of Guangzhou Technology Bureau (No. 12C22021645), Research Projects of Guangdong Science and Technology (2012B050300026), Natural Science Foundation of Guangdong Province (S2012040006593). 18. Cavazzana-Calvo M, Fischer A: Gene therapy for severe combined immunodeficiency: are we there yet? J Clin Invest 2007, 117:1456–1465. 19. Wang Z, Storb R, Lee D, Kushmerick MJ, Chu B, Berger C, Arnett A, Allen J Chamberlain JS, Riddell SR, Tapscott SJ: Immune responses to AAV in canine muscle monitored by cellular assays and noninvasive imaging. Mol Ther 2010, 18:617–624. Author details 1 1Department of Ultrasound Medicine, Key Laboratory for Major Obstetric Diseases of Guangdong Province, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou 510150, China. 2Section of Cardiology, St. Christopher’s Hospital for Children, Drexel University College of Medicine, 3601 A Street, Philadelphia, PA, USA. 20. Wilson JM: Lessons learned from the gene therapy trial for ornithine transcarbamylase deficiency. Mol Genet Metab 2009, 96:151–157. 21. Zheng XZ, Hong LL, Du LF, Wang HP, Qing Gu Q: In vivo and in vitro effects of ultrasound or/and microbubbles on recombinant adeno- associated virus-mediated transgene expression in the retina. Asian Biomed 2009, 3:497–506. 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https://openalex.org/W2100284033	https://www.research-collection.ethz.ch/bitstream/20.500.11850/92299/2/CIG-P.pdf	English	null	CIG-P: Circular Interaction Graph for Proteomics	BMC bioinformatics	2,014	cc-by	3,773	ETH Library Journal Article Rights / license: g Creative Commons Attribution 4.0 International CIG-P: Circular Interaction Graph for Proteomics Christopher K Hobbs1, Michelle Leung1, Herbert H Tsang1* and H Alexander Ebhardt2* Christopher K Hobbs1, Michelle Leung1, Herbert H Tsang1* and H Alexander Ebhardt2* Background: A typical affinity purification coupled to mass spectrometry (AP-MS) experiment includes the purification of a target protein (bait) using an antibody and subsequent mass spectrometry analysis of all proteins co-purifying with the bait (aka prey proteins). Like any other systems biology approach, AP-MS experiments generate a lot of data and visualization has been challenging, especially when integrating AP-MS experiments with orthogonal datasets. Results: We present Circular Interaction Graph for Proteomics (CIG-P), which generates circular diagrams for visually appealing final representation of AP-MS data. Through a Java based GUI, the user inputs experimental and reference data as file in csv format. The resulting circular representation can be manipulated live within the GUI before exporting the diagram as vector graphic in pdf format. The strength of CIG-P is the ability to integrate orthogonal datasets with each other, e.g. affinity purification data of kinase PRPF4B in relation to the functional components of the spliceosome. Further, various AP-MS experiments can be compared to each other. Conclusions: CIG-P aids to present AP-MS data to a wider audience and we envision that the tool finds other applications too, e.g. kinase – substrate relationships as a function of perturbation. CIG-P is available under: http://sourceforge.net/projects/cig-p/ roteomics, Affinity purification coupled to mass spectrometry, Visualization, Integration of datasets Keywords: Proteomics, Affinity purification coupled to mass spectrometry, Visualization, Integra [7], Abacus [8] or a creative relaxed local network scoring scheme [9]. A software package which aids the AP-MS researcher is Cytoscape [10], which is used to integrate, interrogate and filter AP-MS data as well as provide links to functionally annotated databases. The final product of this extensive AP-MS workflow is a high quality, high confidence protein network with edges and nodes frequently presented using the visual output of Cytoscape (see Figure 1A). Although familiar to the AP-MS scientist, these edges and nodes containing dia- grams are not always easy to grasp at first glance by a wider audience. A further limitation of current AP-MS visualization is the abstract integration of orthogonal datasets for relative interpretation of the newly created dataset. For conveying genomic information, Cirocs was introduced by Krzywinski et al. [11]. * Correspondence: herbert.tsang@twu.ca; ebhardt@imsb.biol.ethz.ch 1Applied Research Lab, Faculty of Natural and Applied Sciences, Trinity Western University Canada, Langley, BC, Canada 2Institute of Molecular Systems Biology, Department of Biology, ETH Zürich, Wolfgang-Pauli-Str. 16, 8093 Zürich, Switzerland © 2014 Hobbs et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. SOFTWARE Open Access CIG-P: Circular Interaction Graph for Proteomics Although Circos is very versatile, it also has a very complex input data structure, which includes a very steep learning curve prior to using the program. To simplify the generation of aesthetic comparative proteomics and to overcome the graphical limitation of Cytoscape, we developed Circular Interaction Graph for Proteomics (CIG-P). We believe the circular representation is a very intuitive way of conveying the information con- tent to a wider audience. Through abstracting the data, Originally published in: g y p BMC Bioinformatics 15, https://doi.org/10.1186/1471-2105-15-344 This page was generated automatically upon download from the ETH Zurich Research Collection. For more information, please consult the Terms of use. This page was generated automatically upon download from the ETH Zurich Research Collection. For more information, please consult the Terms of use. Hobbs et al. BMC Bioinformatics 2014, 15:344 http://www.biomedcentral.com/1471-2105/15/344 Background Proteomics using high mass accuracy mass spectrome- ters aims to characterize the proteome of a cell over space and time [1]. A critical sub-discipline of proteo- mics is affinity purification coupled to mass spectrom- etry (AP-MS) [2]. The goal of an AP-MS experiment is to qualitatively and quantitatively characterize protein complexes by either affinity-tagging a protein of interest (bait) or using a bait-specific antibody for affinity puri- fication. Following the enrichment of bait protein, bait-associated proteins will co-purify (termed: prey or interactors) and are identified by subsequent standard bottom-up proteomics approach using high mass accuracy mass spectrometry [3]. The digitized record of an AP-MS experiment is annotated using in silico search engines mapping MS/MS spectra to peptides in the case of shotgun-mass spectrometry [4] or mapping ion chro- matograms to spectral libraries in the case of SWATH- MS [5]. Following the in silico annotation is the critical contaminant filtering to avoid CRAPome annotations [6]. Frequently used filtering algorithms include SAINT Hobbs et al. BMC Bioinformatics 2014, 15:344 http://www.biomedcentral.com/1471-2105/15/344 Page 2 of 5 Figure 1 (See legend on next page.) Figure 1 (See legend on next page.) Figure 1 (See legend on next page.) Page 3 of 5 Hobbs et al. BMC Bioinformatics 2014, 15:344 http://www.biomedcentral.com/1471-2105/15/344 Hobbs et al. BMC Bioinformatics 2014, 15:344 http://www.biomedcentral.com/1471-2105/15/344 (See figure on previous page.) Figure 1 CIG-P circular diagram. On the outer circle, all protein reference sets are placed, separated by color. The width of the colored sections is proportional to the number of proteins they encompass. As scale, the white sections have a width of 3 proteins. A) AP-MS data displayed in circular layout in cytoscape. B) The AP-MS dataset is projected onto a functionally annotated protein dataset. The interactors of SRPK1 were drawn in colored arcs while the kinase substrate data was drawn in black arcs. As seen, the interactors and kinase substrates of SRPK1 are specific to sub-complexes of the spliceosome (see main text for discussion). C) Lenticular function CIG-P diagram (reappearance filtering ON) comparing the interactors of four kinases to interactors of SRPK1. From the black section of SRPK1 (bait protein), colored arcs are drawn representing the high quality protein-protein interactions found in each AP-MS experiment. As seen from the diagram, interactors of SRPK1 and SRPK2 are similar, while SRPK3 and PRPF4B show distinct differences in their interactome. is proportional to three proteins. Implementation For generating circular diagrams, we developed an intui- tive Java based graphical user interface termed CIG-P which uses three input files: an experiment file contain- ing the actual AP-MS experiment, a reference file con- taining a dataset to which the experiment is compared to and lastly a RBG color scheme (see Additional files 1, 2, 3, 4, 5 for examples). In detail, the experiment file contains three comma delimited columns. The first column contains a unique string identifying the bait protein, the second column contains a coding if the drawn arc should be colored (‘ppi’) or black (‘ivtk’), while the third column contains a unique string identi- fying the prey proteins. The reference file is also com- prised of three comma delimited columns whereby columns 1 and 3 are of descriptive nature, while col- umn 2 contains a unique protein identifier string and will be mapped to column 3 of the experiment file. In our case, we use unique uniprot IDs to be consistent within the dataset. The reference file also defines protein sets which are identified similar to the FASTA format initiating the protein set with an ‘>’. Lastly, the color scheme is coded in a highly customizable comma delim- ited RBG format. The number of colors is dependent on the dataset ideally one color per protein set should be used. In case the protein sets defined in the reference file outnumber the colors listed in the color scheme, latter will be cycled until all protein sets are colored. Knowing the RBG color code of each protein set will also make subsequent integration of CIG-P diagrams into presentations easier, as add-on fonts or drawings can take advantage of matching colors. More details on the organization of these files can be found in the (Additional file 2) User_Manual. For the lenticular function CIG-P diagram, the protein sets in the reference input file are defined as high confi- dence prey proteins of individual baits, comparing mul- tiple baits with each other. Alternatively, each protein set can be defined as set of high-confidence prey proteins per condition of the same bait where the cellular system underwent a perturbation. For example, the primary protein set is defined as the prey proteins of a particular bait when the cellular system is treated with the carrier, while all subsequent protein sets are the prey proteins upon stimulating the cellular system with a particular chemical compound. Implementation The resulting series of CIG-P cir- cular diagrams will rapidly visualize the changes in the interactome of the bait as a function of perturbation. CIG-P is also equipped with a reappearance mapping function. If turned OFF, only the first instance of a match is mapped and displayed as arc. The reappearance func- tion OFF can be useful in the above mentioned scenario of a perturbed cellular system whereby the primary protein set contains all prey proteins of the control, while all other protein sets contain prey proteins under perturbation. This set up allows for visualizing which prey proteins are new compared to the control AP-MS experiment (see Additional file 1: Figures S7-S10). On the other hand, with the reappearance function ON all interactions are redundantly drawn, which is important if multiple refer- ence protein sets contain the same protein, e.g. if a certain protein belongs to multiple functionally annotated protein complexes (see Application of CIG-P below). Background The interactions defined in the experiment file are drawn as arcs in the center of the circle (see Figure 1B and C). We believe this layout is quite intuitive and conveys the nature of an AP-MS experiment, whereby all interactions repre- sented by arcs originate from the bait protein. The initial default settings of circle size and arc thickness can be adjusted using the controls in the top left of the CIG-P’s graphical user interphase. Also, new experiment, refer- ence or color schemes can be loaded live into the newly drawn circular diagram. Following below, we present two distinct applications of CIG-P: first the quick visualization of various AP-MS experiments to each other, while the second application focuses on the visual integration of orthogonal proteomics datasets. we further hypothesize that scientist will be able to easier interpret their high quality, high confidence protein networks data. Results and Discussion The output of CIG-P is a circular diagram. On the outer circle of the diagram, both the reference protein sets and the bait protein are placed, latter is flanked by white spaces serving as separator and scale as each white space Page 4 of 5 Hobbs et al. BMC Bioinformatics 2014, 15:344 http://www.biomedcentral.com/1471-2105/15/344 Hobbs et al. BMC Bioinformatics 2014, 15:344 http://www.biomedcentral.com/1471-2105/15/344 Hobbs et al. BMC Bioinformatics 2014, 15:344 http://www.biomedcentral.com/1471-2105/15/344 lack of interactors from the tri-snRNP (U5.U4/U6) (see Additional file 1: Figures S15 and S16). On the contrary, PRPF4B almost exclusively interacts with tri-snRNP as- sociated proteins (see Additional file 1: Figure S18). From the lenticular function CIG-P analysis it was ex- pected that the interactors were quite dissimilar, how- ever, projecting the AP-MS dataset onto an orthogonal functionally fractionated proteomics dataset allows for a rapid functional annotation and visualization of these differences. Great insight into individual AP-MS experiments can be gained by projecting the newly generated data onto orthogonal proteomics datasets. Orthogonal datasets could include native protein complex fractionation tech- niques [12] or functional fractionations and annotations of super-complexes, such as the ribosome, proteasome or spliceosome [13]. Using this type of higher order annota- tion, the individual AP-MS experiment is immediately placed into a wider context for rapid interpretation of the data at hand. Besides rapid comparison of different kinase interac- tors, integration of orthogonal proteomics datasets, CIG-P can also serve to create new working hypothesis: for SRPK1 and SRPK2 not only the prey proteins were determined, but also the in vitro kinase substrates [14]. Hence, we took advantage of CIG-P’s function to either draw colored or black arcs (as defined in the experi- ment file). We define colored arcs as protein-protein interactions and black arcs as protein kinase substrates (see Figure 1B). In the case of SRPK1 we postulate that the kinase binds to 17S U2 related proteins and phosphor- ylates a U1 snRNP protein, presumably promoting a dynamic transition at the onset of the splicing process. To demonstrate the functionality of CIG-P, we visualize data of a published dataset [14] and draw conclusions from our circular diagrams which were not mentioned in the original publication, supporting our initial motivation that abstract visualization can guide scientists to establish new working hypothesis. The original dataset [14] encompasses the interac- tome of the CMGC clade of kinases. Four members of this CMGC clade show many interactions with splicing related proteins. Conclusions CIG-P assists the molecular systems biologist with AP-MS data to rapidly interrogate the high quality high confidence AP-MS prey protein dataset. Various AP-MS experiments can be compared to each other, while the integration of AP-MS data with orthogonal proteomics datasets allows the generation of statements with biological context and intuitive images suitable for a wide audience. Results and Discussion Hence, we will focus on these four kinases: SRPK1 (Uniprot ID: Q96SB4), SRPK2 (P78362), SRPK3 (Q9UPE1) and PRPF4B (Q13523). Although, all kinases mentioned are associated with the splicosome, latter is an extremely dynamic ribonucleic complex cata- lyzing the excision of exons from a primary messenger RNA. To visualize that some of these kinases with over- lapping prey proteins, we used the lenticular function of CIG-P and defined as protein sets (reference file) the preys associated with each kinase. When loading the experiment file of SRPK1 in the non-redundant mapping mode, all 26 interactors are visualized (Additional file 1: Figure S2). To immediately visualize the overlap of the SRPK1 interac- tome with the prey proteins of the other kinases, the re- appearance function of CIG-P was turned on (Additional file 1: Figure S7). From the redundant circular diagram it is apparent that SRPK1 and SRPK2 share a lot of prey proteins, while SRPK3 and PRPF4B have a distinct interactome. Project name: CIG-P Circular Interaction Graph for Proteomics To illustrate the distinct nature of PRPF4B its experi- ment file is loaded into CIG-P from the graphical user interface. It is apparent from the circular representation (see Additional file 1: Figures S5 and S10) that PRPF4B has a distinct interactome presumably acting on a subset of spliceosomal proteins within the splicing cascade. To follow up on commonalities and differences of these four kinases with spliceosomal prey proteins, we set as reference list a protein set derived from extensive functional fractionation of some 300 spliceosomal pro- teins [13]. The projection of AP-MS data onto an or- thogonal proteomics dataset allows scientist to place the AP-MS data into context (see Figure 1B). As already established in the lenticular function CIG-P, SRPK1 and SRPK2 share largely an overlapping network of interac- tors throughout the splicing cycle, except a complete Availability and requirements Availability and requirements Project name: CIG-P Circular Interaction Graph for Proteomics Project home page: http://sourceforge.net/projects/ cig-p/ Operating system: Platform independent Programming language: Processing Other requirements: Java version Java7v45 License: GNU GPL Any restrictions to use by non-academics: N/A Source code is available upon request from herbert. tsang@twu.ca. Additional files Additional file 1: Supporting Figures S1-S18. Additional file 2: User Manual for CIG-P. Additional file 3: Colour_rb.csv (colors). Additional file 4: Complex_resolved.csv (background). Additional file 5: SRPK1.csv (experiment). Project name: CIG-P Circular Interaction Graph for Proteomics Project name: CIG-P Circular Interaction Graph for Proteomics Additional file 1: Supporting Figures S1-S18. Additional file 2: User Manual for CIG-P. Additional file 3: Colour_rb.csv (colors). Additional file 4: Complex_resolved.csv (background). Additional file 5: SRPK1.csv (experiment). References 1. Lamond AI, Uhlen M, Horning S, Makarov A, Robinson CV, Serrano L, Hartl FU, Baumeister W, Werenskiold AK, Andersen JS, Vorm O, Linial M, Aebersold R, Mann M: Advancing cell biology through proteomics in space and time (PROSPECTS). Mol Cell Proteomics 2012, 11(3):O112.017731. 1. Lamond AI, Uhlen M, Horning S, Makarov A, Robinson CV, Serrano L, Hartl FU, Baumeister W, Werenskiold AK, Andersen JS, Vorm O, Linial M, Aebersold R, Mann M: Advancing cell biology through proteomics in space and time (PROSPECTS). Mol Cell Proteomics 2012, 11(3):O112.017731. 2. Gstaiger M, Aebersold R: Applying mass spectrometry-based proteomics to genetics, genomics and network biology. Nature reviews Genetics 2009 10:617–627. 2. Gstaiger M, Aebersold R: Applying mass spectrometry-based proteomics to genetics, genomics and network biology. Nature reviews Genetics 2009, 10:617–627. 3. Gingras AC, Gstaiger M, Raught B, Aebersold R: Analysis of protein complexes using mass spectrometry. Nature Reviews Mol Cell bio 2007, 8:645–654. 3. Gingras AC, Gstaiger M, Raught B, Aebersold R: Analysis of protein complexes using mass spectrometry. Nature Reviews Mol Cell bio 2007, 8:645–654. 4. Duncan DT, Craig R, Link AJ: Parallel tandem: a program for parallel processing of tandem mass spectra using PVM or MPI and X!Tandem. J Proteome Res 2005, 4:1842–1847. 4. Duncan DT, Craig R, Link AJ: Parallel tandem: a program for parallel processing of tandem mass spectra using PVM or MPI and X!Tandem. J Proteome Res 2005, 4:1842–1847. 5. Rost HL, Schmitt U, Aebersold R, Malmstrom L: pyOpenMS: a Python-based interface to the OpenMS mass-spectrometry algorithm library. Proteomics 2014, 14:74–77. 5. Rost HL, Schmitt U, Aebersold R, Malmstrom L: pyOpenMS: a Python-based interface to the OpenMS mass-spectrometry algorithm library. Proteomics 2014, 14:74–77. 6. Mellacheruvu D, Wright Z, Couzens AL, Lambert JP, St-Denis NA, Li T, Miteva YV, Hauri S, Sardiu ME, Low TY, Halim VA, Bagshaw RD, Hubner NC, Al-Hakim A, Bouchard A, Faubert D, Fermin D, Dunham WH, Goudreault M, Lin ZY, Badillo BG, Pawson T, Durocher D, Coulombe B, Aebersold R, Superti-Furga G, Colinge J, Heck AJ, Choi H, Gstaiger M, et al: The CRAPome: a contaminant repository for affinity purification-mass spectrometry data. Nat Methods 2013, 10(8):730–736. 7. Teo G, Liu G, Zhang J, Nesvizhskii AI, Gingras AC, Choi H: SAINTexpress: improvements and additional features in significance analysis of interactome software. J Proteomics 2014, 100:37–43. 8. Funding The development of CIG-P is supported by the faculty start-up grant of Trinity Western University. H.A.E. is a Marie Curie International Incoming Fellow (FP7). Received: 22 April 2014 Accepted: 30 September 2014 Published: 31 October 2014 Received: 22 April 2014 Accepted: 30 September 2014 Published: 31 October 2014 Abbreviations l 13. Agafonov DE, Deckert J, Wolf E, Odenwalder P, Bessonov S, Will CL, Urlaub H, Luhrmann R: Semiquantitative proteomic analysis of the human spliceosome via a novel two-dimensional gel electrophoresis method. Mol Cell Biol 2011, 31:2667–2682. 13. Agafonov DE, Deckert J, Wolf E, Odenwalder P, Bessonov S, Will CL, Urlaub H, Luhrmann R: Semiquantitative proteomic analysis of the human spliceosome via a novel two-dimensional gel electrophoresis method. Mol Cell Biol 2011, 31:2667–2682. CIG-P: Circular interaction graph for proteomics; AP-MS: Affinity purification coupled to mass spectrometry; RGB: Red-green-blue color code; SRPK1-3: Serine/Arginine-Rich Splicing Factor Kinase 1-3; PRPF4B: Pre-MRNA Processing Factor 4. 14. Varjosalo M, Keskitalo S, Van Drogen A, Nurkkala H, Vichalkovski A, Aebersold R, Gstaiger M: The protein interaction landscape of the human CMGC kinase group. Cell Rep 2013, 3:1306–1320. 14. Varjosalo M, Keskitalo S, Van Drogen A, Nurkkala H, Vichalkovski A, Aebersold R, Gstaiger M: The protein interaction landscape of the human CMGC kinase group. Cell Rep 2013, 3:1306–1320. Authors’ contributions CH and ML coded the program and were supervised by HHT. HAE had the initial idea of the project, tested software and wrote the manuscript. All authors read and commented on the manuscript. All authors read and approved the final manuscript. Competing interests The authors declare that they have no competing interests. doi:10.1186/1471-2105-15-344 Cite this article as: Hobbs et al.: CIG-P: Circular Interaction Graph for Proteomics. BMC Bioinformatics 2014 15:344. Additional files Additional file 1: Supporting Figures S1-S18. Additional file 2: User Manual for CIG-P. Additional file 3: Colour_rb.csv (colors). Additional file 4: Complex_resolved.csv (background). Additional file 5: SRPK1.csv (experiment). Page 5 of 5 Hobbs et al. BMC Bioinformatics 2014, 15:344 http://www.biomedcentral.com/1471-2105/15/344 13. Agafonov DE, Deckert J, Wolf E, Odenwalder P, Bessonov S, Will CL, Urlaub H, Luhrmann R: Semiquantitative proteomic analysis of the human spliceosome via a novel two-dimensional gel electrophoresis method. Mol Cell Biol 2011, 31:2667–2682. 14. Varjosalo M, Keskitalo S, Van Drogen A, Nurkkala H, Vichalkovski A, Aebersold R, Gstaiger M: The protein interaction landscape of the human CMGC kinase group. Cell Rep 2013, 3:1306–1320. doi:10.1186/1471-2105-15-344 Cite this article as: Hobbs et al.: CIG-P: Circular Interaction Graph for Proteomics. BMC Bioinformatics 2014 15:344. References Fermin D, Basrur V, Yocum AK, Nesvizhskii AI: Abacus: a computational tool for extracting and pre-processing spectral count data for label-free quantitative proteomic analysis. Proteomics 2011, 11:1340–1345. 9. Hauri S, Wepf A, van Drogen A, Varjosalo M, Tapon N, Aebersold R, Gstaiger M: Interaction proteome of human Hippo signaling: modular control of the co-activator YAP1. Mol Syst Biol 2013, 9:713. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: Submit your next manuscript to BioMed Central and take full advantage of: 10. Shannon P, Markiel A, Ozier O, Baliga NS, Wang JT, Ramage D, Amin N, Schwikowski B, Ideker T: Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res 2003, 13:2498–2504. • Convenient online submission 11. Krzywinski M, Schein J, Birol I, Connors J, Gascoyne R, Horsman D, Jones SJ, Marra MA: Circos: an information aesthetic for comparative genomics. Genome Res 2009, 19:1639–1645. 12. Kristensen AR, Gsponer J, Foster LJ: A high-throughput approach for measuring temporal changes in the interactome. Nat Methods 2012, 9:907–909. 12. Kristensen AR, Gsponer J, Foster LJ: A high-throughput approach for measuring temporal changes in the interactome. Nat Methods 2012, 9:907–909.
https://openalex.org/W3022385915	https://www.biorxiv.org/content/biorxiv/early/2019/06/18/674440.full.pdf	English	null	Accounting for long-range correlations in genome-wide simulations of large cohorts	PLOS genetics	2,020	cc-by	7,626	Coupling Wright-Fisher and coalescent dynamics for realistic simulation of population-scale datasets Dominic Nelson1, Jerome Kelleher2, Aaron P. Ragsdale1, Gil McVean2, and Simon Gravel1 Dominic Nelson1, Jerome Kelleher2, Aaron P. Ragsdale1, Gil McVean2, and Simon Gravel1 1McGill University and Genome Quebec Innovation Centre, Montréal, Québec, Canada 2Big Data Institute, Li Ka Shing Centre for Health Information and Discovery, University of Oxford, Oxford, UK 1McGill University and Genome Quebec Innovation Centre, Montréal, Québec, Canada 2Big Data Institute, Li Ka Shing Centre for Health Information and Discovery, University of Oxford, Oxford, UK 1 Abstract Coalescent simulations are widely used to examine the eﬀects of evolution and demographic history on the genetic makeup of populations. Thanks to recent progress in algorithms and data structures, simulators such as the widely-used msprime [1] now provide genome-wide simulations for millions of individuals. However, this software relies on classic coalescent theory and the corresponding assumptions that sample sizes are small relative to eﬀective population size and that the region being simulated is short. Here we show that coalescent simulations of long regions of the genome exhibit large biases in identity-by-descent (IBD), long-range linkage disequilibrium (LD), and an- cestry patterns, particularly when sample size is large. We present a Wright-Fisher extension to msprime, and show that it produces more realistic distributions of IBD, LD, and ancestry propor- tions, while also addressing more subtle biases of the coalescent. Further, these extensions are more computationally eﬃcient than state-of-the-art coalescent simulations when simulating long regions, including whole-genome data. For shorter regions, eﬃciency and accuracy can be maintained via a ﬂexible hybrid model which simulates the recent past under the Wright-Fisher model and uses coalescent simulations in the distant past. . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted June 18, 2019. ; https://doi.org/10.1101/674440 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted June 18, 2019. ; https://doi.org/10.1101/674440 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted June 18, 2019. ; https://doi.org/10.1101/674440 doi: bioRxiv preprint 1McGill University and Genome Quebec Innovation Centre, Montréal, Québec, Canada 2Big Data Institute, Li Ka Shing Centre for Health Information and Discovery, University of Oxford, Oxford, UK 2 Introduction Coalescent simulations are widely used in the development of statistical tools for genetics research [2–7]. They generate large sequence datasets which can be used to test our understanding of 1 1 . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted June 18, 2019. ; https://doi.org/10.1101/674440 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted June 18, 2019. ; https://doi.org/10.1101/674440 doi: bioRxiv preprint evolution or the ability of statistical tests to identify disease variants and avoid confounding factors. Coalescent theory has been used extensively for this purpose, with Hudson’s ms simulation program [8] having been cited over two thousand times since its publication in 2002. evolution or the ability of statistical tests to identify disease variants and avoid confounding factors. Coalescent theory has been used extensively for this purpose, with Hudson’s ms simulation program [8] having been cited over two thousand times since its publication in 2002. The more recent msprime coalescent simulation software [1] implements Hudson’s original algo- rithm [9], but with a performance increase of several orders of magnitude. This is achieved largely through the introduction of a new data structure, the succinct tree sequence [10, 11], which is extremely eﬃcient at storing genetic variation, and the genetically relevant component of the ge- nealogy of large numbers of samples. For example, simulating a 100 megabase region in a sample of 100,000 individuals generates an 88MB uncompressed succinct tree sequence, whereas the Newick tree format used by ms takes approximately 3.5TB of space [1]. Simulations are useful to the extent that they accurately reﬂect genetic variation within a real sample having undergone a similar demographic history. However, the coalescent is known to be biased relative to the Wright-Fisher model when the sample size is large [12] or for events in the recent past [13]. However, these biases have had limited practical impact because collecting such large empirical data sets was prohibitively costly and the simulation of such large samples was computationally overwhelming. 2 Introduction Both limitations have now been lifted: sequencing datasets now regularly include thousands of sequenced genomes, and msprime can simulate hundreds of thousands of genomes on a laptop computer. The assumptions of the underlying coalescent models should be carefully reexamined in this context. We highlight qualitative and quantitative inaccuracies in coalescent simulations of long regions, due to violated assumptions of the underlying genealogical model. We implement an extension to msprime which corrects the majority of these biases while reducing computational cost relative to coalescent simulations for large samples and long regions. This is accomplished by implementing a backwards-in-time Wright-Fisher model within msprime, which generates biologically plausible genealogies regardless of sample size. Models combining coalescent and Wright-Fisher dynamics can beneﬁt from strengths of both approaches [13], and our implementation also allows for hybrid simulations which combine the advantages of the Wright-Fisher model in recent generations with the eﬃciency of the coalescent in the distant past, maintaining high performance for shorter regions when the Wright-Fisher model alone is less eﬃcient. 2.1 Motivation This work was motivated by our observation that large-scale coalescent simulations resulted in un- realistic relatedness among samples, where nearly every pair of samples were second- or third-degree cousins according to the time to their most recent common ancestor. We traced this phenomenon to samples having more than 2t simulated ancestors at generation t in the past, allowing more recent relatedness than is possible under diploid inheritance. This is a side-eﬀect of Hudson’s coalescent 2 2 . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted June 18, 2019. ; https://doi.org/10.1101/674440 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted June 18, 2019. ; https://doi.org/10.1101/674440 doi: bioRxiv preprint algorithm lacking a diploid population pedigree [12]. Hudson’s coalescent model assumes a small region being simulated [14], and so does not account for multiple simultaneous recombinations during meiosis. Since the time between events in Hudson’s model can be less than a single generation, the per-generation recombination rate in long genomic regions is maintained by multiple single recombinations, but these occur through independent events that involve more than two parental lineages. This is unrealistic, and biases diversity predictions. An independent lineage is created with each event, leading to a large number of new lineages in the timescale of the ﬁrst generation (Figure 1b). This peculiarity of Hudson’s coalescent is negligible in small samples without migration, because simultaneous recombinations have a small eﬀect on the simulated genealogies [13, 15]. In larger samples, or under migration models, recent events induce long-range correlations along the genome due to the diploid population pedigree, and these are not accounted for by Hudson’s coalescent [12]. At whole-genome scale this becomes particularly clear. To highlight the magnitude of the genealogical distortions which can occur, we ﬁrst use both the coalescent and Wright-Fisher models to simulate 10,000 haploid samples in a diploid population of constant size 10,000. Each sample contains 22 chromosomes of realistic lengths. 2.1 Motivation Figure 2 shows that the number of lineages in the coalescent simulation increases very rapidly to reach 10 times the haploid population size 2N (A similar result was shown in [16]). In the Wright-Fisher simulation, the initial growth in number of lineages is much slower and can never exceed those present in the population. We describe some ways these diﬀerences aﬀect variation within simulated cohorts in the Results section. While genealogical distortions are most clear in the ﬁrst few generations, this explosion of lineages also aﬀects genealogies in the more distant past. Figure 2 also shows that, despite rapid coalescence lowering the initial spike in the number of lineages, their number remains above the population size until 150,000 generations in the past. 3.1 Implementation Implementing a discrete backwards-in-time Wright-Fisher model within msprime is conceptually straightforward. To understand the modiﬁcations needed, we ﬁrst outline Hudson’s original al- gorithm to simulate samples under the coalescent model. This brief overview is intended to give context to the modiﬁcations which enable Wright-Fisher simulations to be performed in the same framework. More details of how Hudson’s algorithm is implemented in msprime are given in [1]. framework. More details of how Hudson’s algorithm is implemented in msprime are given in [1]. First, a number of randomly-mating populations are speciﬁed, including eﬀective sizes and migration rates over time. Samples are introduced as haploid lineages within the populations, and the region of the genome being simulated is speciﬁed. The algorithm then constructs the genealogy 3 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted June 18, 2019. ; https://doi.org/10.1101/674440 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted June 18, 2019. ; https://doi.org/10.1101/674440 doi: bioRxiv preprint Parent 1, population 1 Parent 2, population 2 Coalescence Ancestral Material Gametes, population 1 Gametes, population 2 (a) (b) Figure 1: Comparing coalescent and Wright-Fisher lineages. (a) A single backwards-in-time Wright-Fisher gener- ation in our implementation. First, migration moves sample lineages (bottom) between populations. Lineages then draw parents (dotted outlines), and are split by recombination events into the two parental copies of the genome. Each parental copy is then merged into a new lineage, with coalescence events occurring where sample regions overlap (shaded areas). These new lineages are carried on to the next generation. (b) Schematic of lineages simulated under diﬀerent models over a region of length 8 Morgans. Parent 1, population 1 Parent 2, population 2 Coalescence Ancestral Material Gametes, population 1 Gametes, population 2 (a) (b) (b) (a) Figure 1: Comparing coalescent and Wright-Fisher lineages. (a) A single backwards-in-time Wright-Fisher gener- ation in our implementation. First, migration moves sample lineages (bottom) between populations. Lineages then draw parents (dotted outlines), and are split by recombination events into the two parental copies of the genome. 3.1 Implementation Each parental copy is then merged into a new lineage, with coalescence events occurring where sample regions overlap (shaded areas). These new lineages are carried on to the next generation. (b) Schematic of lineages simulated under diﬀerent models over a region of length 8 Morgans. 0 100000 200000 300000 Generations 0 50000 100000 150000 200000 Number of lineages msprime (WF) msprime (Hudson) Figure 2: Number of surviving lineages over time in coalescent and back-in-time Wright-Fisher dynamics. We simulated 10,000 haploid whole genomes with 22 chromosomes of realistic lengths in a population of 10,000 diploid individuals. The method for simulating multiple chromosomes is described in Section 4.1. 0 100000 200000 300000 Generations 0 50000 100000 150000 200000 Number of lineages msprime (WF) msprime (Hudson) Figure 2: Number of surviving lineages over time in coalescent and back-in-time Wright-Fisher dynamics. We simulated 10,000 haploid whole genomes with 22 chromosomes of realistic lengths in a population of 10,000 diploid individuals. The method for simulating multiple chromosomes is described in Section 4.1. 4 4 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted June 18, 2019. ; https://doi.org/10.1101/674440 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted June 18, 2019. ; https://doi.org/10.1101/674440 doi: bioRxiv preprint of each locus within this region by tracing its lineages backwards in time and tracking genomic segments that are ancestral to the sample. To begin, each lineage contains a single ancestral segment spanning the whole simulated genomic region of a sample. As time proceeds backwards, lineages can be split by recombination events (leav- ing the amount of ancestral material unchanged), or participate in common ancestor events, where any overlapping regions coalesce (reducing the amount of ancestral material). The recombination rate depends on the sum of the genetic map distance spanned by ancestral segments carried by all extant lineages, and common ancestor events occur at a rate determined by the number of uncoa- lesced lineages and the eﬀective population size. 4 Results In this section, we ﬁrst highlight qualitative diﬀerences between the coalescent and backwards Wright-Fisher models, and we show that the Wright-Fisher models provide a better description of the data while increasing tractability. We also study the impact of our implementation on previously-studied diﬀerences between coalescent and Wright-Fisher models, such as diﬀering pro- portions of singletons and doubletons [13]. These are shown in Figure S1 and Table S1. We focus here on large discrepancies which, to our knowledge, have not been previously examined. 3.1 Implementation Migration events move haploid lineages between randomly-mating populations, and demographic events modify the number of populations or their size and growth rate parameters. Recombination and common ancestor events are generated at rates depending on the amount of extant ancestral material, and the simulation terminates when every position on the genome has a most recent common ancestor Implementing a back-in-time Wright-Fisher model requires two important changes to Hudson’s algorithm. First, rather than drawing a time to the next event from an exponential distribution, we iterate though discrete generations and draw the events which occur at each time. Second, we modify the way recombination events are carried out, to account for the possibility of multiple re- combinations in a single transmission: we model the number and spatial distribution of breakpoints as a Poisson process, with rate equal to the per-generation recombination rate (i.e., the distance in Morgans). Crossover interference and non-crossover tracts would be straightforward to implement but are not included here. An overview of this model is illustrated in Figure 1a and the detailed order of events occurring at each generation is given in the Supplement. This model ensures that each gamete has a unique diploid parent. 4.1 Distribution of IBD Under the Wright-Fisher model, diploid inheritance constrains the possible gene genealogies [12] and introduces correlations in IBD sharing along long simulated regions: two samples with a recent 5 . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted June 18, 2019. ; https://doi.org/10.1101/674440 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted June 18, 2019. ; https://doi.org/10.1101/674440 doi: bioRxiv preprint common ancestor may be IBD at several distant regions of their genome (for example on diﬀerent chromosomes). In the coalescent, gene genealogies of unlinked loci are constructed independently [12]. This is a poor model for close relatives, since it is unlikely that any two samples will share more than one IBD segment from the same common ancestor, however recent. common ancestor may be IBD at several distant regions of their genome (for example on diﬀerent chromosomes). In the coalescent, gene genealogies of unlinked loci are constructed independently [12]. This is a poor model for close relatives, since it is unlikely that any two samples will share more than one IBD segment from the same common ancestor, however recent. Modelling relatedness patterns is important in large cohorts, where cryptic relatives are common [17, 18]. To illustrate the signiﬁcance of explicitly modelling diploid inheritance in a sample with close relatives, we simulated 500 human haploid whole genomes (chromosome lengths and recom- bination rates are described in the Supplement) in a diploid population of constant size 500 under the coalescent and Wright-Fisher models. We simulate a modestly-sized cohort since the eﬀects we wish to highlight depend only on the ratio of sample size to eﬀective population size, and since it simpliﬁes analysis of the resulting data. Larger cohorts can be simulated very eﬃciently, as shown in Section 4.3. We used the simulated genealogies to extract IBD segments inherited from common ancestors no more than 10 generations in the past. 4.1 Distribution of IBD Closer relatedness means more IBD segments and longer average length, leading to a relationship between number of segments and total length of IBD which is typically used in identifying relative status [17]. Figure 3 shows the qualitative diﬀerence between the two models, with the coalescent model exhibiting far too few IBD segments for closely related samples and poor clustering by TMRCA. By contrast, the Wright-Fisher model shows the expected qualitative distribution of relatedness [17]. An analytical model for the expected number and length of shared ancestry segments (shown as black dots in Figure 3) is provided in the Supplement. The long-range correlations induced by genealogical relatedness can also be measured as linkage disequilibrium between distant loci. This LD is used to estimate sizes of small populations in conservation genetics [19]. Hudson’s coalescent does not capture such LD patterns, whereas the Wright-Fisher extension to msprime predicts the patterns of LD expected under diploid mating (Figure S2). . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted June 18, 2019. ; https://doi.org/10.1101/674440 doi: bioRxiv preprint stronger source of variance [20]. We performed whole-genome simulations to evaluate how well the Wright-Fisher and coalescent models capture variance in ancestry. Figure 4 shows ancestry variance from simulations of 80 haploid samples in a diploid population of size 80, and a single event of 30% admixture at varying time in the past. These parameters were chosen to match those in [20], and the qualitative results depend only on the ratio of sample size to eﬀective population size. The approximate expected values are derived from an argument similar to the one presented in the supplement for IBD sharing and outlined in [20]. The Wright-Fisher model captures both short- and long-term variance in ancestry, as expected. In the coalescent simulations the initial phase of genealogical variance is not present, leading to a 20-fold underestimate of the variance in ancestry. Lacking a diploid population pedigree, whole- genome coalescent simulations of recently admixed populations do not reﬂect the distribution of ancestry expected in a large cohort. 4.2 Ancestry Variance Following Admixture 100 101 102 Number of generations 10 6 10 5 10 4 10 3 10 2 10 1 Variance in ancestry proportion Expected msprime (WF) msprime (Hudson) 100 101 102 Number of generations 10 6 10 5 10 4 10 3 10 2 10 1 Variance in ancestry proportion Expected msprime (WF) msprime (Hudson) Expected msprime (WF) msprime (Hudson) Figure 4: Variance in ancestry after a single admixture event, as a function of time since admixture. Calculated from 80 haploid samples in a diploid population of size 80, with 30% admixture proportions. Error bars show 95% conﬁdence intervals over 50 simulations. 4.2 Ancestry Variance Following Admixture In admixed populations, simulations should capture patterns of ancestry variation among present- day samples. The distribution of ancestry within recently admixed populations can be strongly dependent on pedigree structure, making coalescent simulations of these scenarios problematic. We consider the variance of ancestry proportions following a single pulse of migration. Ancestry variance can be divided into genealogical variance and recombination variance [20]. In the ﬁrst few generations after admixture, variance is driven by genealogical diﬀerences in the number of migrant ancestors of each individual. As time goes on, each present-day individual has more ancestors from the admixed generation, exponentially reducing this source of variance. After roughly 10 genera- tions, variation in the amount of genetic material received from each migrant ancestor becomes a 6 Figure 3: Number of IBD segments between pairs of individuals versus total length of shared IBD segments. 22 chromosomes of realistic lengths, simulated under Wright-Fisher model (top) and coalescent (bottom), compared to expectation under Equations (1) and (2). 500 haploid samples simulated with a diploid population size of 500. Figure 3: Number of IBD segments between pairs of individuals versus total length of shared IBD segments. 22 chromosomes of realistic lengths, simulated under Wright-Fisher model (top) and coalescent (bottom), compared to expectation under Equations (1) and (2). 500 haploid samples simulated with a diploid population size of 500. 7 7 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted June 18, 2019. ; https://doi.org/10.1101/674440 doi: bioRxiv preprint 100 101 102 Number of generations 10 6 10 5 10 4 10 3 10 2 10 1 Variance in ancestry proportion Expected msprime (WF) msprime (Hudson) Figure 4: Variance in ancestry after a single admixture event, as a function of time since admixture. Calculated from 80 haploid samples in a diploid population of size 80, with 30% admixture proportions. Error bars show 95% conﬁdence intervals over 50 simulations. 4.3 Performance The main advantage of msprime over alternate simulators is speed and scalability. This is achieved by eﬃcient algorithms and, especially, new data structures for storing and manipulating ancestral states throughout a simulation. We therefore need to ensure that the present modiﬁcation preserves these advantages. An important part of msprime’s eﬃciency is that Hudson’s algorithm simulates only those events which aﬀect variation within the samples. This means that long stretches of time can be traversed in a single step if they contain no such events. However, Figure 2 shows that the number of lineages 8 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted June 18, 2019. ; https://doi.org/10.1101/674440 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted June 18, 2019. ; https://doi.org/10.1101/674440 doi: bioRxiv preprint 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Simulated length (bp) 1e9 0 100 200 300 400 500 600 Time (s) msprime (WF) msprime (Hudson) hybrid (100 WF generations) hybrid (1000 WF generations) Figure 5: Computation time of Hudson coalescent, Wright-Fisher, and hybrid models. Hybrid models used 100 and 1000 Wright-Fisher generations before switching to the coalescent. Simulations contain from 1 to 22 chromosomes of realistic lengths (using the method described in Section 4.1) in 1000 haploid samples within a diploid population of size 10000. Results for other population sizes are shown in Figure S3. 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Simulated length (bp) 1e9 0 100 200 300 400 500 600 Time (s) msprime (WF) msprime (Hudson) hybrid (100 WF generations) hybrid (1000 WF generations) Figure 5: Computation time of Hudson coalescent, Wright-Fisher, and hybrid models. Hybrid models used 100 and 1000 Wright-Fisher generations before switching to the coalescent. Simulations contain from 1 to 22 chromosomes of realistic lengths (using the method described in Section 4.1) in 1000 haploid samples within a diploid population of size 10000. Results for other population sizes are shown in Figure S3. 4.3 Performance in whole-genome coalescent simulations is so high that the time between events will on average be much less than a single generation. Furthermore, these lineages come at an additional memory and computational cost for the coalescent model. Our Wright-Fisher extension is integrated with msprime’s core simulation framework, and can also easily be combined with coalescent simulations as part of a hybrid model (a hybrid Wright- Fisher/coalescent approach has also been proposed in [13]). This allows us to combine the strengths and eﬃciencies of both models, using the Wright Fisher for robust recent genealogies, and switching to the coalescent in the deeper past when the number of events per generation is low. Figure 5 shows a signiﬁcant performance advantage for pure Wright-Fisher simulations at whole- genome scale, and a modest advantage for slightly shorter regions. For simulations of a small number of chromosomes, the hybrid and coalescent models have nearly identical performance, but the hybrid simulations yield more realistic recent genealogies. Wright-Fisher simulations are therefore faster and provide more biologically plausible outcomes than the pure coalescent approach. 5 Discussion While the Wright-Fisher model may generate a more realistic pedigree than the coalescent model in the recent past, it is still a highly idealized model. Our implementation does not track monogamous couples, for example, and therefore will overestimate the prevalence of half-sibs and underestimate 9 . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted June 18, 2019. ; https://doi.org/10.1101/674440 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted June 18, 2019. ; https://doi.org/10.1101/674440 doi: bioRxiv preprint full sibs compared to a realistic human cohort. Assortative mating and inbreeding are not accounted for, and the migration model, while biologically plausible, is a simpliﬁcation of the real migration process (see implementation details in Supplement). Care should be taken in applications which are particularly sensitive to ﬁne-scale mating or migration patterns. Many of these issues can be addressed by allowing simulations to take place within a pre-speciﬁed pedigree, which is a natural extension to our backwards-in-time Wright-Fisher implementation. Rather than drawing genealogical links at random according to demographic parameters, lineages can simply follow a known pedigree. When reaching a pedigree founder, simulations can then continue by reverting to either the Wright-Fisher or the coalescent models. Real pedigrees of any size could then be used, from extended families up to population-scale [21], or they could be generated with the desired patterns of monogamy or assortative mating in a separate step. We leave such an implementation for future work. Other improvements are also possible. Assigning sexes to parents would allow simulation of the X-chromosome and sex-biased migration. Recombination can be extended to model crossover interference and sex-biased recombination, which have eﬀects on the distribution of IBD [22], as well as non-crossover events. The performance of the hybrid model could also be improved. If the number of Wright-Fisher generations were chosen optimally, it is likely to be more eﬃcient than pure Wright-Fisher simulations in nearly all scenarios. Better guidelines for ﬁnding this optimal value could be developed, or possibly built into the simulation framework itself. 5 Discussion The limitations of the coalescent model have been well-studied, but are generally shown to have modest eﬀects. This is not always the case - we have shown signiﬁcant qualitative and quanti- tative biases in whole-genome simulations of large, complex cohorts. Analysis of such cohorts is challenging, and simulations are a valuable tool for evaluating disease associations and the eﬀects of demography in this context. We have presented here an extension to msprime which corrects major biases and increases performance at whole-genome scale, allowing simulations to continue supporting modern large-scale sequencing eﬀorts. 6 Web Resources • msprime repository: https://github.com/tskit-dev/msprime • msprime repository: https://github.com/tskit-dev/msprime • Documentation (including Wright-Fisher simulations): https://msprime.readthedocs.io • Documentation (including Wright-Fisher simulations): https://msprime.readthedocs.io [1] J. Kelleher, A. M. Etheridge, and G. McVean. “Eﬃcient Coalescent Simulation and Genealog- ical Analysis for Large Sample Sizes”. In: PLoS Comput Biol 12.5 (May 2016), pp. 1–22. References [1] J. Kelleher, A. M. Etheridge, and G. McVean. “Eﬃcient Coalescent Simulation and Genealog- ical Analysis for Large Sample Sizes”. In: PLoS Comput Biol 12.5 (May 2016), pp. 1–22. 10 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted June 18, 2019. ; https://doi.org/10.1101/674440 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted June 18, 2019. ; https://doi.org/10.1101/674440 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted June 18, 2019. ; https://doi.org/10.1101/674440 doi: bioRxiv preprint [2] C. S. Carlson, M. A. Eberle, M. J. Rieder, Q. Yi, L. Kruglyak, and D. A. Nickerson. “Selecting a Maximally Informative Set of Single-Nucleotide Polymorphisms for Association Analyses Using Linkage Disequilibrium”. In: The American Journal of Human Genetics 74.1 (2004), pp. 106–120. [3] B. F. Voight, S. Kudaravalli, X. Wen, and J. K. Pritchard. “A map of recent positive selection in the human genome.” In: PLoS biology 4.3 (2006), e72. [4] R. N. Gutenkunst, R. D. Hernandez, S. H. Williamson, and C. D. Bustamante. “Inferring the joint demographic history of multiple populations from multidimensional SNP frequency data”. In: PLoS Genetics 5.10 (2009). [5] H. Li and R. Durbin. “Inference of human population history from individual whole-genome sequences.” In: Nature 475.7357 (2011), pp. 493–6. [6] N. Li and M. Stephens. “Modeling Linkage Disequilibrium and Identifying Recombination Hotspots Using Single-Nucleotide Polymorphism Data”. In: Genetics 165.4 (2003), pp. 2213– 2233. [7] R. Nielsen, S. Williamson, Y. Kim, M. J. Hubisz, A. G. Clark, and C. Bustamante. “Genomic scans for selective sweeps using SNP data”. In: Genome Research 15.11 (2005), pp. 1566–1575. [8] R. R. Hudson. “Generating samples under a Wright-Fisher neutral model of genetic variation”. In: Bioinformatics 18.2 (2002), pp. 337–338. [9] R. R. Hudson. “Properties of a neutral allele model with intragenic recombination”. In: The- oretical Population Biology 23.2 (1983), pp. References 183–201. [10] J. Kelleher, K. R. Thornton, J. Ashander, and P. L. Ralph. “Eﬃcient pedigree recording for fast population genetics simulation”. In: PLoS computational biology 14.11 (2018), e1006581. [11] J. Kelleher, Y. Wong, P. K. Albers, A. W. Wohns, and G. McVean. “Inferring the ancestry of everyone”. In: BioRxiv (2018), pp. 1–42. [12] J. Wakeley, L. King, B. S. Low, and S. Ramachandran. “Gene genealogies within a ﬁxed pedigree, and the robustness of kingman’s coalescent”. In: Genetics 190.4 (2012), pp. 1433– 1445. [13] A. Bhaskar, A. G. Clark, and Y. S. Song. “Distortion of genealogical properties when the sample is very large.” In: Proceedings of the National Academy of Sciences of the United States of America 111.6 (2014), pp. 2385–90. arXiv: arXiv:1308.0091v1. [14] R. R. Hudson. “Gene genealogies and the coalescent process”. In: Futuyma, D. and Antonovics, J. (eds), Oxford Surveys in Evolutionary Biology. Vol. 7. 1990, pp. 1–44. 11 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted June 18, 2019. ; https://doi.org/10.1101/674440 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted June 18, 2019. ; https://doi.org/10.1101/674440 doi: bioRxiv preprint [15] P. R. Wilton, P. Baduel, M. M. Landon, and J. Wakeley. “Population structure and coalescence in pedigrees: Comparisons to the structured coalescent and a framework for inference”. In: Theoretical Population Biology 115 (2017), pp. 1–12. [16] J. L. Davies, F. Simančík, R. Lyngsø, T. Mailund, and J. Hein. “On recombination-induced multiple and simultaneous coalescent events”. In: Genetics 177.4 (2007), pp. 2151–2160. [17] B. M. Henn, L. Hon, J. M. Macpherson, N. Eriksson, S. Saxonov, I. Pe’er, and J. L. Mountain. “Cryptic distant relatives are common in both isolated and cosmopolitan genetic samples”. In: PLoS ONE 7.4 (2012). [18] V. Shchur and R. Nielsen. “On the number of siblings and p-th cousins in a large population sample”. In: Journal of Mathematical Biology 77.5 (2018), pp. 1–20. [19] R. S. Waples. “A bias correction for estimates of eﬀective population size based on linkage disequilibrium at unlinked gene loci”. [24] A. P. Ragsdale and S. Gravel. “Unbiased estimation of linkage disequilibrium from unphased data”. In: BioRxiv doi:10.1101/557488 (2019). 1.1 Wright-Fisher Implementation Details We describe here the precise order of events happening at each generation in our implementation of the Wright-Fisher model. In the ﬁrst (‘current’) generation, samples are initialized as haploid copies of the region to be simulated (which can later be paired to form diploid individuals). Lineages of each sample are then constructed backwards in time, with each generation explicitly simulated as follows: 1. Migration events are drawn according to the forwards-time rates provided, and migrant lin- eages are moved to their new population. This is equivalent to migration of gametes, as opposed to migration of diploid individuals. A forwards-time event from population i to j moves a lineage from population j to i backwards in time. 2. Demographic events are carried out, such as changes to population sizes or growth rates, mass migrations, or bottlenecks. 3. Each haploid lineage draws a diploid parent within its current population. 4. Recombination occurs, with each breakpoint alternately assigning segments to be inherited from one of the two parental copies of the genome (back-and-forth recombination, see Figure 1b in the main text). 5. Segments inheriting from the same parental copy of the genome are merged into a single lineage, with coalescent events recorded in overlapping regions. When there is a single ancestral lineage at every position in the simulated genome, the simulation terminates. Our whole-genome simulations are performed with a single chromosome of length 28.75 Morgans and 22 ‘eﬀective’ chromosomes of realistic lengths separated by 0.5 Morgans. This is not exactly equivalent to simulating fully independent chromosomes. However, this should not have a qualitative impact on the analyses considered here. References In: Conservation Genetics 7.2 (2006), pp. 167–184. [20] S. Gravel. “Population genetics models of local ancestry”. In: Genetics 191.2 (2012), pp. 607– 619. [21] BALSAC. (2018). BALSAC Population Database: 2016-2017 Annual Report. http://balsac. uqac.ca/english/files/2018/01/BALSAC_RA2017_EN_page_WEB_v2-1.pdf. [22] M. Caballero, D. N. Seidman, T. D. Dyer, D. M. Lehman, J. E. Curran, R. Duggirala, J. Blangero, and A. L. Williams. “Surprising impacts of crossover interference and sex-speciﬁc genetic maps on identical by descent distributions”. In: bioRxiv (2019), p. 527655. [23] W. G. Hill and A. Robertson. “Linkage disequilibrium in ﬁnite populations”. In: Theoretical and Applied Genetics 38.6 (1968), pp. 226–231. [24] A. P. Ragsdale and S. Gravel. “Unbiased estimation of linkage disequilibrium from unphased data”. In: BioRxiv doi:10.1101/557488 (2019). 12 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted June 18, 2019. ; https://doi.org/10.1101/674440 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted June 18, 2019. ; https://doi.org/10.1101/674440 doi: bioRxiv preprint 1.2 Long range linkage disequilibrium For pairs of loci at low recombination distances (r ≪1), it is unlikely for more than a single recombination event to occur in a given meiosis. In this case, the coalescent accurately models LD between linked loci. For larger recombination distances, the probability of multiple recombinations must be consid- ered, and loci only become unlinked under odd number of recombinations. This has probability 13 . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted June 18, 2019. ; https://doi.org/10.1101/674440 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted June 18, 2019. ; https://doi.org/10.1101/674440 doi: bioRxiv preprint P(odd # rec. events\|r) = 1−e−2r 2 , which has a maximum value of 1/2. This leads to non-zero long- range LD, even in the case of fully unlinked loci. The diploid Wright-Fisher captures this, but coalescent estimates decay to zero for increasing r (Figure S2). In diploid individuals, multiple gametes are generated by recombination of the same two parental copies of the genome, and this has consequences on IBD and LD patterns: some classical models of population genetics neglect this and draw pairs of parental chromosomes independently for each gamete ([23]). This also underestimate the amount of linkage disequilibrium among distant sites (Figure S2). Our Wright-Fisher extension to msprime therefore correctly accounts for diploid mating (Figure 1b). 1.3 An approximate model for IBD sharing To provide a simple analytical model for this relationship, we examined a pair of haploid samples sharing a single diploid common ancestor at time t generations in the past. We derived approxima- tions for the number and length of shared haplotypes given t. We can think of the ancestry of each haploid genome as a mosaic formed by copying genomic segments from its 2t−1 possible ancestors. Similarly, a pair of haploid samples can be seen as a mosaic formed by copying from one ancestor for each sample. We can deﬁne paired-ancestry segments as continuous segments having no changes in ancestry in either sample. By this deﬁnition, if each sample has K chromosomes of total length L Morgans, the pair will have on average K + 2Lt paired-ancestry segments. Since each haploid sample has 2t−1 possible ancestors from which to inherit genetic material, a pair of samples will both inherit a paired-ancestry segment from their common ancestor with probability 22t−2. Since the ancestor is diploid, they inherit from the same ancestral copy of the genome with probability 1 2. The probability that a paired-ancestry segment is IBD in the pair is therefore 1 22t−1, and the expected number of IBD segments s between the pair is: s = K + 2Lt 22t−1 . (1) (1) The length of the genome shared, denoted by x, corresponds to L times the probability of having a shared ancestor at any particular locus, which is 1 22t−1, giving: The length of the genome shared, denoted by x, corresponds to L times the probability of having a shared ancestor at any particular locus, which is 1 22t−1, giving: x = L 22t−1. (2) (2) The expected values (s, x) are shown in Figure 3 as black dots for t from 1 to 5 generations, corresponding to half-siblings, ﬁrst cousins, and so on. The expected values (s, x) are shown in Figure 3 as black dots for t from 1 to 5 generations, corresponding to half-siblings, ﬁrst cousins, and so on. 14 14 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted June 18, 2019. ; https://doi.org/10.1101/674440 doi: bioRxiv preprint . 1.3 An approximate model for IBD sharing CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted June 18, 2019. ; https://doi.org/10.1101/674440 doi: bioRxiv preprint Singletons Doubletons Tripletons 200 400 600 800 1000 count model Hudson Wright-Fisher Figure S1: Number of singletons, doubletons, and tripletons simulated under Wright-Fisher and Hudson coalescent models. A 1Mb region was simulated 100 times in 20,000 haploid lineages in a diploid population of 10,000 individuals. Singletons Doubletons Tripletons 200 400 600 800 1000 count model Hudson Wright-Fisher model Hudson Wright-Fisher count Tripletons Figure S1: Number of singletons, doubletons, and tripletons simulated under Wright-Fisher and Hudson coalescent models. A 1Mb region was simulated 100 times in 20,000 haploid lineages in a diploid population of 10,000 individuals. Figure S1: Number of singletons, doubletons, and tripletons simulated under Wright-Fisher and Hudson coalescent models. A 1Mb region was simulated 100 times in 20,000 haploid lineages in a diploid population of 10,000 individuals. Frequency NW F −NH NW F Singletons 0.099131 Doubletons -0.047253 Tripletons 0.010092 Table S1: Relative diﬀerence in mean number of singletons, doubletons, and tripletons under the Wright-Fisher (NW F ) and Hudson (NH) models, from data shown in Figure S1. These results closely match those presented in [13]. 15 . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted June 18, 2019. ; https://doi.org/10.1101/674440 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted June 18, 2019. ; https://doi.org/10.1101/674440 doi: bioRxiv preprint 10 4 10 3 10 2 10 1 100 101 Total recombination rate 10 4 10 3 10 2 10 1 Linkage disequilibrium WF diploid msprime (WF) WF haploid Hill-Robertson (theory) msprime (Hudson) Figure S2: Linkage disequilibrium as measured by σ2 D = E[D2]/E[p(1 −p)q(1 −q)] under diﬀerent simulation and theory models [23]. Simulations were carried out with population size N = 1000 at steady state demography for a single 10M chromosome. 1.3 An approximate model for IBD sharing At fully unlinked loci, the expected value of σ2 D is 1 3N in a diploid model and 1 6N in a haploid model [24]. 10 4 10 3 10 2 10 1 100 101 Total recombination rate 10 4 10 3 10 2 10 1 Linkage disequilibrium WF diploid msprime (WF) WF haploid Hill-Robertson (theory) msprime (Hudson) Figure S2: Linkage disequilibrium as measured by σ2 D = E[D2]/E[p(1 −p)q(1 −q)] under diﬀerent simulation and theory models [23]. Simulations were carried out with population size N = 1000 at steady state demography for a single 10M chromosome. At fully unlinked loci, the expected value of σ2 D is 1 3N in a diploid model and 1 6N in a haploid model [24]. 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Simulated length (bp) 1e9 0 20 40 60 80 100 120 Time (s) msprime (WF) msprime (Hudson) hybrid (100 WF generations) hybrid (1000 WF generations) Figure S3: Computation time of Hudson coalescent, Wright-Fisher, and hybrid models with 100 and 1000 Wright- Fisher generations before switching to the coalescent. Simulations contain from 1 to 22 chromosomes of realistic lengths, using the method described in Section 4.1, in 500 haploid samples within a diploid population of size 500. 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Simulated length (bp) 1e9 0 20 40 60 80 100 120 Time (s) msprime (WF) msprime (Hudson) hybrid (100 WF generations) hybrid (1000 WF generations) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Simulated length (bp) 1e9 0 20 40 60 80 100 120 Time (s) msprime (WF) msprime (Hudson) hybrid (100 WF generations) hybrid (1000 WF generations) Figure S3: Computation time of Hudson coalescent, Wright-Fisher, and hybrid models with 100 and 1000 Wright- Fisher generations before switching to the coalescent. Simulations contain from 1 to 22 chromosomes of realistic lengths, using the method described in Section 4.1, in 500 haploid samples within a diploid population of size 500. 16
https://openalex.org/W2810705029	https://europepmc.org/articles/pmc6099185?pdf=render	English	null	Mechanism Sharing Between Genetic and Gestational Hypoxia-Induced Cardiac Anomalies	Frontiers in cardiovascular medicine	2,018	cc-by	7,188	Mechanism Sharing Between Genetic and Gestational Hypoxia-Induced Cardiac Anomalies Olivia Moumne 1, Rajib Chowdhurry 1, Cassandra Doll 1, Natalia Pereira 1, Mustafa Hashimi 1, Tabor Grindrod 1, James J. Dollar 2, Alberto Riva 3 and Hideko Kasahara 1* Olivia Moumne 1, Rajib Chowdhurry 1, Cassandra Doll 1, Natalia Pereira 1, Mustafa Hashimi 1, Tabor Grindrod 1, James J. Dollar 2, Alberto Riva 3 and Hideko Kasahara 1* 1 Department of Physiology and Functional Genomics, College of Medicine, University of Florida, Gainesville, FL, United States, 2 Department of Pathology, Immunology and Laboratory Medicine and the Emerging Pathogens Institute, University of Florida, Gainesville, FL, United States, 3 Bioinformatics, Interdisciplinary Center for Biotechnology Research, University of Florida, Gainesville, FL, United States Background: Cardiac development is a dynamic process both temporally and spatially. These complex processes are often disturbed and lead to congenital cardiac anomalies that affect approximately 1% of live births. Disease-causing variants in several genetic loci lead to cardiac anomalies, with variants in transcription factor NKX2-5 gene being one of the largest variants known. Gestational hypoxia, such as seen in high-altitude pregnancy, has been known to affect cardiac development, yet the incidence and underlying mechanisms are largely unknown. Citation: Moumne O, Chowdhurry R, Doll C, Pereira N, Hashimi M, Grindrod T, Dollar JJ, Riva A and Kasahara H (2018) Mechanism Sharing Between Genetic and Gestational Hypoxia-Induced Cardiac Anomalies. Front. Cardiovasc. Med. 5:100. doi: 10.3389/fcvm.2018.00100 ORIGINAL RESEARCH published: 13 August 2018 doi: 10.3389/fcvm.2018.00100 Edited by: Jin O-Uchi, University of Minnesota Twin Cities, United States Methods and Results: Normal wild-type female mice mated with heterozygous Nkx2-5 mutant males were housed under moderate hypoxia (14% O2) or normoxia (20.9% O2) conditions from 10.5 days of gestation. Wild-type mice exposed to hypoxia demonstrate excessive trabeculation, ventricular septal defects, irregular morphology of interventricular septum as well as atrial septal abnormalities, which overlap with those seen in heterozygous Nkx2-5 mutant mice. Genome-wide transcriptome done by RNA-seq of a 2-day hypoxic exposure on wild-type embryos revealed abnormal transcriptomes, in which approximately 60% share those from Nkx2-5 mutants without hypoxia. Gestational hypoxia reduced the expression of Nkx2-5 proteins in more than one-half along with a reduction in phosphorylation, suggesting that abnormal Nkx2-5 function is a common mechanism shared between genetic and gestational hypoxia-induced cardiac anomalies, at least at a speciﬁc developing stage. Reviewed by: Zhongzhou Yang, Nanjing University, China Gabrielle C. Geddes, Medical College of Wisconsin, United States Correspondence: Hideko Kasahara hkasahar@uﬂ.edu Specialty section: This article was submitted to Cardiovascular Genetics and Systems Medicine, Specialty section: This article was submitted to Cardiovascular Genetics and Systems Medicine, a section of the journal Frontiers in Cardiovascular Medicine a section of the journal Frontiers in Cardiovascular Medicine Conclusion: The results of our study provide insights into a common molecular mechanism underlying non-genetic and genetic cardiac anomalies. Received: 21 March 2018 Accepted: 02 July 2018 Published: 13 August 2018 Keywords: cardiac anomaly, gestational hypoxia, genetic mutation, mouse models, nkx2-5 Keywords: cardiac anomaly, gestational hypoxia, genetic mutation, mouse models, nkx2-5 RNA-seq and Real-Time Reverse Transcriptase (RT)-PCR p ( ) Total RNAs were isolated from E12.5 wild-type and mutant hearts with or without hypoxia. To maintain hypoxic conditions, hearts were dissected immediately, snap frozen, and stored at −80◦C until RNA isolation. To obtain enough RNA, two to three hearts were combined to prepare a single RNA sample (N = 3 or 4 samples from a total of 8–12 hearts were analyzed for each group). RNA library preparations and sequences were performed at MIT Genome Technology Core (Cambridge, MA). All RNA samples had a RNA integrity number above 8, which was considered high quality. Poly-adenylated RNA via oligo dT puriﬁcation was utilized for library preparation via standard TruSeq protocol (Illumina, San Diego, CA). Reads were single- end and 40 bases pairs long each. The sequence depth was approximately 35 million reads per sample. Considering that the majority of congenital cardiac malformations cannot be linked to speciﬁc genetic etiology, non-genetic eﬀectors of gene regulation need to be further explored (4, 9). Epidemiological studies have indicated that gestational hypoxia, such as seen in high-altitude pregnancies, increase the risk of low intrauterine growth and low birth weight, both of which are known to increase the risk of the fetus developing cardiovascular defects. Throughout the world, about 140 million people live in high altitude environments (elevated above 2,500 meters or 8,000 ft), of whom 400,000 live in the United States (10). Oxygen concentration is decreased from 20.9% at sea level to approximately 15% at 2,500 meters (altitude chart available at https://www.higherpeak.com/altitudechart. html). Not only a high-altitude pregnancy, but also various conditions, such as maternal smoking, congestive heart failure, pulmonary diseases, acute/chronic respiratory tract infections, anemia, preeclampsia, and placental insuﬃciency can cause gestational hypoxia (11). Analysis of RNA sequences was performed as follows: ﬁrst, the quality of the sequence and data was checked using FastQ to detect over-represented K-mers, GC content, and the presence of adaptors. We indexed the MM9 genome from Ensembl using Bowtie2. The raw reads were mapped to the MM9 genome using Tophat. A majority (75–85%; average 82%) of the reads were successfully mapped according to Samstat. After mapping, the gene expression was quantiﬁed with Cuﬄinks software, which normalizes transcript length, number of reads, and sequencing biases by calculating FPKM values (fragments per kilobase of exon model per million mapped reads). INTRODUCTION Congenital cardiac anomalies are the most prevalent birth defects, aﬀecting approximately 1% of live births (1–3). Despite remarkable progress in understanding cardiac development, the mechanisms underlying cardiac maldevelopment in embryos that result in malformations are largely unknown. August 2018 \| Volume 5 \| Article 100 Frontiers in Cardiovascular Medicine \| www.frontiersin.org Genetic and Gestational Hypoxia-Induced Cardiac Anomalies Moumne et al. Cardiac development is a dynamic process both temporally and spatially. Disease-causing variants in several loci have been known to cause cardiac anomalies, and the same nucleotide variant can lead to a wide variation in the type and severity of disease (4). For instance, familial congenital cardiac malformations due to a heterozygous NKX2-5 disease- causing variant are one of the largest sets of genetic mutations related to cardiac malformations (OMIM, NCBI), and currently nearly 40 heterozygous variants have been reported in humans (5–7). Not only in humans, but a varied range of severity of cardiac anomalies was demonstrated in Nkx2-5 mutant mice that have a single point mutation identiﬁed in human patients, and have a nearly identical genetic background by backcrossing (8). These results suggest that non-genetic factors, inﬂuence cardiac malformations. condition, replace bedding, water, and food, and then the cages were returned to the hypoxic chamber until approximately noon on gestation day 18 (E18.5). On the day of delivery, newborn mice at postnatal day 1 were sacriﬁced and hearts were isolated for histological analyses. At gestational day 12.5, or 15.5, mothers were sacriﬁced immediately after moving them from the hypoxic chamber to maintain hypoxic conditions in order to dissect embryonic hearts for RNA isolation or histological analyses. All animal experiments were performed with approval from the University of Florida Institutional Animal Care and Use Committee. RNA-seq and Real-Time Reverse Transcriptase (RT)-PCR A P-value < 0.05 and a fold change of more than 2 relative to control wild-normoxia hearts were considered signiﬁcant. The resulting matrices were reordered based on their expression values using the Euclidean distance and Single Linkage clustering methods, and heatmaps of the diﬀerential expression values (in log2 scale) were generated using the Permut Matrix program. In this study, we examined gestational hypoxia-induced murine cardiac anomalies using a physiological level of hypoxia of 14% oxygen concentration, and found an interaction between non-genetic and genetic cardiac anomalies induced by the abnormal function of Nkx2-5. Frontiers in Cardiovascular Medicine \| www.frontiersin.org Cardiac Anomalies in Newborn Wild-Type and Nkx2-5 Mutant With or Without Gestational Hypoxia area size using the same analytical methods with Image J as reported previously (13). Immunostaining and Western blotting were performed with the following primary antibodies or a cell death detection system: Nkx2-5 pAb (14), GAPDH (MAB374, Millipore, Bedford, MA), phospho-histone H3 (serine 10; Millipore 06-570), and TUNEL (In Situ Cell Death Detection kit, Roche, Basel, Switzerland). Fluorescent microscopic images were obtained using a Axiovert200M (ZEISS, Oberkochen, Germany) attached to CCD camera. Digitalized images were utilized for measurement using Image J software as described (15–17). The newborn mouse hearts were ﬁxed and examined for cardiac anomalies using 5-µm serial tissue sectioning of entire hearts as we described previously (8, 13, 18). Overall Cardiac Anomalies Displayed in Both Wild-Hypoxia and Nkx2-5 Mutant Mice: Ventricular Septal Defects (VSD), Excessive Ventricular Trabeculation and Irregular-Shaped Ventricular Septum Overall Cardiac Anomalies Displayed in Both Wild-Hypoxia and Nkx2-5 Mutant Mice: Ventricular Septal Defects (VSD), Excessive Ventricular Trabeculation and Irregular-Shaped Ventricular Septum Alkaline Phosphatase Treatment of Nkx2-5 To maintain hypoxic conditions, hearts were dissected immediately, snap frozen, and stored at −80◦C until protein puriﬁcation. After rinsing with Tris-buﬀered saline, E12.5 hearts were brieﬂy sonicated in the phosphatase buﬀer (50 mM Tris pH 9.3, 1 mM MgCl2, 0.1 mM ZnCl2, 10% glycerol, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl ﬂuoride), and centrifuged. Ten unit of calf intestinal alkaline phosphatase (CIAP, New England BioLabs, Ipswich, MA) was added to the 20 µl of supernatant and incubated for 30 min at 30◦C. As controls, the sample reaction was performed in the presence of 20 mM Na2HPO4 to inhibit a phosphatase reaction. Representative heart tissue sections obtained from P1 wild- normoxia, wild-hypoxia, mutant-normoxia, and mutant-hypoxia mice are shown in Figure 1B. In contrast to control wild- normoxia mice (n = 14) who did not show any cardiac anomalies, wild-hypoxia mice (n = 16) showed membranous or muscular VSDs, excessive ventricular trabeculation in the right ventricle (RV), and an irregular interventricular septum (Figure 1B, +/+ hypoxia; Table 1A). Of note, ventricular septal formation is completed with the interventricular communication being closed by embryonic day E13.5 to 14 in normal mouse embryos (19). The spectrum of cardiac anomalies displayed in wild-hypoxia mice overlaps with those in mutant-normoxia mice in this study (Figure 1B, +/R52G; Table 1B), similar to our previous study (8). In mutant mice, the penetrance of the VSD between the normoxia and hypoxia groups (73 vs. Animal Models Nkx2-5+/R52G knock-in mice were generated as reported previously (8) and were backcrossed to 129/Sv mice purchased from Charles River Laboratories (Wilmington, MA)(129/SvPasCrl) over 10 generations. Wild-type 129/Sv female mice were bred with Nkx2-5+/R52G males. Embryonic staging was determined by standard methods counting the morning on which the vaginal plug was found as embryonic day 0.5 (E0.5). Around noon on gestation day 10 (E10.5), pregnant female mice with weight gain (12) were placed in the hypoxic chamber (COY Lab Products, Grass Lake, MI) that was connected to nitrogen and oxygen gas. The oxygen content was gradually reduced from 20.9 to 14% over 15 min and the carbon dioxide was absorbed by Carbolime (AliMed, Inc., Dedham, MA). The cages were removed from the hypoxic chamber for approximately 10 to 15 min every day to check the mouse RT-PCR was performed using inventoried TaqMan gene expression assays (Applied Biosystems, Foster City, CA): Nkx2- 5 Mm00657783, Hey2 Mm00469280, Scn5a Mm00451971, KcneI Mm01215533, ANF Mm01255748, and hopx/HOD Mm00558629. Data were normalized to ß-actin expression (no. 4352933E). Duplicate experiments were averaged. Histological Analysis and Western Blotting Serial paraﬃn-embedded tissue sectioning of 5 µm thickness was performed as described previously (13). Two observers examined the digitalized images of the sections and performed quantitative histological measurements of trabecular vs. compact August 2018 \| Volume 5 \| Article 100 Frontiers in Cardiovascular Medicine \| www.frontiersin.org Frontiers in Cardiovascular Medicine \| www.frontiersin.org 2 Moumne et al. Genetic and Gestational Hypoxia-Induced Cardiac Anomalies Cardiac Anomalies in Newborn Wild-Type and Nkx2-5 Mutant With or Without Gestational Hypoxia 79%), excessive ventricular trabeculation (both 100%), and irregular interventricular septum (87 vs. 86%) was not signiﬁcantly diﬀerent (Table 1C). No Changes in Expression of Proliferation and Apoptosis Markers Excessive ventricular trabeculation may be related to abnormal cellular death or proliferations (21, 22). A number of cells positive for the cellular proliferation marker serine 10-phosphorylated histone H3 or the cell-death marker TUNEL, however, was not statistically diﬀerent among the four groups (Figures 2A,B). Excessive Ventricular Trabeculation The ventricular wall is composed of an outer compact layer and an inner trabecular layer. The representative enlarged images of RV showed a thickened trabecular layer with deep intertrabecular recesses (20) in wild-hypoxia and mutants compared to control wild-normoxia (Figure 1C). We quantiﬁed the total area of the trabecular and compact layers and compared the relative ratio to that of randomly selected newborn hearts by two independent observers using the constant criteria as shown in our previous study (8) throughout the analyses (n = 5– 7, Figure 1D). A signiﬁcant increase in the ratio of the RV trabecular layer relative to the total ventricle or the RV compact layer was found in wild-hypoxia and mutants compared to controls. Statistical Analysis Data presented are expressed as mean values plus or minus the standard error of the mean. Results were analyzed by SPSS (version 22) using crosstabs with Fisher’s exact test, non- parametric test, analysis of variance with Fisher’s post-hoc test, or independent T-test. Levene’s test was utilized for equality of variance, and P-values were calculated depending on the assurance of equality. P-values less than 0.05 were considered signiﬁcant. RESULTS Applying Gestational Hypoxia (14% O2) Beginning From Mid-gestation on Wild-Type and Nkx2-5 Mutant Embryos To apply a physiological level of hypoxia, wild-type (+/+) female mice bred with heterozygous Nkx2-5 mutant (+/R52G) male mice were housed in a 14% hypoxic chamber from 10.5 days of gestation, when pregnancy was evident. This mating allowed us to examine the eﬀects of hypoxia on wild-type as well as heterozygous Nkx2-5 mutant embryos without consideration of maternal cardiac defects due to a Nkx2-5 mutation (Figure 1A). The mothers were moved out of the hypoxic chamber on gestational day 18.5 for delivery. Except for hypoxia, other conditions, such as a 12-h day-night cycle, temperature, and food, remained the same between the normoxia and hypoxia groups. Hereafter, the four groups of mice are referred to as wild-normoxia, wild-hypoxia, mutant-normoxia, and mutant- hypoxia. Transcriptome Overlaps Between Wild-Hypoxia and Nkx2-5 Mutant-Normoxia Hearts To examine potential mechanisms underlying cardiac anomalies caused by gestational hypoxia, we performed diﬀerential gene expression analysis of developing hearts 2 days after gestational hypoxia (E12.5) using RNA-seq. Relative to control wild- normoxia hearts, 168 genes were diﬀerentially expressed in wild- hypoxia hearts, and 162 genes were diﬀerentially expressed in mutant-normoxia hearts (Figure 4A, Supplemental Tables 1, 2). Approximately 60% genes overlapped between wild-hypoxia and mutant-normoxia hearts. The expression of 225 transcripts were signiﬁcantly changed in the hypoxia hearts, or Nkx2-5 mutant- normoxia hearts, relative to control wild-normoxia hearts. These diﬀerences were visualized by a heatmap exhibiting with log2 values (Figure 4B). With some exceptions, the clustering of the expression values showed overlapping trends between wild- hypoxia and mutant-normoxia. This suggests the presence of a common mechanism underlying the cardiac anomalies that result from gestational hypoxia and the heterozygous Nkx2- 5 mutation. Expression of Nkx2-5 mRNA was unchanged by gestational hypoxia using RNA-seq (wild-hypoxia vs. wild- normoxia, fold diﬀerence = 1.11, P = 0.23; mutant-hypoxia vs. mutant-normoxia, fold diﬀerence = 0.99, P-value 0.95) and Taqman qRT-PCR (Figure 4C). persistent communications between the left and right atria, permitting postnatal shunting from left-to-right. During embryonic circulation, however, right-to-left shunting of blood through the foramen ovale is essential to circulate the oxygenated blood supplied from the maternal circulation. After birth, when the pulmonary circulation is established, the fossa ovalis is closed by attachment of the ﬂap valve, in other words, the foreshortened primary septum, to its rims (23). During the transition between embryonic to postnatal circulation at P1, a majority of normal mice still demonstrated morphological interatrial communication, with a part of the fossa ovalis still not being sealed by the ﬂap valve (Figure 3A). The size of the fossa ovalis, however, was smaller than the maximum length of the ﬂap valve (Figure 3A), showing the potential that fossa ovalis will be physiologically sealed. Only a few mice showed attachment of the ﬂap valve to the rim to close the fossa ovalis over the entire atrial septum (Figure 3B, closed). Quantitative analyses from a total of 58 hearts showed that size of the fossa ovalis was smaller than the maximum length of ﬂap valve in all control hearts (wild-normoxia), which was reduced to 73% in wild-hypoxia and mutant-normoxia hearts, and further reduced to 55% in mutant-hypoxia hearts (Figure 3C). Atrial Septum Abnormalities W/R52G normoxia) X2 Any malformations 0 (0%) 15 (100%) 0.000 29.0 Excessive trabeculation 0 (0%) 15 (100%) 0.000* 29.0 Ventricular septal defects 0 (0%) 11 (73%) 0.000* 16.5 Irregular-shaped ventricular septum 0 (0%) 13 (87%) 0.000* 22.0 P < 0.05. TABLE 1C \| Cardiovascular malformations, Nkx2-5 mutant normoxia vs. hypoxia. W/R52G normoxia (n = 15) W/R52G hypoxia (n = 14) P(W/R52G normoxia vs. W/R52G hypoxia) X2 Any malformations 15 (100%) 14 (100%) 1.0 NA# Excessive trabeculation 15 (100%) 14 (100%) 1.0 NA# Ventricular septal defects 11 (73%) 11 (79%) 1.0 0.109 Irregular-shaped ventricular septum 13 (87%) 12 (86%) 1.0 0.006 #Since all the animals had any malformations including ventricular noncompaction, X2 cannot be calculated, however there were no statistical differences between two groups (Fisher’s test P = 1.0). TABLE 1C \| Cardiovascular malformations, Nkx2-5 mutant normoxia vs. hypoxia. TABLE 1A \| Cardiovascular malformations, wild-type normoxia vs. hypoxia. TABLE 1C \| Cardiovascular malformations, Nkx2-5 mutant norm A \| Cardiovascular malformations, wild-type normoxia vs. hypoxia rdiovascular malformations, Nkx2-5 mutant normoxia vs. hypoxia. Atrial Septum Abnormalities The most prevalent cardiac anomaly demonstrated in human patients with missense mutations in the NKX2-5 homeodomain is atrial septal defects (6), characterized by August 2018 \| Volume 5 \| Article 100 Frontiers in Cardiovascular Medicine \| www.frontiersin.org 3 Moumne et al. Genetic and Gestational Hypoxia-Induced Cardiac Anomalies RE 1 \| Experimental design of gestational hypoxia (14% O2 saturation) leading to cardiac anomalies that overlap with heterozygous Nkx2-5 mutants. (A) nes of experiments and hypoxic chamber. (B) Representative images of P1 heart sections with simpliﬁed illustrations. Left, wild-type (+/+) normoxia and xia; right, Nkx2-5 mutant (+/R52G) normoxia and hypoxia. (C) Enlarged images of heart sections of the RV. (D) Quantiﬁcation of area size of the total ventricle abecular layer relative to total ventricle, and RV trabecular relative to RV compact layer (mean ± S.E.). LA, left atrium; LV, left ventricle; RA, right atrium; RV, rig cle; and VSD, ventricular septal defect. P < 0.05. FIGURE 1 \| Experimental design of gestational hypoxia (14% O2 saturation) leading to cardiac anomalies that overlap with heterozygous Nkx2-5 mutants. (A) Timelines of experiments and hypoxic chamber. (B) Representative images of P1 heart sections with simpliﬁed illustrations. Left, wild-type (+/+) normoxia and hypoxia; right, Nkx2-5 mutant (+/R52G) normoxia and hypoxia. (C) Enlarged images of heart sections of the RV. (D) Quantiﬁcation of area size of the total ventricle, RV trabecular layer relative to total ventricle, and RV trabecular relative to RV compact layer (mean ± S.E.). LA, left atrium; LV, left ventricle; RA, right atrium; RV, right ventricle; and VSD, ventricular septal defect. P < 0.05. Frontiers in Cardiovascular Medicine \| www.frontiersin.org August 2018 \| Volume 5 \| Article 100 Genetic and Gestational Hypoxia-Induced Cardiac Anomalies Moumne et al. TABLE 1A \| Cardiovascular malformations, wild-type normoxia vs. hypoxia. W/W normoxia (n = 14) W/W hypoxia (n = 16) P (WW normoxia vs. WW hypoxia) X2 Any malformations 0 (0%) 12 (75%) 0.000 20.1 Excessive trabeculation 0 (0%) 10 (63%) 0.000* 13.1 Ventricular septal defects 0 (0%) 4 (25%) 0.045* 5.3 Irregular-shaped ventricular septum 0 (0%) 9 (56%) 0.000* 15.2 P < 0.05. TABLE 1B \| Cardiovascular malformations, wild-type vs. Nkx2-5 mutant (W/R52G) at normoxia. W/W normoxia (n = 14) W/R52G normoxia (n = 15) P (WW normoxia vs. Transcriptome Overlaps Between Wild-Hypoxia and Nkx2-5 Mutant-Normoxia Hearts The diﬀerence between the length of the ﬂap valve relative to the size of fossa ovalis, was highest in control wild-normoxia mice and was signiﬁcantly reduced in mutant-hypoxia mice (Figure 3D, Supplemental Figure 1). Reduction of Nkx2-5 Proteins in Wild-Type Embryos With Gestational Hypoxia Expression of Nkx2-5 protein, however, was reduced nearly one- half by gestational hypoxia relative to controls (Figures 4D,E). The majority of Nkx2-5 proteins from control hearts appeared to migrate into a single higher molecular weight. In contrast, Nkx2-5 proteins were separated into two distinct bands by hypoxia (Figure 4D), due to a reduction in phosphorylation (Figure 4F). mRNA expression of several known downstream targets of Nkx2-5 was reduced in wild-hypoxia and mutant hearts (Figure 4G). To note, Nkx2-5 acts not only as an activator but August 2018 \| Volume 5 \| Article 100 Frontiers in Cardiovascular Medicine \| www.frontiersin.org Frontiers in Cardiovascular Medicine \| www.frontiersin.org 5 Moumne et al. Genetic and Gestational Hypoxia-Induced Cardiac Anomalies FIGURE 2 \| No changes in expression of proliferation and apoptosis markers. (A) Representative images of phospho-histone 3 staining (left) and TUNEL staining of RV wall tissue sections (right). Arrowheads indicate positively stained cells. (B) Summarized data (mean ± S.E.) of phospho-histone 3 positive nuclei and TUNEL positive nuclei in entire hearts relative to the area size obtained from multiple hearts. Total area size examined in each group is shown. FIGURE 2 \| No changes in expression of proliferation and apoptosis markers. (A) Representative images of phospho-histone 3 staining (left) and TUNEL staining of RV wall tissue sections (right). Arrowheads indicate positively stained cells. (B) Summarized data (mean ± S.E.) of phospho-histone 3 positive nuclei and TUNEL positive nuclei in entire hearts relative to the area size obtained from multiple hearts. Total area size examined in each group is shown. of these cardiac anomalies overlaps with cardiac anomalies genetically induced by a heterozygous Nkx2-5 mutation in mice and humans. Gestational hypoxia reduces the expression of the Nkx2-5 protein by nearly one-half, and a genome- wide screening of mRNA shows that approximately 60% of dysregulated genes are overlapped between wild-hypoxia and mutant-normoxia relative to controls. To note, a 50% reduction of Nkx2-5 in heterozygous knockout mice leads to cardiac anomalies, including VSDs and ASDs (8, 26–28). In our study, VSDs were observed in 33% of heterozygous Nkx2-5 knockout mice (8), with this incidence in agreement with other studies (26– 28), whereas VSDs were observed in 82% of heterozygous Nkx2-5 knock-in mice having the same genetic background (8). also as a repressor depending on the context of the target genes in mouse mid-embryonic hearts (13). Reduction of Nkx2-5 Proteins in Wild-Type Embryos With Gestational Hypoxia The expression of mRNA or protein of several important cardiac transcription factors, including Tbx5, Gata4, Mef2c, Hand1 and 2, was examined in E12.5 hearts by RNA-seq or Western blotting. The expression of these transcription factors was unchanged by gestational hypoxia or the heterozygous Nkx2- 5 mutant (Supplemental Table 3, Supplemental Figure 2). Overall, there were phenotypic similarities between wild- hypoxia and Nkx2-5 mutant hearts, and Nkx2-5 proteins were reduced nearly by one-half after gestational hypoxia. Frontiers in Cardiovascular Medicine \| www.frontiersin.org DISCUSSION g g g The expression of Nkx2-5 proteins was reduced in embryonic hearts after 2 days of hypoxia without changing mRNA expression. Under various environmental stressors including hypoxia, all organisms respond and defend themselves to survive (29). Because proteins catalyze most cellular processes, rapid changes in protein levels are critical. There are many post-transcriptional steps at which cellular protein levels can be regulated, including abnormal RNA processing such as exporting to the cytoplasm, mRNA processing, and localization, translation, and post-translational modiﬁcations of proteins, as well as protein degradation (29). Nkx2-5 proteins are highly Although gestational hypoxia and a mutation in Nkx2-5 in mice have been shown to cause congenital heart disease (8, 24, 25), we report for the ﬁrst time to our knowledge that non-genetic and genetic cardiac anomalies share a common mechanism relating to abnormal function of Nkx2-5, at least at a speciﬁc developing stage, E12.5. Moderate gestational hypoxia, namely 14% oxygen saturation, induces cardiac anomalies in wild-type mice, such as VSDs, excessive ventricular trabeculation, and irregular interventricular septum morphology. The spectrum Frontiers in Cardiovascular Medicine \| www.frontiersin.org August 2018 \| Volume 5 \| Article 100 6 Moumne et al. Genetic and Gestational Hypoxia-Induced Cardiac Anomalies FIGURE 3 \| Comparison of interatrial communication on P1 hearts between three experimental groups and control (wild-normoxia). (A) Representative histological sections from P1 control hearts. Asterisk indicates open interactial communication. The size of fossa ovalis and the length of ﬂap valve are indicated. (B) Representative histological sections from P1 hearts demonstrating closed fossa ovalis (left), open fossa ovalis where the size of fossa ovalis is smaller than the length of ﬂap valve (middle), and open fossa ovalis where the size of the fossa ovalis is larger than the length of the ﬂap valve (right). (C) Ratio of hearts showing the size of the fossa ovalis as smaller than the length of the ﬂap valve. The number of mice examined is indicated. (D) The difference between the size of the fossa ovalis and the length of ﬂap valve. P < 0.05. FO, fossa ovalis. FIGURE 3 \| Comparison of interatrial communication on P1 hearts between three experimental groups and control (wild-normoxia). (A) Representative histological sections from P1 control hearts. Asterisk indicates open interactial communication. The size of fossa ovalis and the length of ﬂap valve are indicated. Frontiers in Cardiovascular Medicine \| www.frontiersin.org DISCUSSION (A) RNA-seq data n E12.5 wild-hypoxia hearts and 162 genes in normoxia Nkx2-5 mutant hearts relative to the wild-normoxia hearts (n = 3 or 4 samples from each ession of 225 transcripts that were signiﬁcantly changed in the wild-hypoxia hearts, or Nkx2-5 mutant hearts, relative to control wild-normoxia d by heatmap exhibiting with log2 values. Upregulation (magenta), downregulation (green), and mean gene expression (black). (C) Real-time mRNA relative to ß-actin in E12.5 hearts from four groups. (D) Western blotting demonstrating Nkx2-5 and GAPDH proteins in wild-normoxia and (E) Quantitative data for Nkx2-5 protein expression relative to GAPDH. (F) Western blotting demonstrates that the addition of phosphatase (CIAP) higher molecular weight band to a lower molecular weight band, which was inhibited by the addition of Na2HPO4 in the reaction performed al-time RT-PCR demonstrates the expression of several known Nkx2-5 targets normalized to ß-actin in four groups. Mean ± S.E. P < 0.05. ﬀected by complex compensatory mechanisms wild normoxia; our study could have missed initial responses FIGURE 4 \| Gestational hypoxia led to a reduction of Nkx2-5 proteins and changes in mRNA expression overlapping with the Nkx2-5 mutant. (A) RNA-seq data showed 166 genes in E12.5 wild-hypoxia hearts and 162 genes in normoxia Nkx2-5 mutant hearts relative to the wild-normoxia hearts (n = 3 or 4 samples from each group). (B) The expression of 225 transcripts that were signiﬁcantly changed in the wild-hypoxia hearts, or Nkx2-5 mutant hearts, relative to control wild-normoxia hearts was visualized by heatmap exhibiting with log2 values. Upregulation (magenta), downregulation (green), and mean gene expression (black). (C) Real-time RT-PCR of Nkx2-5 mRNA relative to ß-actin in E12.5 hearts from four groups. (D) Western blotting demonstrating Nkx2-5 and GAPDH proteins in wild-normoxia and wild-hypoxia hearts. (E) Quantitative data for Nkx2-5 protein expression relative to GAPDH. (F) Western blotting demonstrates that the addition of phosphatase (CIAP) resulted in shifting a higher molecular weight band to a lower molecular weight band, which was inhibited by the addition of Na2HPO4 in the reaction performed side-by-side. (G) Real-time RT-PCR demonstrates the expression of several known Nkx2-5 targets normalized to ß-actin in four groups. Mean ± S.E. P < 0.05. without being aﬀected by complex compensatory mechanisms. Nevertheless, a limited number of transcripts, namely about 150 genes, were diﬀerentially expressed relative to control wild-normoxia; our study could have missed initial responses that occured earlier than 2 days of hypoxia. DISCUSSION (B) Representative histological sections from P1 hearts demonstrating closed fossa ovalis (left), open fossa ovalis where the size of fossa ovalis is smaller than the length of ﬂap valve (middle), and open fossa ovalis where the size of the fossa ovalis is larger than the length of the ﬂap valve (right). (C) Ratio of hearts showing the size of the fossa ovalis as smaller than the length of the ﬂap valve. The number of mice examined is indicated. (D) The difference between the size of the fossa ovalis and the length of ﬂap valve. *P < 0.05. FO, fossa ovalis. or ventricular noncompaction, is a cardiomyopathy with persistence of the trabecular layer, which can lead to both diastolic and systolic dysfunction (20, 33–35). Mechanisms leading to excessive ventricular trabeculation by gestational hypoxia and the Nkx2-5 mutation need further investigation. For instance, excessive ventricular trabeculation was more evident in the right ventricle compared to the left by gestational hypoxia in P1 hearts. The reduction in Nkx2-5 proteins, however, was not apparently diﬀerent between right and left ventricles in E12.5 hearts (Supplemental Figure 3). This might suggest that the hypoxia on heart anomalies may involve other factors, such as Isl1, acting in the secondary heart ﬁeld (36). phosphorylated in hearts in vivo (30), but were reduced after exposure to hypoxia, suggesting that protein modiﬁcations, including phosphorylation, may be involved. There are open questions as to whether a reduction of the Nkx2-5 protein is a defensive mechanism in cardiac development, and how it is reduced under hypoxia. During normal cardiac development, the ventricular trabecular layer is formed on approximately E10.5, coinciding with an increase of in the ventricular myocardial mass (31, 32), likely to facilitate the exchange of oxygen and nutrients from the blood locating in the ventricular cavities. As normal cardiac development progresses, development of discrete coronary arteries allows the outer compact layers to thicken, with the trabecular layer becoming less obvious, but this is altered during gestational hypoxia. Increased trabeculation, RNAseq was examined after 2 days of hypoxia with the intention of ﬁnding early responses that lead to cardiac anomalies August 2018 \| Volume 5 \| Article 100 7 Moumne et al. Genetic and Gestational Hypoxia-Induced Cardiac Anomalies onal hypoxia led to a reduction of Nkx2-5 proteins and changes in mRNA expression overlapping with the Nkx2-5 mutant. ACKNOWLEDGMENTS We are grateful to Drs. Charles Wood, Robert Anderson, Miguel Zárate and the MIT Genome Technology Core for valuable suggestions and technical support. We would also like to thank Corey Astrom, ELS, for her editorial expertise and assistance with this manuscript. RNAseq data will be available at NCBI GEO repository database with the accession number GSE114532. We introduced hypoxia beginning at gestation day 10.5, when pregnancy was conﬁrmed by weight gain (12) and abdominal expansion. In humans, increased levels of human chorionic gonadotropin is a sensitive marker for pregnancy that is detectable shortly after pregnancy occurs, however, these tests for mice are not currently available to the research (12). FUNDING This work was supported by the NIH grant (1R21 HD090608), and the University Florida Opportunity Fund (to HK). This work was supported by the NIH grant (1R21 HD090608), and the University Florida Opportunity Fund (to HK). DISCUSSION Alternatively, this stage may be too early to explain the cardiac phenotypes August 2018 \| Volume 5 \| Article 100 Frontiers in Cardiovascular Medicine \| www.frontiersin.org 8 Genetic and Gestational Hypoxia-Induced Cardiac Anomalies Moumne et al. examined in P1 hearts. This is the same for heterozygous Nkx2- 5 mutant mice in which cardiac development will be most likely aﬀected prior to E12.5. however, there was no signiﬁcant diﬀerence in the incidence of cardiac anomalies, including VSDs, which was demonstrated in 73% of mutant-normoxia mice (n = 15) and 79% of mutant- hypoxia mice (n = 14). Our study agrees with the previous studies showing that gestational hypoxia causes cardiac anomalies when exposed to lower oxygen levels. For instance, 10.5% oxygen saturation between E10.5 and E13.5 led to VSDs with an incidence of 33% (n = 5 out of 15 mice) and ventricular non-compaction (24). The susceptibility of mouse hearts to hypoxia appeared stage-dependent, in which E10.5 embryos have the highest susceptibility between E10.5 and E18.5 (24). Outﬂow tract anomalies, such as double outlet right ventricle, were displayed under ∼10.5% hypoxia (24); but not in our hypoxic conditions using the 129/Sv mouse strain. Extreme hypoxia, namely 5.5% oxygen saturation for 8 h, led mice with the C57BL/6 background to cardiac anomalies (25). However, this low level of hypoxia will not be applicable, even on the highest mountain, Mt. Everest (22,800 feet or 6,960 meters, 6.9% oxygen saturation), and adult mice died under 5.5% oxygen saturation within 1 to 2 h under our experimental conditions using the 129/Sv mouse strain. In summary, we report that moderate chronic gestational hypoxia leads to cardiac anomalies that overlap with heterozygous Nkx2-5 mutant mice accompanied by a reduction in Nkx2-5 proteins. Non-genetic and genetic cardiac anomalies share a common mechanism regarding the abnormal function of Nkx2-5. Just as important, this ﬁnding is likely to provide insights into the common molecular mechanisms underlying non-genetic and genetic cardiac anomalies. Such insights would potentially allow for the future development of speciﬁc therapeutic strategies for patients suﬀering from a wide-ranges of congenital cardiac anomalies. SUPPLEMENTARY MATERIAL We initiated this study to test whether the severity of genetic cardiac anomalies induced by the heterozygous Nkx2-5 mutation will be worsened by a combination of genetic and environmental eﬀects (41), i.e., gestational hypoxia. Expression of several Nkx2-5 downstream targets were aﬀected by gestational hypoxia, We initiated this study to test whether the severity of genetic cardiac anomalies induced by the heterozygous Nkx2-5 mutation will be worsened by a combination of genetic and environmental eﬀects (41), i.e., gestational hypoxia. Expression of several Nkx2-5 downstream targets were aﬀected by gestational hypoxia, The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fcvm. 2018.00100/full#supplementary-material AUTHOR CONTRIBUTIONS Experiments were designed and performed by OM, RC, CD, NP, MH, TG, JD, AR, and HK. The manuscript was prepared by OM and HK. Under extreme hypoxia, such as 1–5% O2 in cell culture, transcription factor hypoxia-induced factor 1 alpha (Hif1α) protein is stabilized and its short half-life is extended (36–38). Several studies showed Hif1α plays a critical role in cardiac development with induction of Nkx2-5 transcription using Hif1α knockdown, knockout or cobalt chloride, which elicits hypoxia- like responses (36, 39, 40). Under 14% hypoxia starting from E10.5, however, there were no changes in expression of Nkx2-5 mRNA 2 days and 8 days of exposure of hypoxia, or Hif1α protein within 8 days of exposure of hypoxia relative to the age-matched normoxic condition (Supplemental Figure 4). 8. Ashraf H, Pradhan L, Chang EI, Terada R, Ryan NJ, Briggs LE, et al. A mouse model of human congenital heart disease: high incidence of diverse cardiac anomalies and ventricular noncompaction produced by heterozygous nkx2- 5 homeodomain missense mutation. Circ Cardiovasc Genet. (2014) 7:423–33. doi: 10.1161/CIRCGENETICS.113.000281 7. Benson DW. Genetic origins of pediatric heart disease. Pediatr Cardiol. (2010) 31:422–9. doi: 10.1007/s00246-009-9607-y REFERENCES 5. Scott EM, Carter AM, Grant PJ. 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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. No use, distribution or reproduction is permitted which does not comply with these terms. 26. Biben C, Weber R, Kesteven S, Stanley E, McDonald L, Elliott DA, et al. Cardiac septal and valvular dysmorphogenesis in mice heterozygous for mutations in the homeobox gene nkx2-5. Circ Res. (2000) 87:888–95. doi: 10.1161/01.RES.87.10.888 August 2018 \| Volume 5 \| Article 100 Frontiers in Cardiovascular Medicine \| www.frontiersin.org 10
https://openalex.org/W2951290121	https://www.frontiersin.org/articles/10.3389/fgene.2019.00639/pdf	English	null	Proband Whole-Exome Sequencing Identified Genes Responsible for Autosomal Recessive Non-Syndromic Hearing Loss in 33 Chinese Nuclear Families	Frontiers in genetics	2,019	cc-by	6,580	ORIGINAL RESEARCH doi: 10.3389/fgene.2019.00639 published: 17 July 2019 Proband Whole-Exome Sequencing Identified Genes Responsible for Autosomal Recessive Non- Syndromic Hearing Loss in 33 Chinese Nuclear Families Shushan Sang 1,2†, Jie Ling 1,3,4†, Xuezhong Liu 1,5,6, Lingyun Mei 1,2, Xinzhang Cai 1,2, Taoxi Li 1,2,7, Wu Li 1,2, Meng Li 1,2, Jie Wen 1,2, Xianlin Liu 1,2, Jing Liu 1,2, Yalan Liu 1,2, Hongsheng Chen 1,2, Chufeng He 1,2* and Yong Feng 1,2,7,8* Shushan Sang 1,2†, Jie Ling 1,3,4†, Xuezhong Liu 1,5,6, Lingyun Mei 1,2, Xinzhang Cai 1,2, Taoxi Li 1,2,7, Wu Li 1,2, Meng Li 1,2, Jie Wen 1,2, Xianlin Liu 1,2, Jing Liu 1,2, Yalan Liu 1,2, Hongsheng Chen 1,2, Chufeng He 1,2* and Yong Feng 1,2,7,8* Edited by: Musharraf Jelani, Islamia College University, Pakistan Specialty section: This article was submitted to Genetic Disorders, a section of the journal Frontiers in Genetics Specialty section: This article was submitted to Genetic Disorders, a section of the journal Frontiers in Genetics Specialty section: This article was submitted to Genetic Disorders, a section of the journal Frontiers in Genetics Received: 24 February 2019 Accepted: 18 June 2019 Published: 17 July 2019 Received: 24 February 2019 Accepted: 18 June 2019 Published: 17 July 2019 Keywords: proband-WES, ARNSHL, nuclear families, similar phenotype, molecular diagnosis Edited by: Musharraf Jelani, Islamia College University, Pakistan 1 Department of Otolaryngology, Xiangya Hospital, Central South University, Changsha, China, 2 Key Laboratory of Otolaryngology Major Diseases Research of Hunan Province, Changsha, China, 3 Institute of Molecular Precision Medicine, Xiangya Hospital, Central South University, Changsha, China, 4 Hunan Key Laboratory of Molecular Precision Medicine, Changsha, China, 5 Department of Otolaryngology, University of Miami Miller School of Medicine, Miami, FL, United States, 6 Dr. John T. Macdonald Foundation Department of Human Genetics, University of Miami Miller School of Medicine, Miami, FL, United States, 7 Hunan Jiahui Genetics Hospital, Changsha, China, 8 National Clinical Research Center for Geriatric Disorders, Xiangya Hospital, Central South University, Changsha, China Islamia College University, Pakistan Reviewed by: Abdelaziz Tlili, University of Sharjah, United Arab Emirates Muhammad Ansar, Quaid-i-Azam University, Pakistan Correspondence: Chufeng He hechufeng@csu.edu.cn Yong Feng fengyong_hn@hotmail.com †These authors share first authorship. Reviewed by: Abdelaziz Tlili, University of Sharjah, United Arab Emirates Muhammad Ansar, Quaid-i-Azam University, Pakistan Autosomal recessive non-syndromic hearing loss (ARNSHL) is a highly heterogeneous disease involving more than 70 pathogenic genes. However, most ARNSHL families have small-sized pedigrees with limited genetic information, rendering challenges for the molecular diagnosis of these patients. Therefore, we attempted to establish a strategy for identifying deleterious variants associated with ARNSHL by applying proband whole- exome sequencing (proband-WES). Aside from desiring to improve molecular diagnostic rates, we also aimed to search for novel deafness genes shared by patients with similar phenotype, making up for the deficiency of small ARNSHL families. In this study, 48.5% (16/33) families were detected the pathogenic variants in eight known deafness genes, including 10 novel variants identified in TMPRSS3 (MIM 605551), MYO15A (MIM 602666), TMC1 (MIM 606706), ADGRV1 (MIM 602851), and PTPRQ (MIM 603317). Apart from six novel variants with a truncating effect (nonsense, deletion, insertion, and splice-site), four novel missense variants were not found in 200 unrelated control population by using Sanger sequencing. It is important to note that none of novel genes were shared across different pedigrees, indicating that a larger sample size might be needed. Proband-WES is a cost-effective and precise way of identifying causative variants in nuclear families with ARNSHL. This economical strategy may be appropriated as a clinical application to provide molecular diagnostics, genetic counseling, and individualized health maintenance measures for patients with ARNSHL at hearing clinics. Correspondence: Chufeng He hechufeng@csu.edu.cn Yong Feng fengyong_hn@hotmail.com *Correspondence: Chufeng He hechufeng@csu.edu.cn Yong Feng fengyong_hn@hotmail.com †These authors share first authorship. Edited by: Edited by: Musharraf Jelani, Islamia College University, Pakistan WES Agilent SureSelect Human All Exon v6 (60 Mb) Kit (Agilent Technologies, Santa Clara, CA, USA) was used for WES, and sequencing was conducted on a HiSeq X10 platform (Illumina, San Diego, CA, USA) in an in-solution hybridization capture system to obtain all coding exons and flanking region sequences according to the manufacturer’s standard protocol. g One microgram of genomic DNA from proband was fragmented into 180–200 bp libraries which were modified, ligated to adaptors, and purified for subsequent PCR amplification. Amplified products were then hybridized to a custom array using the SureSelectXT Target Enrichment System (G7530-90000, Agilent). The hybridized DNA was eluted using magnetic beads, and the final enriched DNA fragments were then re-amplified by bridge-PCR and sequenced simultaneously on an Illumina HiSeq X10 System with 100-bp paired-end sequencing. In present study, we recruited affected sib pair pedigrees with similar phenotypes and performed WES only on probands of each family (so-called proband-WES) to search for candidate deafness-causing variants. Subsequently, we applied Sanger sequencing to verify which variants co-segregate with phenotype among the first degree relatives of the probands. The raw sequencing data was filtered to remove low- quality reads [the ratio of bases with low quality scores (less than 19) > 50% and/or ambiguous nucleotide rate >5% and/or adapter contamination >5 bp] and then turned into clean reads. These clean reads were then aligned to the reference human genome sequence (hg19) using the Burrows-Wheeler Aligner (BWA) (Li and Durbin, 2010). The GATK Software (https://www. broadinstitute.org/gatk/) was used for base quality re-calibration and variants calling (McKenna et al., 2010). We also used ANNOVAR software to annotate each variant, including gene name, genomic region, and function. Citation: Sang S, Ling J, Liu X, Mei L, Cai X, Li T, Li W, Li M, Wen J, Liu X, Liu J, Liu Y, Chen H, He C and Feng Y (2019) Proband Whole-Exome Sequencing Identified Genes Responsible for Autosomal Recessive Non-Syndromic Hearing Loss in 33 Chinese Nuclear Families. Front. Genet. 10:639. doi: 10.3389/fgene.2019.00639 July 2019 \| Volume 10 \| Article 639 Frontiers in Genetics \| www.frontiersin.org 1 Proband Based WES for ARNSHL Sang et al. Frontiers in Genetics \| www.frontiersin.org y The workflow of this study is shown in Figure 1. y The workflow of this study is shown in Figure 1. BACKGROUND The emergence of whole-exome sequencing (WES) offers an unprecedented opportunity to explore causative mutations in patients suffering from extremely heterogeneous genetic disorders (Li et al., 2018), such as ARNSHL. In present study, we recruited affected sib pair pedigrees with similar phenotypes and performed WES only on probands of each family (so-called proband-WES) to search for candidate deafness-causing variants. Subsequently, we applied Sanger sequencing to verify which variants co-segregate with phenotype among the first degree relatives of the probands. Most families with ARNSHL have small-sized pedigrees with an affected sib pair, affected parent–child pair, or a single affected child (Bademci et al., 2016). Despite the fact that linkage analysis has succeeded in mapping an abundance of genetic diseases over past few decades (Vardarajan et al., 2018), compared to studies associated with large-sized pedigrees, nuclear families with limited generations provide less information for genetic mapping and yield lower statistical power to discern quantitative trait loci (QTL) by linkage analysis (Dyer et al., 2001). Large unsolved families with ARNSHL urgently require a cost-effective method to detect the genetic etiology. The emergence of whole-exome sequencing (WES) offers an unprecedented opportunity to explore causative mutations in patients suffering from extremely heterogeneous genetic disorders (Li et al., 2018), such as ARNSHL. Subjects j We recruited 33 nuclear families with ARNSHL from the Otolaryngology Department of Xiangya Hospital, Central South University, China. To ensure the pedigrees from a homogeneous population with ARNSHL, we used the following inclusion criteria: 1) the probands had other affected sibling(s); 2) patients’ parents were asymptomatic; 3) all affected individuals suffered from congenital or prelingual deafness with a severity ranging from severe to profound; and 4) HL was the sole symptom of all patients. All experiments were approved by the ethics committee of Xiangya Hospital, Central South University. Written informed consent was provided by all participants or designated guardians of affected children prior to participation in this study.f BACKGROUND history investigation, somatoscopy, and temporal bone image examination via high-resolution computed tomography (HRCT) and magnetic resonance imaging (MRI) when necessary, so as to exclude syndromic and secondary HL. As part of routine examination, audiological evaluations including auditory brainstem response (ABR), multiple auditory steady-state evoked responses (ASSR), and distortion production otoacoustic emissions (DPOAE) were performed on all affected children. Severity of hearing impairment according to ABR data was classified as subtle (16~25 dB), mild (26~40 dB), moderate (41~70 dB), severe (71~90 dB), or profound (≥90 dB). Clinical and audiological evaluations indicated that all recruited individuals had severe to profound NSHL, without the evidence of syndromic, secondary, or auditory neuropathy. Peripheral blood samples of all probands and their family members (including parents and siblings) were collected and used to extract genomic DNA according to standard procedures for sequencing and molecular analysis. Hearing loss (HL) is one of the most common sensory disorders, affecting nearly 328 million adults and 32 million children worldwide (WHO, 2017). It is estimated that approximately 30,000 Chinese infants are born with congenital non-syndromic HL (NSHL) annually (Ouyang et al., 2009). For congenital or early-onset HL, genetic factor is considered to be the dominant etiology (Nance, 2003; Wang et al., 2018), especially for those with a family history. Among hereditary cases, approximately 70% are NSHL, for which hearing impairment is the exclusive phenotype (Niu et al., 2017; Deng et al., 2018). It is reported that almost 80% of NSHL cases involve autosomal-recessive inheritance with high genetic heterogeneity (Angeli et al., 2012). The major symptom of autosomal-recessive NSHL (ARNSHL) is bilaterally symmetric, severe-to-profound, and prelingual sensorineural HL, which is known to be caused mainly by monogenic mutations (Wu et al., 2016; Deng et al., 2018). 2016; Deng et al., 2018). Most families with ARNSHL have small-sized pedigrees with an affected sib pair, affected parent–child pair, or a single affected child (Bademci et al., 2016). Despite the fact that linkage analysis has succeeded in mapping an abundance of genetic diseases over past few decades (Vardarajan et al., 2018), compared to studies associated with large-sized pedigrees, nuclear families with limited generations provide less information for genetic mapping and yield lower statistical power to discern quantitative trait loci (QTL) by linkage analysis (Dyer et al., 2001). Large unsolved families with ARNSHL urgently require a cost-effective method to detect the genetic etiology. Variant Evaluationh In this study, average coverage of the targeted regions (exons and adjacent 20 bps introns from splice sites) were 99%, 97%, and 94% for the 10X, 30X, and 50X reads, respectively (Supplementary Table 2). The candidate variants were annotated on the basis of their pathogenicity information in the ClinVar (http://www.ncbi.nlm. nih.gov/clinvar), the DVD and HGMD database. Pathogenic prediction scores were calculated for non-synonymous variants to assess the influence of amino acid substitution on protein structure and function with Polyphen2 (http://genetics.bwh. harvard.edu/pph2), SIFT (http://sift.jcvi.org), MutationTaster (http://mutationassessor.org), and MutationAssessor (http:// mutationassessor.org). Only when pathogenic prediction scores were generated by two or more tools, were the non-synonymous variants considered pathogenic. We reclassified variants based on the American College of Medical Genetics and Genomics (ACMG) guidelines into five categories: pathogenic (P), likely pathogenic (LP), uncertain significance (US), likely benign (LB), and benign (B) (Richards et al., 2015). According to the filtering criteria mentioned above, 48.5% (16/33) families were found to carry homozygous or compound heterozygous variants in the DAGL. All detected variants were predicted to be P or LP by the ACMG guidelines and met the phenotype–genotype co-segregation. A total of 18 different variants were detected in eight known deafness genes. The known deafness genes with variants detected in this study were GJB2 (MIM 121011; 21.2%), TMPRSS3 (MIM 605551; 6.1%), MYO15A (MIM 602666; 6.1%), TMC1 (MIM 606706; 3%), ADGRV1 (MIM 602851; 3%), PTPRQ (MIM 603317; 3%), SLC26A4 (MIM 605646; 3%), and OTOF (MIM 603681; 3%) (Table 1). Among them, 55.6% (10/18) were novel variants that apart from six novel variants with a truncating effect (nonsense, deletion, insertion, and splice-site), the other four were missense variants detected in TMPRSS3 and MYO15A, respectively, which were undetected in 200 unrelated control subjects.t Homozygous and putatively compound heterozygous variants that were detected in the DAHL or predicted to be “P” by the ACMG guidelines were given priority to further analysis. In unsolved families, homozygous and putatively compound heterozygous variants predicted to be “LP” or “US” by the ACMG guidelines were chosen for further verification. j A novel homozygous frame-shift mutation and a novel homozygous missense mutation in TMPRSS3 were identified in two nuclear families, respectively. Both of the affected siblings presented congenital, profound ARNSHL. The TMPRSS3 c.432delA (p.Q144fs) mutation was predicted to disturb protein spatial structure by destroying the scavenger receptor cysteine- rich (SRCR) domain and the serine protease domain. Variants Filtrationh The analysis began with quality control (QC) including the coverage and average read depth of all coding exons and intron- exon adjacent regions, genotype quality scores, and QualByDepth (Q/D) scores. The low-quality variants (read depth <10-fold, quality score <25, Q/D score <5) were excluded from downstream f Detailed clinical evaluation of each affected subject was performed by an otologist and a geneticist, including medical July 2019 \| Volume 10 \| Article 639 2 Proband Based WES for ARNSHL Sang et al. FIGURE 1 \| Overall workflow of the proband whole-exome sequencing (proband-WES) pipeline. O/D, Phred-like quality score divided by depth; MAF, minor a frequency. July 2019 \| Volume 10 \| Article 639 Frontiers in Genetics \| www.frontiersin.org 3 Sang et al. Proband Based WES for ARNSHL analysis. The remaining variants were filtered according to minor allele frequency (MAF) <1% in multiple databases including the Exome Aggregation Consortium (ExAC) (http:// exac.broadinstitute.org/), the 1000 Genome Project (http:// browser.1000genomes.org), and gnomAD (http://gnomad. broadinstitute.org/). Variants with an allele frequency > 0.5% in the Annoroad Typical Chinese Genomes (ATCG database) were also filtered out. In addition, we produced a deafness-associated gene list (DAGL) by searching the Deafness Variation Database (DVD; http://deafnessvariationdatabase.org/), OMIM database (http://www.omim.org/), and HGMD database (http://www. hgmd.cf.ac.uk/ac/) with keywords “hearing loss” or “deafness” (Supplementary Table 1). The filtered variants were further annotated according to whether they appeared in the DAGL or not. Among the filtered variants, all homozygous and putatively compound heterozygous mutations were selected as candidate pathogenic variants. HL. Finally, we also tested the novel missense variants identified in this study by Sanger sequencing and excluded those which have been detected in 200 unrelated control subjects. Variant Evaluationh The TMPRSS3 c.T551C (p.L184S) mutation resulted in an amino acid substitution at an extremely conservative site in the SRCR domain. The 3D molecular model showed that the TMPRSS3 c.432delA (p.Q144fs) mutation destroyed spatial structure of the protein by affecting the SRCR and the trypsin-like serine protease domain; for the TMPRSS3 c.T551C (p.L184S) mutation, the mutant-type serine formed three extra hydrogen bonds with histidine at position 186 and formed an extra hydrogen bond with serine at position 187, which may perturb the protein structure (Figure 2). Both of the homozygous mutations [c.432delA (p.Q144fs) and c.T551C (p.L184S)] occurred in the highly conserved SRCR Structure Modelingf To evaluate the effect of the novel missense variants detected in TMPRSS3 and MYO15A on the structure and function of the proteins, three-dimensional (3D) models of the wild-type and mutant protein structures were built. Since there is no homology model for the protein structures of TMPRSS3 and MYO15A in the Protein Data Bank (PDB), the 3D molecular structures were constructed by the I-TASSER server creating five models per protein structure (Roy et al., 2010; Roy et al., 2012) and the optimal models were visualized using PyMOL software. Frontiers in Genetics \| www.frontiersin.org Sanger Sequencing Sanger sequencing was used to confirm candidate variants to determine whether they co-segregated with HL in family members. Primers for the exons of all candidate variants were designed using Primer3 online software (http://primer3.ut.ee/) based on the human genome reference sequence and manufactured by Bioshon Corporation, Changsha, China. The genomic DNA of candidate variants was amplified with primer pairs and then purified by agarose gel electrophoresis following the manufacturer’s protocol. The purified products were sequenced on an ABI 3100 DNA Analyzer (Thermo Fisher Scientific, Waltham, MA, USA). Sequencing data was analyzed using Lasergene version 7.1 (DNASTAR, Madison, WI, USA).t After sequencing, only those variants co-segregated with disease status in all family members were deemed responsible for July 2019 \| Volume 10 \| Article 639 Frontiers in Genetics \| www.frontiersin.org 4 Sang et al. TABLE 1 \| The detailed information of pedigrees with pathogenic variants identified in this study. Family ID Gene name mRNA Variation Aa Change Zygosity Alle Frq. gnomAD SIFT Polyphen2 Mutation Taster ACMG Reference HL533 TMPRSS3 NM_024022.2 c.551T > C p.L184S Hom. 1.62E-05 0.003 0.99 1 LP Novel# M21968 TMPRSS3 NM_024022.2 c.432delA p.Q144fs Hom. 3.61E-05 . . . P Novel M21958 MYO15A NM_016239.3 c.3833A > C p.Q1278P Comp.Het. NA 0.053 0.939 0.967 LP Novel# MYO15A NM_016239.3 c.9876G > T p.W3292C 1.44E-05 0.003 1 1 LP Novel# HL1822 MYO15A NM_016239.3 c.4351G > A p.D1451N Comp.Het. 1.22E-05 0.006 1 1 LP PMID: 17546645 MYO15A NM_016239.3 c.6461G > A p.C2154Y NA 0 1 1 LP Novel# D008 TMC1 NM_138691.2 c.150delT p.N50fs Comp.Het. NA . . . P Novel TMC1 NM_138691.2 c.1224+2T > C NA NA . . 1 P Novel HL1121 ADGRV1 NM_032119.3 c.3232G > T p.E1078X Comp.Het. NA . . 1 P Novel ADGRV1 NM_032119.3 c.2057_2063del p.A686fs NA . . . P Novel HL1222 PTPRQ NM_001145026.1 c.552delC p.D184fs Hom. NA . . . P Novel M19411 OTOF NM_194248.2 c.5713-2A > G NA Comp.Het. 1.63E-05 . . 1 LP PMID: 22575033 OTOF NM_194248.2 c.5197G > A p.E1733K 4.06E-06 0.002 1 1 P PMID: 19250381 D033 GJB2 NM_004004.5 c.235delC p.L79fs Hom. 0.000451 . . . P PMID: 27308839 D040 GJB2 NM_004004.5 c.439G > A p.E147K Comp.Het. 1.22E-05 0.001 1 1 P PMID: 14676473 GJB2 NM_004004.5 c.235delC p.L79fs 0.000451 . . . P PMID: 27308839 D048 GJB2 NM_004004.5 c.235delC p.L79fs Hom. 0.000451 . . . P PMID: 27308839 HL051 GJB2 NM_004004.5 c.235delC p.L79fs Hom. 0.000451 . . . #: were novel missense variants that were not found in 200 unrelated control subjects. NA, not available; Comp.Het., compound heterozygote. NA, not available; Comp.Het., compound heterozygote. Sanger Sequencing P PMID: 27308839 M20989 GJB2 NM_004004.5 c.235delC p.L79fs Hom. 0.000451 . . . P PMID: 27308839 M21006 GJB2 NM_004004.5 c.235delC p.L79fs Hom. 0.000451 . . . P PMID: 27308839 M20960 GJB2 NM_004004.5 c.428G > A p.R143Q Comp.Het. NA 0.001 1 1 P PMID: 11313763 GJB2 NM_004004.5 c.235delC p.L79fs 0.000451 . . . P PMID: 27308839 HL476 SLC26A4 NM_000441.1 c.1546dupC p.F515fs Comp.Het. 1.99E-05 . . . P PMID: 10874637 SLC26A4 NM_000441.1 c.919-2A > G NA 3.65E-04 . . . P PMID: 10874637 #: were novel missense variants that were not found in 200 unrelated control subjects. NA not available; Comp Het compound heterozygote TABLE 1 \| The detailed information of pedigrees with pathogenic variants identified in this study. July 2019 \| Volume 10 \| Article 639 Proband Based WES for ARNSHL Proband Based WES for ARNSHL Sang et al. FIGURE 2 \| Pedigree information, variation spectrum and three-dimensional protein modeling of TMPRSS3. (A) are pedigree charts, DNA sequence chromatograms of the two families with TMPRSS3 mutations as well as the conservative prediction of the c.551T>C (p.Leu184Ser) mutation. (B) is the mutant spectrum of TMPRSS3. (C) is the 3D models showing that the p.Gln144fs mutation destroys the spatial structure of protein (yellow) and the p.Leu184Ser mutation makes Ser184 form three extra hydrogen bonds with His186 and formed an extra hydrogen bond with Ser187. FIGURE 2 \| Pedigree information, variation spectrum and three-dimensional protein modeling of TMPRSS3. (A) are pedigree charts, DNA sequence chromatograms of the two families with TMPRSS3 mutations as well as the conservative prediction of the c.551T>C (p.Leu184Ser) mutation. (B) is the mutant spectrum of TMPRSS3. (C) is the 3D models showing that the p.Gln144fs mutation destroys the spatial structure of protein (yellow) and the p.Leu184Ser mutation makes Ser184 form three extra hydrogen bonds with His186 and formed an extra hydrogen bond with Ser187 FIGURE 2 \| Pedigree information, variation spectrum and three-dimensional protein modeling of TMPRSS3. (A) are pedigree charts, DNA sequence chromatograms of the two families with TMPRSS3 mutations as well as the conservative prediction of the c.551T>C (p.Leu184Ser) mutation. (B) is the mutant spectrum of TMPRSS3. (C) is the 3D models showing that the p.Gln144fs mutation destroys the spatial structure of protein (yellow) and the p.Leu184Ser mutation makes Ser184 form three extra hydrogen bonds with His186 and formed an extra hydrogen bond with Ser187. Sanger Sequencing domain of the TMPRSS3 protein, disturbing the function of this transmembrane protein to activate the ENaC sodium channel and interact with extracellular molecules. MYO15A motor domain revealed that the c.A3833C (p.Q1278P) mutation weakened the interaction of glutamine 1278 with other amino acids by decreasing the number of hydrogen bonds, which might have a detrimental effect on ATP hydrolysis and actin-binding stability. For the c.G6461A (p.C2154Y) mutation, the cysteine at position 2154 was replaced by tyrosine with a longer side chain, which is predicted to add two extra hydrogen bonds. For the c.G9876T9 (p.W3292C) mutation, tryptophan as a hydrophobic amino acid at position 3292 was surrounded by multiple hydrophobic amino acids that would damage the network of hydrophobic interaction after turning into cysteine (Figure 3). Two compound heterozygous mutations in MYO15A were detected with the following genotypes: c.A3833C (p.Q1278P)/c. G9876T (p.W3292C) occurred in family M21958 and c.G4351A (p.D1451N)/c.G6461A (p.C2154Y) in family HL1822. Multiple sequence alignment (MSA) identified that the four variants in MYO15A detected in this study were located at highly conserved sites. Three novel variants, c.A3833C (p.Q1278P), c.G6461A (p.C2154Y), and c.G9876T (p.W3292C), resided in the motor domain, the first MyTH4 domain, and the second FERM domain, respectively (Figure 3). Protein modeling revealed that hydrophobic proline was substituted for a hydrophilic amino acid at position 1278 and decreased the number of hydrogen bonds by two compared to the wild-type residue, which may affect the function of adjacent ATP-binding pockets. The motor domain comprises the actin and ATP-binding sites and acts as a molecular dynamic component. The 3D structure modeling of the Interestingly, a compound heterozygous mutation in ADGRV1 with a novel nonsense mutation and a novel frameshift mutation was detected in family HL1121. Their asymptomatic parents carried a heterozygous mutation in ADGRV1, respectively. Mutations in ADGRV1 were related to Usher syndrome 2C. The two affected siblings may be too young to present retinal degeneration and vestibular dysfunction (Table 1). July 2019 \| Volume 10 \| Article 639 Frontiers in Genetics \| www.frontiersin.org 6 Proband Based WES for ARNSHL Sang et al. FIGURE 3 \| Pedigree information, variation spectrum, and three-dimensional protein modeling of MYO15A. (A) are pedigree charts, DNA sequence chromatogram and the conservative prediction of four MYO15A mutations which is found in two different families. (B) is the mutant spectrum of MYO15A. Sanger Sequencing (C) is the 3D models showing that the p.Gln1278Pro mutation shortens the side chain and deletes the hydrogen bonds with Gln1275 and Glu1274, which may affect the function of adjacent ATP binding pocket (red); (D) the p.Cys2154Tyr mutation lengthens the side chain and adds hydrogen bond with Pro2078 and Leu2074, and (E) the p.Trp3292Cys mutation shortens the side chain and destructs the hydrophobic core. NT, not tested. FIGURE 3 \| Pedigree information, variation spectrum, and three-dimensional protein modeling of MYO15A. (A) are pedigree charts, DNA sequence chromatograms, and the conservative prediction of four MYO15A mutations which is found in two different families. (B) is the mutant spectrum of MYO15A. (C) is the 3D models showing that the p.Gln1278Pro mutation shortens the side chain and deletes the hydrogen bonds with Gln1275 and Glu1274, which may affect the function of adjacent ATP binding pocket (red); (D) the p.Cys2154Tyr mutation lengthens the side chain and adds hydrogen bond with Pro2078 and Leu2074, and (E) the p.Trp3292Cys mutation shortens the side chain and destructs the hydrophobic core. NT, not tested. July 2019 \| Volume 10 \| Article 639 Frontiers in Genetics \| www.frontiersin.org 7 Proband Based WES for ARNSHL Sang et al. satisfied with autosomal recessive inheritance pattern based on the data of proband-WES, which require further verification in pedigree members. Moreover, plenty of databases gathering all detected variants and several tools predicting the pathogenicity of variants offer powerful resources for genetic diagnosis. Many reference databases, such as the 1000 Genomes Project (1000 Genomes Project Consortium et al., 2015), the exome aggregation consortium (ExAC) (Walsh and Thomson, 2017), and the HapMap Project (Willer et al., 2006), provide vast information for identifying human genetic variations and serve as key components in genetic research by achieving sequence alignment and genotype imputation, evaluating MAF of variants, and filtering potential neutral variants. Multiracial samples and data from these databases allow for broad sharing and provide a wide representation of human genetic variation, which allows for the identification of connections between variants and hereditary diseases, making it easy to filter variants and make up for the drawbacks of nuclear families for linkage analyses. Furthermore, the ACMG developed a detailed set of guidelines for the interpretation of the functional significance of sequence variants generated by next-generation sequencing (Richards et al., 2015), facilitating the precise interpretation of variants pathogenicity. DISCUSSION It is challenging to detect causative mutations in ARNSHL due to: 1) small family size with limited generations, 2) high clinical and genetic heterogeneity, and 3) a large portion of non-recurrent variants as a result of large population mobility and low rates of consanguineous marriage. Therefore, the present study aimed to establish a strategy for detecting pathogenic variants causing ARNSHL and identify new genes shared by various pedigrees with similar phenotype, and then provide genetic counseling and health guidance for those individuals with deafness-causing variants identified.h i The emergence of next-generation sequencing (NGS) facilitates the discovery of new causative genes and the elucidation of the genetic mechanisms of hereditary diseases. Targeted sequencing panel, WES, and whole-genome sequencing (WGS) are all applications of NGS. However, there is a major limitation of targeted sequencing panel in which only few known genes can be detected and panel requires revalidation after the addition of new genes, making it unsuitable for highly heterogeneous diseases in which new disease-causing genes are constantly being identified (Bademci et al., 2016). It is well known that new deafness-causing genes have been identified every year, which raises an obstacle for the application of panel in clinical deafness etiology detection. Though WGS is the most comprehensive means of detection genetic etiology at present, its high cost makes it unsuitable for population-based studies of Mendelian disease. WES has been widely used as an alternative method to detect causative variants in genetic diseases, especially for Mendelian disorders (Lee et al., 2014). Though less than 2% of the entire human genome contains protein-coding regions, approximately 85% of pathogenic mutations in Mendelian diseases have been identified in these regions (Botstein and Risch, 2003). WES is capable of sequencing nearly all exons in the protein-coding regions of the human genome (Ng et al., 2009) and has innovated etiologic research in the field of human genetic disorders with comprehensive genetic analysis and the identification of novel disorder-causing genes, especially for diseases with extreme heterogeneity. Lee et al. reported that the greater number of disease-causing genes of a Mendelian disease that has been identified, the higher molecular diagnostic rate, could be achieved using WES (Lee et al., 2014). There are over 70 genes with more than 100 loci associated with ARNSHL (http://hereditaryhearingloss.org/); screening these loci would allow to achieve a high molecular diagnostic rate in the deafness cohort. Sanger Sequencing Although several candidate new deafness genes were identified in this study, there were no such genes shared among unrelated pedigrees. Our results indicate that a larger sample size might be needed in further study. Frontiers in Genetics \| www.frontiersin.org July 2019 \| Volume 10 \| Article 639 CONCLUSION ARNSHL patients with similar phenotypes may carry the same genetic factors, which can be identified by proband-WES with population-based studies. In the present study, 48.5% (16/33) of nuclear families with ARNSHL yielded a molecular diagnosis. Namely, the strategy of proband-WES works well to identify genetic etiology for families with ARNSH, which is the basis of providing genetic counseling and personalized health maintenance measures and preventing the transmission of deafness genes. ARNSHL patients with similar phenotypes may carry the same genetic factors, which can be identified by proband-WES with population-based studies. In the present study, 48.5% (16/33) of nuclear families with ARNSHL yielded a molecular diagnosis. Namely, the strategy of proband-WES works well to identify genetic etiology for families with ARNSH, which is the basis of providing genetic counseling and personalized health maintenance measures and preventing the transmission of deafness genes. DATA AVAILABILITY with Usher syndrome during puberty. Our results allow for the recommendation to acquire Braille alphabet competency in advance of the onset of blindness, which would improve social- life adaptability and quality of life after becoming speech- and hearing-impaired, as well as blindness. The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher. In the current study, three known deafness genes (GJB2, TMPRSS3, and MYO15A) were detected in several pedigrees, suggesting that the strategy of proband-WES can be successfully applied to search for novel ARNSHL genes shared by multiple families with similar phenotype. However, no new gene was found to be shared by multiple families, which may be due to the limited sample size. As a result, we plan to expand the sample size in future. Additionally, there are still some pedigrees that have not identified a genetic etiology, which may be due to: 1) effect of environmental factors or combined effect of environmental and genetic factors and 2) variants located in intron areas or other types of variants that are beyond the detectability of WES. DISCUSSION Therefore, patients with ARNSHL are considered to be appropriate for WES because there is no exclusive genetic test available for this highly genetically heterogeneous disorder. WES for nuclear families can be conducted as trio-WES (both the proband and his/her related parents sequenced simultaneously) to identify de novo variants or as proband- WES (only the proband sequenced). De novo variants occurring in the proband are frequently heterozygous and likely result in autosomal dominant inheritance. In fact, two non-twin siblings have little chance of generating the same de novo variant causing deafness. A large-scale clinical study showed that, except for de novo variants, trio-WES did not yield a higher molecular diagnostic rate than proband-WES (Lee et al., 2014). Therefore, we performed proband-WES on the nuclear families with ARNSHL to detect potentially causative variants combined with parents-child Sanger sequencing (both parents and their siblings were sequenced simultaneously) to verify the candidate variants. In the present study, the strategy of proband-WES yielded a high molecular diagnostic rate (16 of 33 cases, 48.5%), indicating that our methods succeeded in identifying the genetic etiology for a large portion of ARNSHL families. It is worth noting that some putatively compound heterozygous variants were proven by Sanger sequencing to be transmitted from one of the parents (data not shown), suggesting that these pseudo-compound heterozygous variants were benign and therefore should be removed from consideration as pathogenic in the affected subjects. Therefore, any compound heterozygous variants tested in the proband must be validated as having been transmitted from both parents. g In family HL1121, we detected a novel c.G3232T (p.E1078X) nonsense variant and a novel c.2057_2063del (p.A686fs) frameshift variant in ADGRV1, which segregated with hearing loss in this family. ADGRV1, a gene that is crucial for the development of hair cells, can result in Usher syndrome 2C, which is characterized by congenital moderate-to-severe hearing loss, retinal degeneration in the second decade of life or later, and normal vestibular function (Yan et al., 2018). This intriguing finding may be associated with the fact that these affected members might be too young to present retinal degeneration, which generally begins to appear in patients Determining a method by which to determine the pathogenicity of variants is one of the major obstacles for proband-WES. Fortunately, there are limited candidate variants July 2019 \| Volume 10 \| Article 639 8 Sang et al. Proband Based WES for ARNSHL SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fgene.2019.00639/ full#supplementary-material AUTHOR CONTRIBUTIONS SS is responsible for the design of this study, acquisition, analysis and interpretation of data for the work, as well as drafting the work; JL revised the draft critically for important intellectual content; CH and YF provided approval for publication of the content; WL, ML, JW, XLL, JL, and YL collect the detailed information and blood samples of pedigrees; HS performs audiometric tests on all patients as well as their family members; XZL, XC, LM and TL agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. According to the results of the present study, the strategy of proband-WES is a cost-effective and highly efficient testing procedure for ARNSHL families. Thus, this strategy can be applied to numerous ARNSHL patients at hearing clinics to yield a genetic diagnosis. Acquiring a genetic diagnosis of HL may offer a wealth of benefits such as making individualized health instruction, providing genetic counseling, and evaluating the recurrence rate of HL, offering detailed information for pre-natal diagnosis and preimplantation genetic diagnosis (PGD), as we have validated in a previous study (Deng et al., 2018). ACKNOWLEDGMENTS We greatly thank the pedigree members for agreeing to participate in this study. We also appreciate the help and advice of our colleagues. This study was supported by grants from the National Natural Science Foundation of China (Grants No. 81470705, 81771023, 81771024), the Major State Basic Research Development Program of China (973 Program) (Grants no. 2014CB541702), and in part by the China Postdoctoral Science Foundation (Grants No. 2018M632999). Nance, W. E. (2003). The genetics of deafness. Ment. Retard. Dev. Disabil. Res. Rev. 9, 109–119. doi: 10.1002/mrdd.10067 REFERENCES Dyer, T. D., Blangero, J., Williams, J. T., Goring, H. H., and Mahaney, M. C. (2001). The effect of pedigree complexity on quantitative trait linkage analysis. Genet. Epidemiol. 21 Suppl 1, S236–S243. doi: 10.1002/gepi.2001.21.s1.s236 1000 Genomes Project Consortium, Auton, A., Brooks, L. D., Durbin, R. M., Garrison, E. P., Kang, H. M., et al. (2015). A global reference for human genetic variation. Nature 526, 68–74. doi: 10.1038/nature15393 Lee, H., Deignan, J. L., Dorrani, N., Strom, S. P., Kantarci, S., Quintero-Rivera, F., et al. (2014). 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Biochem. 47, 1883–1897. doi: 10.1159/000491068 Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Roy, A., Kucukural, A., and Zhang, Y. (2010). I-TASSER: a unified platform for automated protein structure and function prediction. Nat. Protoc. 5, 725–738. doi: 10.1038/nprot.2010.5 Roy, A., Yang, J., and Zhang, Y. (2012). COFACTOR: an accurate comparative algorithm for structure-based protein function annotation. Nucleic Acids Res. 40, W471–W477. July 2019 \| Volume 10 \| Article 639 REFERENCES doi: 10.1093/nar/gks372 Copyright © 2019 Sang, Ling, Liu, Mei, Cai, Li, Li, Li, Wen, Liu, Liu, Liu, Chen, He and Feng. 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. Vardarajan, B. N., Beecham, G. W., and Haines., J. L. (2018). Pedigree selection and information content. Curr. Protoc. Hum. Genet. 97, e56. doi: 10.1002/cphg.56h Walsh, R., and Thomson, K. L. (2017). Reassessment of Mendelian gene pathogenicity using 7,855 cardiomyopathy cases and 60,706 reference samples. Genet. Med. 19, 192–203. doi: 10.1038/gim.2016.90 July 2019 \| Volume 10 \| Article 639 Frontiers in Genetics \| www.frontiersin.org 10
https://openalex.org/W2955119366	https://www.nature.com/articles/s41598-019-45943-0.pdf	English	null	Asymmetric quantum correlations in the dynamical Casimir effect	Scientific reports	2,019	cc-by	7,871	Asymmetric quantum correlations in the dynamical Casimir effect Xue Zhang1,2, Hui Liu1,2, Zhihai Wang1,2 & Taiyu Zheng1,2 Considering the current available experimental studies on the dynamical Casimir effect (DCE) in superconducting microwave waveguides, we study asymmetric quantum correlations in microwave radiation. The asymmetric quantum correlations are created by the presence of detuning in the DCE. We study the asymmetric quantum steering and determine the parameter regions of one- and two-way quantum steering. It shows that steering from Bob to Alice is more difficult than steering from Alice to Bob. Moreover, we find regions that represent states that, although entangled, cannot be used for teleporting coherent states; however, the steerable states are appropriate for quantum teleportation. We investigate how the teleportation fidelity functions as an indicator of the quality of EPR steering in the DCE. Received: 26 December 2018 Accepted: 19 June 2019 Published: xx xx xxxx Received: 26 December 2018 Accepted: 19 June 2019 Published: xx xx xxxx The dynamical Casimir effect (DCE) was theoretically predicted in 19701. Pairs of particles created due to time-dependent external conditions can be achieved in very different setups, e.g., by a changing boundary condi- tion1,2 or the index of refraction of the medium3,4. In recent years, considerable attention has been given to DCE by many physicists5–11. There is a vast body of literature on theoretical5,6 and experimental7–11 aspects of DCE.ii h In 2011, the DCE was first confirmed by an experiment constructed with a superconducting circuit termi- nated by a superconducting quantum interference device (SQUID)11. A more general revision about the DCE in the superconducting circuit has been reported in 20127. The superconducting circuits are not only used in the experimental demonstration for the DCE, but also may be to verify the analogue Hawking radiation7 and to realize quantum computers. Moreover, in a array made of N superconducting open stripline waveguides, the entangled NOON states have been created12. The microwave radiation produced by the DCE in superconducting circuits therefore has high potential of being distinctly nonclassical. In experiments in 2011, pairs of photons gen- erated by the DCE revealed quantum entanglement13 and quantum discord14. We will investigate the other useful forms of quantum correlations in the experiments. q p On one hand, we focus on the asymmetric nature which is caused by the detuning in the DCE. We will employ Einstein-Podolsky-Rosen (EPR) steering15–20 to explore the asymmetric nature of the bipartite Gaussian system. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports 1Center for Quantum Sciences and School of Physics, Northeast Normal University, Changchun, 130024, China. 2Center for Advanced Optoelectronic Functional Materials Research, and Key Laboratory for UV-Emitting Materials and Technology of Ministry of Education, Northeast Normal University, Changchun, 130024, China. Correspondence and requests for materials should be addressed to T.Z. (email: zhengty@nenu.edu.cn) DCE in Superconducting Waveguides p g g In 2011, an experimental observation of the DCE was reported11 in a superconducting waveguide (SW)39,40. The experiment demonstrated that the DCE produces a two-mode squeezed state13,41–44. The electromagnetic field in the SW can be characterized by the flux operator Φ x t ( , ). Quantizing the electromagnetic field by the flux opera- tor Φ x t ( , ) and solving the massless, one-dimensional Klein-Gordon wave equation, x t x t ( , ) ( , ) 0 xx tt 2 υ ∂Φ − ∂Φ = − , the the flux operator Φ x t ( , ) can be expressed in the form13,39,40 ˆ ˆ x t S d a e b e ( , ) 4 [ ], (1) i t kx i t kx 0 ( ) ( )  ∫ π ω ω Φ = + ω ω ω ω − + − − (1) where ωˆa and ωˆb are the annihilation operators for incoming and outgoing photons of the SW, respectively. Here, k is the wave number, and S0 is the characteristic impedance. The DCE can be studied by the scattering theory. The time-dependent boundary condition brings about an effective length and mixes the independent incoming and outgoing modes. The effective length can be written in the form L t E t L ( ) ( /2 ) ( ) , (2) J eff 0 2 0 π = Φ (2) L0, Φ = h 0 / e (2 ) and = Φ E t E t ( ) [ ( )] J J ext represent the characteristic inductance per unit length of the wave- guide, the magnetic flux quantum and the flux-dependent effective Josephson energy, respectively. Φ t( ) ext is the time-dependent applied magnetic flux through the SQUID. For a sinusoidal modulation with driving frequency d ω / π 2 and normalized amplitude , we obtain  ω = + E t E t ( ) [1 sin ] J J d 0 and an effective length modulation ampli- tude  δ = L L (0) eff eff . In the perturbative regime, the outgoing modes are correlated with frequencies ω+, ω− (ω ω ω + = + − d)39. In previous studies, the authors considered a symmetric situation by setting d ω ω ω = = + − /2. DCE in Superconducting Waveguides To investigate the effect of asymmetry, we introduce the detuning δω, by setting ω ω = ± d/ δω ± 2 . Noting ω = ± ± a a( ) and ω = ± ± b b( ), the relationship between the incoming and outgoing operators is written as ˆ ˆ ˆ = − − ± ± + b a ifa , (3) (3) where f L L (0) (4) eff eff δ ω ω υ ω ω υ = = . + − + −  (4) Let us consider the CM6 of the bipartite Gaussian CV system in the DCE. All the Gaussian properties can be obtained by the CM14,21,45 Let us consider the CM6 of the bipartite Gaussian CV system in the DCE. All the Gaussian properties can be obtained by the CM14,21,45 V R R R R R R 1 2 , (5) = 〈 + 〉−〈 〉〈 〉 αβ α β β α α β (5) where ˆ ˆ ˆ ˆ R X P X P ( , , , ) , (6) T = − − + + (6) is the vector of the field quadratures with elements: vector of the field quadratures with elements: is the vector of the field quadratures with elements: is the vector of the field quadratures with elements: X b b P i b b ( )/ 2 , ( )/ 2 (7) = + = − − . ± ± ± + ± ± ± + ˆ ˆ ˆ ˆ ˆ ˆ (7) arly, the quadratures of the input fields are expressed as Similarly, the quadratures of the input fields are expressed as res of the input fields are expressed as = + = − − . ± ± ± + ± ± ± + X a a P i a a ( )/ 2 , ( )/ 2 (8) 0 0 ˆ ˆ ˆ ˆ ˆ ˆ (8) We suppose that the input fields are in a quasi-vacuum state confirmed by a small number of thermal photons nth + , n th −. Then, the CM of the input fields is We suppose that the input fields are in a quasi-vacuum state confirmed by a small number of thermal photons nth + , n th −. Asymmetric quantum correlations in the dynamical Casimir effect In ref.21, EPR steering was discussed in continuous variable (CV) systems, but the effect of detuning was not taken into account. The asymmetric nature of the system is caused by detuning in the DCE, and the asymmetrical non- localities are receiving considerable attention22–29 for special tasks in quantum information processing, quantum metrology30, quantum state merging31–33, remote state preparation34 one-sided device-independent quantum key distribution35, and entanglement verification36. In contrast to symmetric systems, we show how one can produce a desired EPR state to fulfill a given directional quantum task by adding asymmetric amounts of detuning to each subsystem in the DCE. We also find the regions that represent one-way steerable states or two-way steerable states. On the other hand, we relate the entanglement and EPR steering to the teleportation fidelity34,37,38. We show that some states lying in the area are entangled, but they cannot be used as a quantum resource for teleportation. The teleportation fidelity is directly related to the strength of EPR steering. Therefore, the teleportation fidelity becomes an indicator of the quality of the EPR steering in the DCE.h The paper is organized as follows: In Sec. II, we review the DCE in superconducting circuits and derive the covariance matrix (CM) of the system. In Sec. III, we employ different entanglement markers in terms of the CM. We also include a detailed discussion on the relationship between the different entanglement markers and the teleportation fidelity for the DCE. We summarize the research in Sec. IV. 1Center for Quantum Sciences and School of Physics, Northeast Normal University, Changchun, 130024, China. 2Center for Advanced Optoelectronic Functional Materials Research, and Key Laboratory for UV-Emitting Materials and Technology of Ministry of Education, Northeast Normal University, Changchun, 130024, China. Correspondence and requests for materials should be addressed to T.Z. (email: zhengty@nenu.edu.cn) Scientific Reports \| (2019) 9:9552 \| https://doi.org/10.1038/s41598-019-45943-0 1 www.nature.com/scientificreports/ DCE in Superconducting Waveguides Then, the CM of the input fields is V n n m m 0 0 0 0 0 0 0 0 0 0 0 0 , (9) 0 0 0 0 0 =       (9) The elements in Eq. (9) can be calculated. The elements in Eq. (9) can be calculated. The elements in Eq. (9) can be calculated. = + = + . − + n n m n 1/2 , 1/2 (10) 0 th 0 th = + . + m n 1/2 (10) 0 th (10) where ( ) n e 1 th 1 kT  = − ± − ω± , d ω ω = ± / δω ± 2 with the detuning δω. Similarly, we can obtain the CM of the output fields where ( ) n e 1 th 1 kT  = − ± − ω± , d ω ω = ± / δω ± 2 with the detuning δω. Similarly, we can obtain the CM of the output fields where ( ) n e 1 th 1 kT  = − ± − ω± , d ω ω = ± / δω ± 2 with the detuning δω. Similarly, we can obtain the CM of the output fields Scientific Reports \| (2019) 9:9552 \| https://doi.org/10.1038/s41598-019-45943-0 Scientific Reports \| (2019) 9:9552 \| https://doi.org/10.1038/s41598-019-45943-0 2 www.nature.com/scientificreports/ Figure 1. The PHS criterion for entanglement as a function of the detuning δω and the driving amplitude  for a temperature of T = 30 mK in (a) and the detuning δω and the temperature for the driving amplitude = .0 3  in (b). The dashed lines in (a,b) represent E 0 25 NT = . , which is the critical value of the PHS criterion. The small parameter < . f 0 078 is well within the perturbative analysis. The plots are realized with the experimental parameters 20 GHz d ω π = , Leff(0) = 0.5 mm, and v = 1.2 × 108 m/s. Figure 1. The PHS criterion for entanglement as a function of the detuning δω and the driving amplitude  for a temperature of T = 30 mK in (a) and the detuning δω and the temperature for the driving amplitude = .0 3  in (b). The dashed lines in (a,b) represent E 0 25 NT = . Asymmetric Quantum Correlations in the DCE Th d d h h h b Asymmetric Quantum Correlations in the DCE Th d d h h h b Asymmetric Quantum Correlations in the DCE Th d d h h h b l l h l y The DCE can produce microwave radiation, which exhibits non-local interaction properties, in the virtual par- ticles of the quantum vacuum13,14,21,45. Therefore, the microwave radiation must possess quantum correlations. The theory of quantum correlations has been well developed in quantum optics, and the quantum nature of a Gaussian state can be test by many markers. In the following, we will discuss the Peres-Horodecki-Simon (PHS) criterion for entanglement, the EPR criterion for one- and two-way steering, and the teleportation fidelity in the DCE. We focus on two points: the asymmetric nature caused by the detuning in the DCE and how the teleporta- tion fidelity works as an indicator in CV teleportation protocols. The PHS Criterion for Entanglement. Entanglement is the edge of quantum correlation. The PHS crite- rion has been proven to be the necessary and sufficient condition for the entanglement of bipartite Gaussian CV systems46, and it can be expressed simply in terms of the CM elements. = + + − − > . E n m c nm c ( 2 ) 4( ) 1 4 (13) NT 2 2 2 2 2 (13) If Eq. (13) is satisfied, the bipartite Gaussian CV state will be entangled. We will use this PHS criterion to study the DCE radiation. By solving Eq. (13), we can get the range of f to ensure that the state is entangled. Taking a Taylor series expansion of f, + nth and n th −, we obtain If Eq. (13) is satisfied, the bipartite Gaussian CV state will be entangled. We will use this PHS criterion to study the DCE radiation. By solving Eq. (13), we can get the range of f to ensure that the state is entangled. Taking a Taylor series expansion of f, + nth and n th −, we obtain > + + . + − + − f n n n n 2 2 5( ) (14) th th th th (14) For the sake of a comparison with the results of ref.45, we consider no detuning. We can write ω ω ω = = + − d /2, and then n n n th th th = = + − . DCE in Superconducting Waveguides , which is the critical value of the PHS criterion. The small parameter < . f 0 078 is well within the perturbative analysis. The plots are realized with the experimental parameters 20 GHz d ω π = , Leff(0) = 0.5 mm, and v = 1.2 × 108 m/s. =    − −    V n c n c c m c m 0 0 0 0 0 0 0 0 , (11) (11) n n f n m n f n c f n n 1/2 (1/2 ), 1/2 (1/2 ), (1 ) (12) th 2 th th 2 th th th = + + + = + + + = + + . − + + − + − (12) Asymmetric Quantum Correlations in the DCE Th d d h h h b When both < → E 1/2 B A and < → E 1/2 A B are satisfied, two-way EPR steering is con- firmed; i.e., the DCE state with f f f max { , } B A A B > → → can be used to produce two-way steering. i B A A B → → In Fig. 2(a), we observe that the EPR steering from B to A vanishes with the detuning 0 18 d δω ω > . for any driving amplitude at T = 30 mK. Figure 2(c) reveals that a certain value of the driving amplitude ( 0 12 > .  ) is required to overcome the detuning and gives rise to EPR steering from A to B. We also find that thermal noise tends to suppress EPR steering from both → EB A and → EA B in Fig. 2(b,d), but in Fig. 2(c,d), we observe that EPR steering from A to B is highly robust to temperature and detuning. The Teleportation Fidelity in the DCE. In this section, we focus on how to evaluate the quality of entan- glement in CV teleportation protocols. The PHS criterion is incapable of measuring entanglement in a quantita- tive manner. In contrast to the PHS criterion, the fidelity  of CV teleportation for a Gaussian state only depends on the entanglement quality of the resource itself. Accordingly, the fidelity  of teleportation is employed as a benchmark of an entangled state. Using the Gaussian state described by the CM in Eq. (11),  can be written as50 (19) m n c (1 2 ) (19) 1 = + + − . − Equation (19) shows that when 1/2 ≤  , the teleportation protocol of a Gaussian state can be completed implemented with classical strategies; in contrary, if the resource is entangled,  will be larger than 1/2. Thus, the fidelity  is an indicator of the quality of the entanglement, and the state can be used as a quantum resource for teleportation. In other words, > 1/2  requires f to exceed the threshold value > − + + + − − f n n 1 ( 1) th th 1/2. Considering no detuning condition, we can write n n n th th th = = + − . Asymmetric Quantum Correlations in the DCE Th d d h h h b → E n c m 1 2 (15) B A 2 (15) We note that, being based on conditional variances, the EPR criterion is asymmetric under swapping subsys- ems. Then, the EPR steering criterion from Alice to Bob can be recast by exchanging m and n in Eq. (15), = − < . → E m c n 1 2 (16) A B 2 (16) To satisfy the EPR steering criterion < → E 1/2 B A (or E 1/2 A B < → ) requires f to exceed the threshold value given by → fB A (or → fA B). Solving the inequalities, we can obtain f n n n n 2 (1 2 ) (2 1)(4 3) , (17) B A th th th th > + + + → − + − + > + + + . → + − + − f n n n n 2 (1 2 ) (2 1)(4 3) (18) A B th th th th (17) (18) In the case of = = + − n n n th th th, the threshold values → fB A and fA B → are the same. We note that f f f n 2 /3 B A A B th = = > → → for  n 1 th , which is consistent with the result reported in ref.21. n the case of = = + − n n n th th th, the threshold values → fB A and fA B → are the same. We note that f f n 2 /3 A B th = = > → for  n 1 th , which is consistent with the result reported in ref.21. B A A B Figure 2 shows the behaviour of the EPR steering with respect to the driving amplitude, temperature and detuning. In contrast to symmetric systems (ω ω = + −), we introduce directional EPR steering to test the quantum correlations in the DCE. The presence of detuning causes asymmetric steering and makes steering from A to B easier than from B to A, as illustrated in Fig. 2. The sensitivity of the steering parameter → EA B is asymmetrical, and one-way steering is evident. Asymmetric Quantum Correlations in the DCE Th d d h h h b When  n 1 th , the above inequality can be further simplified to f nth > . This result is consistent with the result reported in ref.45.hhi For the sake of a comparison with the results of ref.45, we consider no detuning. We can write ω ω ω = = + − d /2, and then n n n th th th = = + − . When  n 1 th , the above inequality can be further simplified to f nth > . This result is consistent with the result reported in ref.45.hhi The PHS criterion for the DCE is shown in Fig. 1. The figure visualizes the PHS criterion as a function of tem- perature, detuning, and driving amplitude. We observe that for a temperature of T = 30 mK, the PHS criterion is always larger than 1/4 for any sensible values of the driving amplitude and the detuning. In the case of a fixed  0 3 = . , we find that the entanglement vanishes at high temperatures within the given detuning regime, and the Scientific Reports \| (2019) 9:9552 \| https://doi.org/10.1038/s41598-019-45943-0 3 www.nature.com/scientificreports/ threshold value is almost 68 mK. We also observe that thermal noise tends to suppress entanglement, and the driving amplitude  tends to promote entanglement. threshold value is almost 68 mK. We also observe that thermal noise tends to suppress entanglement, and the driving amplitude  tends to promote entanglement. The EPR Criterion for Steering. Steering is a significant form of quantum correlations47,48 and is expressed asymmetrically between Alice and Bob. Asymmetric steering describes the idea of action at a distance of the EPR state, which allows Alice to adjust Bob’s state by means of local measurements15. On the operational side, the asymmetric steering can be useful for providing security in one-sided device-independent quantum key dis- tribution schemes35. In this section, we focus on the asymmetric steering caused by the detuning condition in the DCE. We will employ EPR steering to explore the asymmetric nature of the bipartite Gaussian CV state. Mathematically, the EPR criterion can be calculated in terms of CM elements, that is, from the result of Q. Y. He49. The EPR steering criterion from Bob to Alice can be expressed by: = − < . Asymmetric Quantum Correlations in the DCE Th d d h h h b The EPR criterion for steering from B to A as a function of the detuning δω and the driving amplitude  in (a) and of the detuning δω and the temperature T in (b). The dashed lines in (a,b) represent E 0 5 A B = . → , which is the critical value of the EPR steering criterion from B to A. The EPR criterion for steering from A to B as a function of the detuning δω and the driving amplitude  in (c) and of the detuning δω and the temperature T in (d). The dashed lines in (c,d) represent E 0 5 A B = . → , which is the critical value of the EPR steering criterion from A to B. The small parameter f 0 078 < . is well within the perturbative analysis. The parameters are the same as those in Fig. 1. Figure 3. The teleportation fidelity as a function of the detuning δω and the driving amplitude  in (a) and of the detuning δω and the temperature T in (b). The dashed lines in (a,b) represent = .0 5, which is the critical value of the teleportation fidelity. The small parameter < . f 0 078 is well within the perturbative analysis. The parameters are the same as those in Fig. 1. Figure 3. The teleportation fidelity as a function of the detuning δω and the driving amplitude  in (a) and of the detuning δω and the temperature T in (b). The dashed lines in (a,b) represent = .0 5, which is the critical value of the teleportation fidelity. The small parameter < . f 0 078 is well within the perturbative analysis. The parameters are the same as those in Fig. 1. Witnesses. By focusing on the DCE, we will employ four different witnesses to determine whether the states exhibit entanglement or steering. Witnesses. By focusing on the DCE, we will employ four different witnesses to determine whether the states exhibit entanglement or steering. Asymmetric Quantum Correlations in the DCE Th d d h h h b When  n 1 th , the above inequality can be further simplified to f nth > . This result is the same with the result of the PHS criterion. From this perspective, the entangled states can be used for teleporting coherent states. However, considering the detuning condition, the threshold values are different of the fidelity and the PHS criterion. The entangled states are not appropriate for quantum teleportation. We can see that the detuning creates an important influence on the fidelity.ii li In Fig. 3(a), we find that, in the case of δω ω > .0 226 d, the fidelity is always less than 0.5 for any driving ampli- tude. The fidelity is always less than 0.5 with  0 138 < . for any detuning. In other words, below the region of the dashed line in Fig. 3(a), the states are not appropriate for the quantum teleportation. Figure 3(b) reveals that the fidelity is robust to detuning when the temperature is below 34 mK. Scientific Reports \| (2019) 9:9552 \| https://doi.org/10.1038/s41598-019-45943-0 4 www.nature.com/scientificreports/ Figure 2. The EPR criterion for steering from B to A as a function of the detuning δω and the driving amplitude  in (a) and of the detuning δω and the temperature T in (b). The dashed lines in (a,b) represent E 0 5 A B = . → , which is the critical value of the EPR steering criterion from B to A. The EPR criterion for steering from A to B as a function of the detuning δω and the driving amplitude  in (c) and of the detuning δω and the temperature T in (d). The dashed lines in (c,d) represent E 0 5 A B = . → , which is the critical value of the EPR steering criterion from A to B. The small parameter f 0 078 < . is well within the perturbative analysis. The parameters are the same as those in Fig. 1. igure 2. The EPR criterion for steering from B to A as a function of the detuning δω and the driving amplitude i ( ) d f h d i δ d h T i (b) Th d h d li i ( b) E 0 Figure 2. Asymmetric Quantum Correlations in the DCE Th d d h h h b w nm c n m c w n c m w m c n w m n c 4( ) ( 2 ) 1 4 , 1 2 , 1 2 , 1 2 (1 2 ) (20) PHS 2 2 2 2 2 B A 2 A B 2 1 = − − + + + = − − = − − = − + + − . → → −  w nm c n m c w n c m w m c n w m n c 4( ) ( 2 ) 1 4 , 1 2 , 1 2 , 1 2 (1 2 ) PHS 2 2 2 2 2 B A 2 A B 2 1 = − − + + + = − − = − − = − + + − . → → −  (20) Scientific Reports \| (2019) 9:9552 \| https://doi.org/10.1038/s41598-019-45943-0 5 www.nature.com/scientificreports/ Figure 4. Plots of the different witnesses of Eq. (20) as entanglement markers. The witnesses as a function of the driving amplitude  for a temperature of T = 30 mK in (a). The witnesses have threshold values of  above which they are less than zero. The threshold values are ≈ .0 0068  for the teleportation fidelity, 0 0027 ≈ .  for entanglement, 0 053 ≈ .  for directional one-way steering from A to B, and 0 26 ≈ . for directional one-way steering from B to A. In (b), the witnesses as a function of the detuning δω for a driving amplitude = .  0 3 and a temperature of T = 30 mK. The teleportation fidelity and steering from B to A vanish for the given detunings 0 33 d δω ω ≈ . and δω ω ≈ .0 11 d, respectively. However, the entanglement and steering from A to B do not disappear in this parameter regime. The witnesses as a function of the temperature T for (c) = .0 3 and (d) 0 8 = . . In (c), the witness of the teleportation fidelity is zero at ≈ T 69 mK, and the entanglement vanishes at ≈ T 74 mK. The temperature region of directional one-way steering from A to B is T 31 mK 47 mK < < , and the region of two-way steering is T 31 mK < . Asymmetric Quantum Correlations in the DCE Th d d h h h b In summary, each witness corresponds to the above criteria [see Eqs (13), (15), (16) and (19)], which can be reduced to In summary, each witness corresponds to the above criteria [see Eqs (13), (15), (16) and (19)], which can be reduced to In summary, each witness corresponds to the above criteria [see Eqs (13), (15), (16) and (19)], which can be educed to w w w w 0 entanglement, 0 steering (B A), 0 steering (A B), 0 entanglement (21) PHS B A A B       < ⇔ < ⇔ → < ⇔ → < ⇒ . → → w w w w 0 entanglement, 0 steering (B A), 0 steering (A B), 0 entanglement (21) PHS B A A B       < ⇔ < ⇔ → < ⇔ → < ⇒ . → → (21) The first three criteria are sufficient and necessary to confirm entanglement or steering for the DCE. The fourth criterion is only sufficient (not necessary) to confirm entanglement. fii In Fig. 4 we compare the behaviours of the witnesses in Eq. (20) for realistic experimental parameters. We observe that the teleportation fidelity offers a more restrictive condition with respect to the PHS criterion in the given parameter regions. In other words, we find the regions representing states that, even if entangled, are not suitable for teleportation. From the perspective of teleportation, the above entanglement can be used for locally transforming the two subsystems, but it is not helpful for teleportation. If the states can be used as a quantum resource for teleportation, we will have <  w 0. Interestingly, we also find that the EPR steering criterion is stricter than the teleportation fidelity criterion for any given value. If the states are steerable, the states will always be used for teleportation. The teleportation fidelity of the state is directly related to the strength of EPR steering.fi ph pi y y g g Moreover, the presence of detuning creates asymmetric steering, making steering from B to A more difficult than from A to B, as illustrated in Fig. 4. We also find the parameter regions of directional one-way steering from A to B and two-way steering in this regime. In Fig. Asymmetric Quantum Correlations in the DCE Th d d h h h b In (d), the threshold temperature is replaced by ≈ T 107 mK for entanglement and ≈ T 101 mK for teleportation fidelity. The temperature regions of directional one-way steering from A to B and two-way steering are displaced to T 47 mK 68 mK < < and < T 46 mK, respectively. The parameters are: ω π = 20 GHz d , Leff(0) = 0.5 mm, δω ω = .0 1 d, and v = 1.2 × 108 m/s. Figure 4. Plots of the different witnesses of Eq. (20) as entanglement markers. The witnesses as a function of the driving amplitude  for a temperature of T = 30 mK in (a). The witnesses have threshold values of  above which they are less than zero. The threshold values are ≈ .0 0068  for the teleportation fidelity, 0 0027 ≈ .  for entanglement, 0 053 ≈ .  for directional one-way steering from A to B, and 0 26 ≈ . for directional one-way steering from B to A. In (b), the witnesses as a function of the detuning δω for a driving amplitude = .  0 3 and a temperature of T = 30 mK. The teleportation fidelity and steering from B to A vanish for the given detunings 0 33 d δω ω ≈ . and δω ω ≈ .0 11 d, respectively. However, the entanglement and steering from A to B do not disappear in this parameter regime. The witnesses as a function of the temperature T for (c) = .0 3 and (d) 0 8 = . . In (c), the witness of the teleportation fidelity is zero at ≈ T 69 mK, and the entanglement vanishes at ≈ T 74 mK. The temperature region of directional one-way steering from A to B is T 31 mK 47 mK < < , and the region of two-way steering is T 31 mK < . In (d), the threshold temperature is replaced by ≈ T 107 mK for entanglement and ≈ T 101 mK for teleportation fidelity. The temperature regions of directional one-way steering from A to B and two-way steering are displaced to T 47 mK 68 mK < < and < T 46 mK, respectively. The parameters are: ω π = 20 GHz d , Leff(0) = 0.5 mm, δω ω = .0 1 d, and v = 1.2 × 108 m/s. Asymmetric Quantum Correlations in the DCE Th d d h h h b 4(b), the detuning also tends to suppress entanglement and steering, but entanglement and steering from A to B are almost insensitive to detuning in this regime. In Fig. 4(c,d), we find that thermal noise tends to suppress entanglement and steering, but the temperature threshold values of the four witnesses increase with the driving amplitude. For the driving amplitude 0 8 = . , the entangle- ment and teleportation fidelity are robust to thermal noise within 100 mK, and two-way steering is robust to thermal noise within 46 mK. 6 Scientific Reports \| (2019) 9:9552 \| https://doi.org/10.1038/s41598-019-45943-0 www.nature.com/scientificreports/ Conclusions In conclusion, we have analyzed asymmetric quantum correlations of the DCE in a superconducting circuit ter- minated by a SQUID. We studied the ability of the DCE to generate one- and two-way steering, forms of quantum correlation that are stronger than entanglement and essential for quantum technologies. We found the param- eter regions of one- and two-way steering. The presence of detuning causes asymmetric steering, resulting in directional steering from Alice to Bob that is easier than from Bob to Alice. Moreover, we have theoretically investigated how the fidelity of CV teleportation of the DCE varies with the parameters. 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https://openalex.org/W2749573973	https://europepmc.org/articles/pmc5577251?pdf=render	English	null	Phylogeny and expression pattern analysis of TCP transcription factors in cassava seedlings exposed to cold and/or drought stress	Scientific reports	2,017	cc-by	10,440	Ning Lei2, Xiang Yu 3,4, Shuxia Li1, Changying Zeng1, Liangping Zou1, Wenbin Liao1 & Ming Peng1 The TCP transcription factors usually act as integrators of multiple growth regulatory and environmental stimuli. However, little is known about this gene family in the important tropical crop cassava (Manihot esculenta). In this study, 36 TCP genes were identified and renamed based on cassava whole-genome sequence and their sequence similarity with Arabidopsis TCPs. Typical TCP domains were detected in these proteins by multiple sequence alignment analysis. Evolutionary analysis indicated that MeTCPs could be divided into 8 subgroups, which was further supported by gene structure and conserved motif analyses. qRT-PCR analysis revealed tissue-specific and hormone-responsive expression patterns of MeTCP genes. Moreover, with global expression and promoter analysis, we found that MeTCPs showed similar or distinct expression patterns under cold and/or drought stress, suggesting that they might participate in distinct signaling pathways. Our study provides the first comprehensive analysis of TCP gene family in the cassava genome. The data will be useful for uncovering the potential functions of MeTCP genes, and their possible roles in mediating hormone and abiotic stress responses in cassava. As sessile organism, plant growth and yield are strongly influenced by environmental stimuli such as cold and drought1. To respond and adapt to these conditions, plants develop various mechanisms at both physio- logical and biochemical levels2. It has been well established that many adaptation processes are regulated by stress-responsive gene expression1, 3. Transcription factors (TFs), which are a diverse family of regulatory proteins with DNA-binding domains, play a central role in mediating various aspects of cellular processes by regulating gene expression through interacting with cis-elements in the promoter regions of various downstream genes4, 5. Series studies previously have uncovered a group of TF genes, such as AP2/ERF, MYB and bZIP, which participate in various stress-induced physiological processes and regulatory networks in higher plants6, 7.iti p y g p g y g p TCP genes encode plant-specific transcription factors, which are named after the first four functionally identified members: TB1 (TEOSINTE BRANCHED 1) in Zea mays, CYC (CYCLOIDEA) in Antirrhinum majus, and PCF1 and PCF2 (PROLIFERATING CELL FACTORS 1 and 2) in Oryza sativa8. Typically, N-terminus of this class of transcription factors exhibits a highly conserved TCP domain, which contains a non-canonical basic-Helix-Loop-Helix (bHLH) structure involved in DNA binding, protein-protein interaction and protein nuclear localization9, 10. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Received: 7 April 2017 Accepted: 26 July 2017 Published: xx xx xxxx Phylogeny and expression pattern analysis of TCP transcription factors in cassava seedlings exposed to cold and/or drought stress Received: 7 April 2017 Accepted: 26 July 2017 Published: xx xx xxxx Received: 7 April 2017 Accepted: 26 July 2017 Published: xx xx xxxx Ning Lei2, Xiang Yu 3,4, Shuxia Li1, Changying Zeng1, Liangping Zou1, Wenbin Liao1 & Ming Peng1 Ning Lei2, Xiang Yu 3,4, Shuxia Li1, Changying Zeng1, Liangping Zou1, Wenbin Liao1 & Ming Peng1 Results Id tifi Identification of TCP genes in cassava. In order to extensively identify cassava TCP genes, two search strategies were used in this study: first, we used the protein sequences of all 24 TCP genes from Arabidopsis33 as BLASTp queries to perform multiple searches against the latest whole genome of cassava (http://www.phytozome. net/cassava); then, the TCP domain (PF03634)46 was employed as query to perform a blast search against the same cassava genome database. After discarding redundant sequences, 36 candidate TCP genes were identified in cassava, and further conserved domain detection confirmed that all the identified TCPs harbor the conserved TCP domain that is the basic characteristics of this family. Due to the lack of standard annotation designated to the 36 cassava TCP genes, we named them MeTCP2 to MeTCP23 according to the Arabidopsis TCP proteins with highest sequence similarity. The length of the 36 newly identified TCP proteins varied from 73 to 562 amino acid residues and the relative molecular weight ranged from 8.3 to 58.1 kDa, with isoelectric points in the range of 5.53–10.08. The grand average of hydropathy (GRAVY) of this family genes showed that all of the proteins had a negative value, indicating that all the MeTCP proteins were hydrophilic (Table 1). Phylogenetic analysis of the MeTCP genes. To characterize the evolutionary and phylogenetic rela- tionships between cassava TCP genes and other known TCPs, an unrooted Neiboring-Joining (NJ) tree was con- structed on the basis of multiple sequence alignment of 36 MeTCP complete protein sequences with 24 and 22 TCP protein sequences from Arabidopsis and rice, respectively (Fig. 1). At deep nodes, the phylogenetic relation- ship was unclear and the bootstrap values were low as a result of relatively large number of sequences. To verify the reliability of our phylogenetic tree, we also build the phylogenetic trees of TCP transcription family with Minimal Evolution methods (Fig. S1). The tree topologies were robust within different tree-building methods, except at the deep nodes. Considering these results, the NJ tree was employed for further study. Based on the bootstrap value of clade and topology of the tree, the MeTCP proteins could be distributed into 8 distinct groups, designated as Group A to Group H. In general, TCPs from cassava have closer relationships with the TCPs from dicot plant Arabidopsis than that from monocot plant rice, which is accord with the current understanding of plant evolutionary history. Ning Lei2, Xiang Yu 3,4, Shuxia Li1, Changying Zeng1, Liangping Zou1, Wenbin Liao1 & Ming Peng1 Based on the homology of the TCP domains, TCP proteins can be further divided into two major subfamilies, Class I (represented by the rice PCF proteins) and class II (represented by CYC and TB1)8, 11. The DNA-Binding site selection assays revealed that the consensus binding sequences for these two classes are slightly different, but overlapping with GGNCCC sequences. The DNA binding sequence for class I is 1Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou, 571101, China. 2Institute of Tropical Agriculture and Forestry, Hainan University, Haikou, 570228, China. 3National Key Laboratory of Plant Molecular Genetics and National Center for Plant Gene Research (Shanghai), Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200032, China. 4Present address: Department of Biology, University of Pennsylvania, Philadelphia, PA, 19104, USA. Ning Lei, Xiang Yu and Shuxia Li contributed equally to this work. Correspondence and requests for materials should be addressed to M.P. (email: pengming@itbb.org.cn) SCIENTIfIC REPOrTS \| 7: 10016 \| DOI:10.1038/s41598-017-09398-5 1 www.nature.com/scientificreports/ GGNCCCAC while class II prefer to bind the DNA motif G(T/C)GGNCCC10. Both class I and class II include TCPs that can function as transcriptional activators and repressors12. GGNCCCAC while class II prefer to bind the DNA motif G(T/C)GGNCCC10. Both class I and class II include TCPs that can function as transcriptional activators and repressors12. p p Increasing evidences have indicated that proteins of TCP family take part in the regulation of many biological processes during plant growth and development, including plant architecture12, 13, leaf morphogenesis14–16, hor- mone pathways13, 17–19 and response to environmental stimuli among various species20–22. For example, studies in Arabidopsis suggest that AtTCP14 appears to function in regulating embryonic growth of seeds23. AtTCP15, along with its closest homolog AtTCP14, regulates cell proliferation in the developing leaf blade and specific floral tissues, and also modulates gibberellins and auxin responses17, 24. The expression of a repressor form of AtTCP11 caused pleiotropic developmental alterations25. AtTCP2, AtTCP3, AtTCP4, AtTCP10 and AtTCP24 have been earlier identified as targets of miRNA319, and are required for leaf development, petal growth, cell wall synthesis and JA synthesis14, 26, 27. Creeping bentgrass (Agrostis stolonifera) plants overexpressing Osa-miR319a, in which four putative target genes, AsPCF5, AsPCF6, AsPCF8, and AsTCP14 were down-regulated, displayed morpholog- ical changes and exhibited enhanced drought and salt tolerance associated with increased leaf wax content and water retention20. Ning Lei2, Xiang Yu 3,4, Shuxia Li1, Changying Zeng1, Liangping Zou1, Wenbin Liao1 & Ming Peng1 In rice, overexpression of OsTCP19 led to developmental abnormalities like fewer lateral root formation and contributed to better stress tolerance21. formation and contributed to better stress tolerance . Widely cultivated in tropical area, cassava is considered as one of the most important economic crops world- wide, providing starch for food, feed and energy production28–30. Cassava can effectively utilize light, heat and water resource; it is resistant to drought but sensitive to low temperature31. Cold and drought stress severely limit cassava plant growth and yield32. Thus, uncovering the mechanisms underlying the resistance of cassava to these stresses may provide candidate genes for genetic improvement of stress resistance for cassava. Previously, we performed strand specific RNA sequencing for cassava TMS60444 shoot apices and young leaves under cold and PEG-induced drought stress, providing an excellent resource for analysis of stress responsive genes globally. To date, numerous TCP family members have been identified in various species33–45. However, no evidence is available regarding the TCP family in cassava. Due to the critical role of TCP transcription factors in the control of plant development and abiotic stress responses, we performed for the first time the comprehensive analysis of the MeTCP transcription factor family in cassava. In the present study, a total of 36 non-redundant MeTCP transcrip- tion factor encoding genes were identified in cassava genome and were subsequently subjected to a systematic analysis, including phylogenetic relationships, gene structure, conserved motif and expression pattern among different tissues and hormone treatments. We also analyzed the expression of the MeTCP genes under normal growth conditions and various abiotic stressors. On the basis of the expression profiles of MeTCP genes and the phylogenetic analysis among the TCP domain proteins in Arabidopsis, rice and cassava, the functions of MeTCPs were predicted. Taken together, our genome-wide analysis of MeTCP gene family will contribute to future studies on the functional characterization of MeTCP proteins in cassava, as well as the identification and comprehensive analysis of the TCP transcription factor family in other species. Widely cultivated in tropical area, cassava is considered as one of the most important economic crops world- wide, providing starch for food, feed and energy production28–30. Cassava can effectively utilize light, heat and water resource; it is resistant to drought but sensitive to low temperature31. Cold and drought stress severely limit cassava plant growth and yield32. Ning Lei2, Xiang Yu 3,4, Shuxia Li1, Changying Zeng1, Liangping Zou1, Wenbin Liao1 & Ming Peng1 Thus, uncovering the mechanisms underlying the resistance of cassava to these stresses may provide candidate genes for genetic improvement of stress resistance for cassava. Previously, we performed strand specific RNA sequencing for cassava TMS60444 shoot apices and young leaves under cold and PEG-induced drought stress, providing an excellent resource for analysis of stress responsive genes globally.i g p g y p g g y To date, numerous TCP family members have been identified in various species33–45. However, no evidence is available regarding the TCP family in cassava. Due to the critical role of TCP transcription factors in the control of plant development and abiotic stress responses, we performed for the first time the comprehensive analysis of the MeTCP transcription factor family in cassava. In the present study, a total of 36 non-redundant MeTCP transcrip- tion factor encoding genes were identified in cassava genome and were subsequently subjected to a systematic analysis, including phylogenetic relationships, gene structure, conserved motif and expression pattern among different tissues and hormone treatments. We also analyzed the expression of the MeTCP genes under normal growth conditions and various abiotic stressors. On the basis of the expression profiles of MeTCP genes and the phylogenetic analysis among the TCP domain proteins in Arabidopsis, rice and cassava, the functions of MeTCPs were predicted. Taken together, our genome-wide analysis of MeTCP gene family will contribute to future studies on the functional characterization of MeTCP proteins in cassava, as well as the identification and comprehensive analysis of the TCP transcription factor family in other species. SCIENTIfIC REPOrTS \| 7: 10016 \| DOI:10.1038/s41598-017-09398-5 Results Id tifi Additionally, the TCP genes showed an interspersed distribution in most clades, which is consistent with those found in previous analyses of TCP in G. raimondii and Citrullus lanatus35, 43, indicating that the TCP family expanded before the divergence of the lineages. However, the TCP genes were not evenly distributed in some clades, such as the largest clade Group H has the maximum 7 members, whereas Group C contains only 3 MeTCP genes from cassava, suggesting the existence of a diversified MeTCP family in cassava with diverse functions (Fig. 1). Remarkably, many Arabidopsis TCP genes had more than three counterparts in cassava, such as MeTCP11, MeTCP15, MeTCP18 and MeTCP20, indicating that MeTCP genes duplicated after the divergence of cassava and Arabidopsis. Results Id tifi It also suggests that higher number of genes in cas- sava as compared to Arabidopsis is the result of more gene duplication events in cassava or higher frequency of SCIENTIfIC REPOrTS \| 7: 10016 \| DOI:10.1038/s41598-017-09398-5 2 www.nature.com/scientificreports/ Gene ID Name Length(aa) MW(Da) PI Gravy Genomic locus Manes.01G187000.1 MeTCP15a 392 42214.9 9.32 −0.643 Chromosome01:28455200..28457428 forward Manes.01G263300.1 MeTCP9a 366 38916.33 8.94 −0.283 Chromosome01:33561357..33563642 forward Manes.01G020700.1 MeTCP18a 387 44030.35 7.93 −0.773 Chromosome01:3502035..3504116 forward Manes.01G094200.1 MeTCP13a 350 38533.91 8.37 −0.667 Chromosome01:21880606..21882675 forward Manes.02G055900.1 MeTCP13b 361 39880.76 9.06 −0.63 Chromosome02:4209171..4211393 forward Manes.02G066400.1 MeTCP12 383 42814.84 9.78 −0.569 Chromosome02:4925458..4927250 reverse Manes.02G194200.1 MeTCP23a 425 45005.56 7.98 −0.572 Chromosome02:15997522..15999455 forward Manes.04G016700.1 MeTCP11d 240 26058.47 8.98 −0.394 Chromosome04:1959602..1961886 forward Manes.04G016800.1 MeTCP11b 155 16551.84 9.45 −0.415 Chromosome04:1966346..1966813 forward Manes.04G088500.1 MeTCP20a 312 33848.59 7.22 −0.782 Chromosome04:22580287..22581884 reverse Manes.05G041000.1 MeTCP9b 346 36898.79 7.8 −0.325 Chromosome05:2911279..2913198 forward Manes.05G100100.1 MeTCP15b 388 41706.51 8.23 −0.582 Chromosome05:8461910..8463076 reverse Manes.05G123700.1 MeTCP20c 73 8314.64 10.08 −1.034 Chromosome05:14666983..14667397 forward Manes.05G119300.1 MeTCP18b 372 42181.38 8.12 −0.748 Chromosome05:12207088..12208206 reverse Manes.06G072800.1 MeTCP18c 416 47024.77 9.48 −0.879 Chromosome06:18738590..18740594 forward Manes.06G083400.1 MeTCP5a 340 37976.22 8.47 −0.611 Chromosome06:19670814..19672682 reverse Manes.06G093900.1 MeTCP15c 396 42673.9 7.37 −0.73 Chromosome06:20627278..20631189 forward Manes.06G141800.1 MeTCP19 358 37747 5.53 −0.478 Chromosome06:24669381..24671688 reverse Manes.07G022400.1 MeTCP8a 550 56832.22 7.55 −0.646 Chromosome07:2093408..2096799 forward Manes.08G009200.1 MeTCP11a 185 19802.37 6.97 −0.497 Chromosome08:675290..675847 reverse Manes.09G051000.1 MeTCP16 403 43569.25 7.58 −0.11 Chromosome09:6817330..6819454 forward Manes.10G120400.1 MeTCP8b 562 58079.71 8.46 −0.621 Chromosome10:23214679..23218733 reverse Manes.11G083000.1 MeTCP20b 307 32859.71 8.99 −0.671 Chromosome11:11404469..11406607 forward Manes.11G108500.1 MeTCP7 273 27679.86 9.72 −0.271 Chromosome11:20090517..20092799 forward Manes.11G149000.1 MeTCP11c 198 21355.38 9.43 −0.335 Chromosome11:26034972..26035568 reverse Manes.12G007700.1 MeTCP20d 299 32367.12 9.41 −0.702 Chromosome12:722180..724162 reverse Manes.13G008300.1 MeTCP20e 282 30554.12 9.52 −0.656 Chromosome13:815757..817107 reverse Manes.13G138300.1 MeTCP3b 343 37809.47 5.9 −0.782 Chromosome13:26570479..26573043 reverse Manes.14G058400.1 MeTCP3a 336 36987.57 5.87 −0.699 Chromosome14:4620482..4621572 reverse Manes.14G077200.1 MeTCP15d 398 42850.16 7.39 −0.716 Chromosome14:6245053..6246249 reverse Manes.14G086500.1 MeTCP5b 387 43276.03 7.21 −0.713 Chromosome14:6949867..6951866 forward Manes.14G097000.1 MeTCP18d 474 52697.96 9.31 −0.782 Chromosome14:7827437..7828861 reverse Manes.15G091000.1 MeTCP4 422 45753.27 6.17 −0.636 Chromosome15:6736704..6738513 reverse Manes.15G123800.1 MeTCP2a 481 52167.1 7.86 −0.886 Chromosome15:9374446..9382168 reverse Manes.17G072800.1 MeTCP2b 481 52472.3 7.06 −0.926 Chromosome17:21184852..21198102 reverse Manes.18G103100.1 MeTCP23b 425 45159.97 6.81 −0.563 Chromosome18:9144633..9147156 forwardi Table 1. TCP genes identified in cassava genome. retaining copies after duplication. Group C contained three cassava TCPs, two Arabidopsis members but there were no TCP from rice, implying this group was either acquired after the divergence of monocots and dicots or lost in rice. Remarkably, 5 of the group H members (AtTCP2, AtTCP3, AtTCP4, AtTCP10, and AtTCP24) are post-transcriptionally targeted by miRNA319 in Arabidopsis. Results Id tifi A total of 36 MeTCPs from cassava, 24 AtTCPs from Arabidopsis and 22 OsTCPs from rice were used to construct the Neighbor-Joining tree by MEGA 6.0 with 1000 bootstrap based on the full length sequences of TCPs. The eight subgroups are indicated with different colors Figure 1. Phylogenetic relationships of TCP transcription factors from cassava, Arabidopsis and rice. A total of 36 MeTCPs from cassava, 24 AtTCPs from Arabidopsis and 22 OsTCPs from rice were used to construct the Neighbor-Joining tree by MEGA 6.0 with 1000 bootstrap based on the full length sequences of TCPs. The eight subgroups are indicated with different colors. Figure 1. Phylogenetic relationships of TCP transcription factors from cassava, Arabidopsis and rice. A total of 36 MeTCPs from cassava, 24 AtTCPs from Arabidopsis and 22 OsTCPs from rice were used to construct the Neighbor-Joining tree by MEGA 6.0 with 1000 bootstrap based on the full length sequences of TCPs. The eight subgroups are indicated with different colors. showed that the only motif that hit for the database was the conserved TCP domain (motif 1). TCP domain was found in all MeTCPs, except MeTCP20c, which contained a truncated TCP domain. In general, MeTCP proteins clustered in same subgroup share similar motif composition, while high divergence was observed among different subgroups. This observation indicated that the MeTCP members within the same subgroup may have redundant functions and that some motifs may contribute to the specific function of that subfamily, which is in agreement with the previous report35, 41. According to 36 MeTCP sequence features within the TCP domain, we determined that MeTCPs from Group A, B, C, D and E belong to class I subfamily while the other MeTCPs belong to class II subfamily, as for all species so far. As reported earlier, the group members belonged to class I subfamily have extended homology C-terminal from the TCP domain, while the class II subfamily has an extended basic region, and all groups have internally conserved, but distinct loop region sequences25, 51. The motif analysis also showed that sequence conservation outside the TCP domain was low and sequence length on both sides of the TCP domain varied greatly, resulting in proteins ranging from 73 (MeTCP20c) to 563 (MeTCP8b) amino acids. Results Id tifi The closest homologs of these Arabidopsis genes in cassava are these five genes: MeTCP2a, MeTCP2b, MeTCP3a, MeTCP3b and MeTCP4, all containing putative tar- get site of miR31947, 48. This suggests that regulation of leaf development by a redundant set of miRNA-regulated homologous TCP genes occurs in cassava. retaining copies after duplication. Group C contained three cassava TCPs, two Arabidopsis members but there were no TCP from rice, implying this group was either acquired after the divergence of monocots and dicots or lost in rice. Remarkably, 5 of the group H members (AtTCP2, AtTCP3, AtTCP4, AtTCP10, and AtTCP24) are post-transcriptionally targeted by miRNA319 in Arabidopsis. The closest homologs of these Arabidopsis genes in cassava are these five genes: MeTCP2a, MeTCP2b, MeTCP3a, MeTCP3b and MeTCP4, all containing putative tar- get site of miR31947, 48. This suggests that regulation of leaf development by a redundant set of miRNA-regulated homologous TCP genes occurs in cassava. Gene structure and conserved motifs of cassava TCPs. To further examine the structural features of cassava TCP genes, we investigated the exon/intron structures of individual MeTCP genes by alignment of cDNA sequences and corresponding genomic DNA sequences. Additionally, we also built an unrooted phylogenetic tree with MeTCP protein sequences (Fig. 2a), to determine whether the gene structure of MeTCPs is consistent with the phylogenetic subfamily. As illustrated in Fig. 2b, the number of introns of MeTCP genes varied from 0 to 4. For instance, 32 out of 36 MeTCP genes had no intron, while the other MeTCP genes possess 1–3 introns, with the exception of MeTCP16 containing four introns. As expected, most of MeTCP genes in the same subfamily showed similar exon-intron distribution patterns in terms of exon length and intron number, which supports their close evolutionary relationship and the classification of subgroup. However, MeTCP genes in group H showed great variability in intron number and exon length. y g To obtain more insights into the diversity of motif compositions among MeTCPs, conserved motifs were predicted by using MEME program49, and annotated by ScanProsite program50. A total of 20 conserved motifs in the MeTCP proteins, designated as motif 1 to motif 20, were captured by MEME (Fig. 2c, Fig. S2). The results SCIENTIfIC REPOrTS \| 7: 10016 \| DOI:10.1038/s41598-017-09398-5 3 www.nature.com/scientificreports/ Figure 1. Phylogenetic relationships of TCP transcription factors from cassava, Arabidopsis and rice. Results Id tifi For example, MeTCP20c, the smallest predicted protein, is probably truncated by a frame shift mutation that cause premature termination, since sequence homology with Arabidopsis TCP20 extends well beyond the stop codon. This result is similar to SlTCP27, which encode the smallest TCP protein with 113 amino acids in tomato36. Although MeTCP20c lacks the conserved C-terminal part of the TCP domain, which may alter its DNA binding ability, experimental evidences are required to establish the precise role of truncated TCP domain in the regula- tion of MeTCP20c activity. Expression analysis of MeTCP genes in different tissues. To investigate the potential functions as well as to identify probable functional redundancy through similar expression patterns for the cassava TCP genes, the detection of their expression were carried out in different tissues including root, leaf, stem and shoot apex using qRT-PCR. As shown in Fig. 3, it is apparent that the expression levels in different tissues vary widely between the cassava TCP genes, as well as between different tissues for individual TCP genes, indicating functional SCIENTIfIC REPOrTS \| 7: 10016 \| DOI:10.1038/s41598-017-09398-5 4 www.nature.com/scientificreports/ Figure 2. The gene structure and conserved protein motifs of MeTCP genes according to the phylogenetic relationship. (a) The unrooted phylogenetic tree of all TCP genes in cassava was constructed using Neighbor- Joining method and the bootstrap test was performed with 1,000 iterations. (b) The gene structure with exon/ intron organization of TCP genes of cassava. The orange boxes represent 5′-UTR or 3′-UTR, red boxes represent exons and blue lines indicate introns. (c) The conserved protein motifs in the TCP family were identified using MEME program. Each motif is indicated with a specific color. Figure 2. The gene structure and conserved protein motifs of MeTCP genes according to the phylogenetic relationship. (a) The unrooted phylogenetic tree of all TCP genes in cassava was constructed using Neighbor- Joining method and the bootstrap test was performed with 1,000 iterations. (b) The gene structure with exon/ intron organization of TCP genes of cassava. The orange boxes represent 5′-UTR or 3′-UTR, red boxes represent exons and blue lines indicate introns. (c) The conserved protein motifs in the TCP family were identified using MEME program. Each motif is indicated with a specific color. specialization among TCP gene family members in cassava plant development. Of them, some genes were exclu- sively highly expressed in a specific tissue. Results Id tifi For example, MeTCP20e and MeTCP11d genes exhibited higher transcriptional abundance in roots as compared to other organs; MeTCP2a, MeTCP3a, MeTCP5b, MeTCP8a, MeTCP13a and MeTCP20d showed specifically high expression in leaves, implying their specific roles in the corresponding tissues. Opposite to the tissue-specific expression pattern of MeTCP genes, many genes were more widely and less specifically expressed, such as MeTCP9b, MeTCP13b, MeTCP20a, MeTCP20b, and MeTCP23a genes, implying that these genes may play regulatory roles at multiple development stages. However, further stud- ies are still needed to unravel the divergent roles of MeTCP genes.i specialization among TCP gene family members in cassava plant development. Of them, some genes were exclu- sively highly expressed in a specific tissue. For example, MeTCP20e and MeTCP11d genes exhibited higher transcriptional abundance in roots as compared to other organs; MeTCP2a, MeTCP3a, MeTCP5b, MeTCP8a, MeTCP13a and MeTCP20d showed specifically high expression in leaves, implying their specific roles in the corresponding tissues. Opposite to the tissue-specific expression pattern of MeTCP genes, many genes were more widely and less specifically expressed, such as MeTCP9b, MeTCP13b, MeTCP20a, MeTCP20b, and MeTCP23a genes, implying that these genes may play regulatory roles at multiple development stages. However, further stud- ies are still needed to unravel the divergent roles of MeTCP genes.i g g To address the conservation and specificity of TCP expression pattern beyond species, we compared the expression level of homologous TCP gene pairs in these four tissues between Arabidopsis and cassava. We found a subset of TCP genes were positively correlated with pearson correlation coefficient (PCC) higher than 0.3 between Arabidopsis and cassava (Fig. S3–4, such as MeTCP2a/b and AtTCP2, MeTCP5a/b and AtTCP5, MeTCP8a/b and AtTCP8, MeTCP13a/b and AtTCP13, MeTCP15b/c and AtTCP15, MeTCP19 and AtTCP19 et al., indicating func- tional conserved expressional pattern of these genes. In contrast, some TCP genes show no correlation or negative SCIENTIfIC REPOrTS \| 7: 10016 \| DOI:10.1038/s41598-017-09398-5 5 www.nature.com/scientificreports/ Figure 3. Heatmap representation for expression profiles of 36 MeTCP genes across different tissues. The expression levels of MeTCP genes were obtain through quantitative real-time PCR. MeACTIN was used as the reference gene. Figure 3. Heatmap representation for expression profiles of 36 MeTCP genes across different tissues. The Figure 3. Heatmap representation for expression profiles of 36 MeTCP genes across different tissues. The expression levels of MeTCP genes were obtain through quantitative real-time PCR. MeACTIN was used as the reference gene. Results Id tifi correlation between Arabidopsis and cassava, such as MeTCP9a/b and AtTCP9, MeTCP11a/b/c/d and AtTCP11, suggesting their functions have been diversely changed in different species. correlation between Arabidopsis and cassava, such as MeTCP9a/b and AtTCP9, MeTCP11a/b/c/d and AtTCP11, suggesting their functions have been diversely changed in different species. Expression patterns of MeTCP genes in response to hormone treatments. Multiple studies have been reported that TCP proteins regulate plant development and environmental stress adaption by mediating hormone biogenesis and response17–19, 21. To study the total effect of plant hormones on MeTCP genes, the expres- sion levels of 36 MeTCP genes were detected in response to abscisic acid (ABA), gibberellin (GA3), indole acetic acid (IAA), jasmonate acid (JA), zeatin (ZT) and 6-benzylaminopurine (6-BA) hormone treatments by quantita- tive RT-PCR (Fig. 4, Supplemental Table S1). In general, hormone treatments resulted in a wide variety of MeTCP gene expression profiles. In JA treatment, 15 and 7 MeTCP genes were obviously induced and inhibited, respec- tively. Of them, the most up-regulated gene was MeTCP20e, and the most down-regulated gene was MeTCP3a. Similarly, 6-BA and ZT treatment led to 12 and 13 MeTCP genes were obviously induced, 11 and 5 MeTCP genes were inhibited, respectively. In GA treatment, most of MeTCPs were significantly induced and only two MeTCPs were inhibited. MeTCP7 and MeTCP23b were found to be most up-regulated. As for IAA treatment, 10 and 5 MeTCP genes showed dramatic increase and decrease, respectively. MeTCP7 and MeTCP16 went through the largest increase and decrease, respectively. ABA plays a crucial role in the adaptive response of plants to abiotic stresses52. We found 22 members showed strong sensitivity toward ABA, indicating that these genes may be regulated by ABA signal pathway. Among them, 19 genes had relative high levels of transcript abundance after ABA treatment. Notably, most of MeTCP genes responded to at least one treatment; Particularly, MeTCP11b, SCIENTIfIC REPOrTS \| 7: 10016 \| DOI:10.1038/s41598-017-09398-5 6 www.nature.com/scientificreports/ Figure 4. Heatmap representation for expression patterns of MeTCP genes under various hormone treatments. The expression profile data of MeTCP genes under JA, 6-BA, GA, ZT, ABA and IAA were obtain through quantitative real-time PCR. MeACTIN was used as the reference gene. Figure 4. Heatmap representation for expression patterns of MeTCP genes under various hormone treatments. The expression profile data of MeTCP genes under JA, 6-BA, GA, ZT, ABA and IAA were obtain through quantitative real-time PCR. Results Id tifi However, in discordant response class, SCIENTIfIC REPOrTS \| 7: 10016 \| DOI:10.1038/s41598-017-09398-5 7 www.nature.com/scientificreports/ Figure 5. Expression profiles of MeTCP genes in leaves and shoot apices after cold and drought treatment. The transcript data generated from three replicates of RNA-seq data. The relative expression values were log2 transformed. The bar represents relative expression values. Figure 5. Expression profiles of MeTCP genes in leaves and shoot apices after cold and drought treatment. The transcript data generated from three replicates of RNA-seq data. The relative expression values were log2 transformed. The bar represents relative expression values. three genes (MeTCP15d, MeTCP15b and MeTCP16) showed increased expression pattern under cold treatment, whereas down-regulated after drought treatment. By contrast, the expression levels of four genes (MeTCP2b, MeTCP19, MeTCP13a and MeTCP13b) were induced by drought treatment, but were repressed or unaltered after cold treatment. Meanwhile, MeTCP8a/5a and MeTCP20a/20b/9b/18d/3b were specifically response to cold and drought stress, respectively. Generally, the expression levels of MeTCP genes in response to cold and drought stresses were dramatically changed, implying their putative roles in stress tolerance. three genes (MeTCP15d, MeTCP15b and MeTCP16) showed increased expression pattern under cold treatment, whereas down-regulated after drought treatment. By contrast, the expression levels of four genes (MeTCP2b, MeTCP19, MeTCP13a and MeTCP13b) were induced by drought treatment, but were repressed or unaltered after cold treatment. Meanwhile, MeTCP8a/5a and MeTCP20a/20b/9b/18d/3b were specifically response to cold and drought stress, respectively. Generally, the expression levels of MeTCP genes in response to cold and drought stresses were dramatically changed, implying their putative roles in stress tolerance. Expression analysis of MeTCP genes under various abiotic stress treatments. To further assess the response of MeTCP genes to various abiotic stresses and related signaling pathway at transcriptional lev- els, 12 MeTCP genes (MeTCP20c, MeTCP18a, MeTCP20e, MeTCP11a, MeTCP11b, MeTCP18b, MeTCP11c, MeTCP12, MeTCP18c, MeTCP4, MeTCP3a and MeTCP15c) induced or repressed by cold and drought stresses based on RNA-seq data were chosen for further examination of their expression patterns after cold, drought and salt treatments (Fig. 6). We found most of the analyzed genes exhibited differential expression in response to at least one stress treatment, implying their putative roles in these stresses tolerance. Overall, in the cold, drought and salt conditions, expression of MeTCP20c, MeTCP18a, MeTCP20e and MeTCP11a genes were significantly up-regulated, and the largest expressional change of these genes were usually observed when responding to cold and/or drought treatments. Results Id tifi MeACTIN was used as the reference gene. MeTCP12 and MeTCP21 responded to all hormone treatments, indicating these genes might play pivotal roles in the cross-talk of hormones, which would be candidates for further research in the field. However, we also found MeTCP11d, MeTCP20a and MeTCP23a were not able to respond to any treatments. Taken together, these results suggest the complicated regulatory mechanism of MeTCP genes in response to hormone treatments in cassava. MeTCP12 and MeTCP21 responded to all hormone treatments, indicating these genes might play pivotal roles in the cross-talk of hormones, which would be candidates for further research in the field. However, we also found MeTCP11d, MeTCP20a and MeTCP23a were not able to respond to any treatments. Taken together, these results suggest the complicated regulatory mechanism of MeTCP genes in response to hormone treatments in cassava. Expression profiles of MeTCP genes in response to cold and/or drought stress. Plants are fre- quently challenged by abiotic stressors such as cold and drought. Recent studies have suggested that TCP pro- teins are widely involved in signaling and response to environmental stimuli18, 22. However, information on the involvement of TCP proteins in stress responses in cassava is limited. To investigate the potential roles of MeTCP genes in response to abiotic stresses, cassava seedlings of TMS60444 genotypes were subjected to cold (4 °C) and PEG (20% PEG 6000)-induced drought stress and then the leaves tissues were sampled to extract RNA for sub- sequent RNA-seq analysis. According to the transcriptome data, 18 (50%) and 24 (66.7%) MeTCP genes showed significantly change (fold change >2) under cold and drought treatment, respectively. Among them, 7 (38.9%) and 11 (61.1%) MeTCP genes were up- and down-regulated by cold, respectively; 10 (41.7%) and 14 (58.3%) MeTCP genes were up- and down-regulated by drought, respectively (Fig. 5, Supplemental Table S2). These results also showed that the number of MeTCP genes down-regulated by cold and drought was greater than that were up-regulated, suggesting the comprehensive response of MeTCP genes to cold and/or drought at transcrip- tional levels. There were 23 MeTCP genes in total differential expressed under both cold and drought treatments, which were categorized into 4 different classes: concordant response to cold and drought, discordant response, cold-specific and drought-specific. Among concordant response, MeTCP20c/20e/11a and MeTCP18b/11c/12 were co-induced and co-repressed by two kinds of stresses, respectively. Results Id tifi This observation is well consistent with the RNA-seq data. 6 genes (MeTCP11b, MeTCP18b, MeTCP11c, MeTCP12, MeTCP4 and MeTCP3a) were down-regulated following cold and drought treatments, among which, MeTCP12 underwent the greatest change of mRNA expression levels. Interestingly, the expression levels of all these MeTCP genes showed obviously increase under salt treatment, suggesting they may play different roles in response to the three stresses. In addition, not too many changes were observed in MeTCP18c and MeTCP15c genes when any of three stresses were carried out. These data show the potential of some MeTCP genes for enhancing adversity resistant capacity in cassava. Analysis of the regulatory cis-elements in the promoter of cold- and drought-responsive MeTCPs. The altered expression of 12 MeTCPs that were co-induced/repressed by cold and drought stresses indicates that they may be regulated by key stress regulatory genes. In order to elucidate the mechanism of transcriptional regulation of these genes, analysis of their promoter region was performed for the cis-elements. A 1.5 kb sequence upstream to the open reading frame of MeTCP20c, MeTCP18a, MeTCP20e, MeTCP11a, MeTCP11b, MeTCP18b, MeTCP11c, MeTCP12, MeTCP18c, MeTCP4, MeTCP3a and MeTCP15c was identified and subjected to MEME analysis. A number of common cis-acting elements were identified in the proximal 8 SCIENTIfIC REPOrTS \| 7: 10016 \| DOI:10.1038/s41598-017-09398-5 www.nature.com/scientificreports/ e.com/scientificreports/ Figure 6. Confirmation of the expression patterns of cold- and drought-responsive MeTCPs using qRT- PCR. The expression patterns of MeTCP under cold, drought and salt stress. The values shown are the means ± standard deviation of three replicates. MeACTIN was used as the reference gene. Figure 7. The conserved DNA sequence motifs analysis of cold- and drought-responsive MeTCP promoters. The conserved motifs in the TCP promoters were identified using MEME program. Each motif is indicated with a specific color. ure.com/scientificreports/ Figure 6. Confirmation of the expression patterns of cold- and drought-responsive MeTCPs using qRT- PCR. The expression patterns of MeTCP under cold, drought and salt stress. The values shown are the means ± standard deviation of three replicates. MeACTIN was used as the reference gene. Figure 6. Confirmation of the expression patterns of cold- and drought-responsive MeTCPs using qRT- PCR The expression patterns of MeTCP under cold, drought and salt stress The values shown are the Figure 6. Confirmation of the expression patterns of cold- and drought-responsive MeTCPs using qRT- PCR. The expression patterns of MeTCP under cold, drought and salt stress. Results Id tifi The values shown are the means ± standard deviation of three replicates. MeACTIN was used as the reference gene. Figure 7. The conserved DNA sequence motifs analysis of cold- and drought-responsive MeTCP promoters. The conserved motifs in the TCP promoters were identified using MEME program. Each motif is indicated with a specific color. Figure 7. The conserved DNA sequence motifs analysis of cold- and drought-responsive MeTCP promoters. The conserved motifs in the TCP promoters were identified using MEME program. Each motif is indicated with a specific color. promoters (Fig. 7, Fig. S5). All the identified promoters had motif 1, and most of them also contained motif 3 and 10, except for MeTCP20e, MeTCP11b, MeTCP11c, MeTCP12 and MeTCP18c, suggesting that these commonly present cis-acting elements may be involved in stress response. Notably, motifs such as 9, 13, 14 and 15 are more present in the promoters of MeTCP which are down-regulated by both cold and drought stresses, suggesting that these motifs may play key roles in the regulation of MeTCP members. promoters (Fig. 7, Fig. S5). All the identified promoters had motif 1, and most of them also contained motif 3 and 10, except for MeTCP20e, MeTCP11b, MeTCP11c, MeTCP12 and MeTCP18c, suggesting that these commonly present cis-acting elements may be involved in stress response. Notably, motifs such as 9, 13, 14 and 15 are more present in the promoters of MeTCP which are down-regulated by both cold and drought stresses, suggesting that these motifs may play key roles in the regulation of MeTCP members. www.nature.com/scientificreports/ Similar to the gene structure, most subgroups of MeTCPs also exhibited conserved motif composition, with several motifs observed in some MeTCP subgroups, such as motif 4 and 17 for subgroup D, motif 7 for subgroup A (Fig. 2c). These unique motifs may contribute to the specific function of these subgroup members. In general, the majority of MeTCP genes in the same subfamilies are evolutionarily conserved, which supports their close evolutionary relationship and the classification of subgroups.h y pi g p The expression pattern analysis of MeTCP genes helps us to assess their possible functions and provide a solid foundation for future functional studies. Generally, similar to the previous study, MeTCP genes exhibited greatly differential expression patterns across a variety of tissues, not only among subgroups but members within the same subgroups (MeTCP5a and MeTCP5b, MeTCP13a and MeTCP13b), suggesting that these MeTCP genes may function diversely in various tissues (Fig. 3). On the contrary, some MeTCP genes with extremely high sequence identity (MeTCP2a and MeTCP2b, MeTCP18c and MeTCP18d) showed conserved expression patterns (Fig. 3), implying they may play a redundant role in regulating plant growth. p y g y y p y g g p g To date, the role of plant hormones in regulating plant growth, development, and abiotic stress responses by modulating gene expression is well established52, 55. To our knowledge, although the relationship between TCP proteins and hormones has been widely known, such as cytokinins, JA and GA, the dynamic and spatially expression patterns of MeTCP genes response to various hormones was still obscure. Our current results revealed that the majority of MeTCP genes detected here displayed distinct changes under different hormone treatments. To expect, most of MeTCP were up-regulated or down-regulated by cytokinins (ZT and 6-BA). MeTCP15b and MeTCP23b, orthologs of AtTCP15 and AtTCP23, respectively, had high expression levels under ZT and 6-BA treatments. In Arabidopsis, cytokinin treatments induce TCP15 transcription and promote TCP15 (and TCP14) protein activation by post-translational modification, which in turn promote cytokinin responses56. MeTCP4, MeTCP20b, and MeTCP20e, orthologs of AtTCP4 and AtTCP20, respectively, had altered expression patterns under JA. In young leaves of Arabidopsis, AtTCP20 repressed the transcription of LIPOXYGENASE2 (AtLOX2) gene, which is involved in JA synthesis and promotes leaf senescence, while this negative control is antagonized by AtTCP4 as the leaf matures19. www.nature.com/scientificreports/ www.nature.com/scientificreports/ transcription that play very important roles during plant development and abiotic stress responses22. To our knowledge, although a range of TCP family members have been described in various species35, 36, 40–45, 54, no studies have been performed on TCP genes in cassava. Additionally, the mechanisms of cassava responds to abiotic stress are poorly understood. This background knowledge prompted us to identify the full complement and expression profile of this important gene family during development and under abiotic stresses in cassava.i p pi p g y g p In the present study, a comprehensive set of 36 non-redundant TCP-encoding genes were identified and characterized from the current version of the cassava genome. Previous studies have identified 24 TCP genes in Arabidopsis33, 22 in rice34, 38 in cotton35, 30 in tomato36, and 27 in watermelon43. The amplification of TCP gene family members in cassava can be explained by its larger genome size (~760 Mb) compared to that of Arabidopsis (~125 Mb) and gene duplication events in this family. Evolutionary analysis indicated that the cassava TCPs could be clustered into 8 subgroups, which is minimal different with previous evolutionary classification of TCPs in cotton and watermelon35, 43 (Fig. 1). The phylogenetic tree showed obvious differences in number of MeTCPs and ratio of MeTCPs/AtTCPs among subgroups. The change in the ratio of MeTCPs/AtTCPs suggested that the MeTCP family had undergone lineage-specific expansion and functional divergence during the course of evolu- tion. Phylogenetic tree also showed that group C contained three cassava members and two Arabidopsis members, but there were no TCP from rice. On contrast, TCPs in subgroup A expanded in monocots but not in Arabidopsis, indicating that TCP genes of these subgroups expanded in a species-specific manner from common ancestral genes that were present prior to the diversification of the monocot and dicot lineages. The classification of TCP protein was further supported by gene structure analysis and conserved protein motif analysis. Gene structure analysis showed that the majority of MeTCP genes within the same subgroup exhibited very similar gene struc- ture in terms of exon length and intron number. Furthermore, conserved protein motif analysis indicated that all the MeTCP proteins contained typical TCP domain, except MeTCP20c, which contains a truncated TCP domain. SCIENTIfIC REPOrTS \| 7: 10016 \| DOI:10.1038/s41598-017-09398-5 Discussion Discussion Adverse environmental conditions, such as cold and drought stress, impose severe effects on the plant growth, development and limit crop productivity and yield53. TCP transcription factors are a class of plant-specific SCIENTIfIC REPOrTS \| 7: 10016 \| DOI:10.1038/s41598-017-09398-5 9 Materials and Methods d ifi i d bi i f Identification and bioinformatics analysis of candidate genes. To identify potential members of the cassava TCP protein family, The Arabidopsis TCP protein sequences were used as seed queries in BLASTp searches against the cassava database (Phytozome: http://www.phytozome.net/cassava.php)57. The TCP domain (PF03634, Pfam; http://pfam.sanger.ac.uk/)46 was also employed as query to perform a blast search against the same genome database. The identified MeTCP proteins were renamed as MeTCP2 to MeTCP23 according to the Arabidopsis TCP proteins with highest sequence similarity. Information on MeTCP genes, including exons and introns number, open reading frame (ORF) and amino acid (AA) lengths, was obtained from Phytozome data- base. The molecular weight, theoretical isoelectric point (PI) and grand average of hydropathy (GRAVY) of the MeTCP proteins were investigated using ExPASy online tools (http://web.expasy.org/protparam/). Analysis of phylogenetic relationships and gene structure. Multiple sequence alignments were applied to confirm the conserved domains of predicted MeTCP proteins. The Clustal × 2.058 was employed to align the full-length MeTCP proteins from cassava, Arabidopsis and rice. Then, the bootstrap neighbor-joining evolutionary tree was created by MEGA 6.0 software59 with 1000 bootstrap replicates based on the sequence alignments. The exon-intron organization of MeTCP genes was determined by comparing the coding DNA sequence (CDS) with its corresponding genomic sequences using the Gene Structure Display Server (GSDS) software (http://gsds.cbi.pku.edu.cn/)60. Identification of conserved motif of MeTCP proteins and promoters. By using the Multiple Expectation maximization for Motif Elicitation (MEME) program (http://meme.nbcr.net/meme/cgi-bin/meme.cgi), the conserved motifs in full-length cassava MeTCP protein sequences were identified with the following param- eters: maximum number of motifs was 20 and the optimum width of motifs was set between 10 and 5049. The identified protein motifs were further annotated with ScanProsite50. MeTCP promoter sequences in cassava were submitted to online MEME program for identification of conserved motifs. The optimized MEME parameters were as follows: any number of repetitions and maximum number of motifs-15. Plant materials and hormone/stress treatment. Cassava (Manihot esculenta) cultivar (TMS60444) was used in the present study. Segments cut from cassava stems were inserted into MS plates in a greenhouse at 26 ± 2 °C, with a photoperiod of 16 h light and 8 h dark. All hormone and environmental treatments were conducted when uniform-sized seedlings developed two fully opened trifoliate leaves (approximately two weeks after sowing). Materials and Methods d ifi i d bi i f For hormone treatment, 14-day-old cassava seedlings were soaked in liquid MS medium with 100 μM indole acetic acid(IAA), 100 μM gibberellin (GA3), 100 μM Methyl jasmonate (MeJA), 100 μM abscisic acid (ABA),100 μM zeatin (ZT) and 100 μM, 6-benzylaminopurine (6-BA) for 3 h, respectively, and then the young leaves and shoot apex from at least ten separate seedlings/plants were harvested. Seedlings soaked in liquid MS medium without any hormone were used as control. For cold treatment, seedlings were placed at 4 °C for 24 h, and then the young leaves and shoot apex were collected for RNA isolation. For drought and salt stress treatment, cassava seedlings were treated with 20% PEG6000 and 100 mM NaCl, and harvested at 6 h after treatment, respec- tively. In all cases, parallel and untreated plants at the same stage were used as controls. All samples harvested were flash-frozen in liquid nitrogen, and stored at −80 °C until RNA isolation. RNA isolation and expression analysis. Total RNA was extracted from 0.1 g of tissue by using Plant RNA kit (OMEGA), following the manufacturer’s instructions. Reverse transcription reactions were performed using 5 μg of RNA with PrimeScript RT reagent kit with gDNA Eraser (TIANGEN, Beijing, China). Quantitative reverse transcription PCR (qRT-PCR) was performed as described elsewhere61, the PCR conditions were as fol- lows: pre-incubation at 94 °C for 5 min, followed by 40 cycles at 94 °C for 10 s, 60 °C for 10 s, 72 °C for 30 s. After amplication was complete, a melting curve was obtained by holding at 95 °C for 5 s and then at 65 °C for 15 s, followed by heating slowly at 0.1 °C/s to 95 °C. Real-time PCR was performed with a Bio-Rad real-time thermal cycling system using SYBR® Premix Ex Taq™ II (TaKaRa, Japan) to assess gene expression levels. The relative expression levels of each gene were calculated by the 2-ΔΔCt method. The cassava actin gene was used as internal control for normalization. The primers used are listed in Supplemental Table S3. MeTCP genes that were up- or down-regulated by at least two-fold were considered as differentially expressed. The experiments were performed in triplicate. Expression correlation analysis of level between cassava and Arabidopsis. The expression levels of AtTCPs in different tissues were extracted from the microarray data62. www.nature.com/scientificreports/ It is noteworthy that 22 MeTCPs were response to ABA, indicating these genes might function as key mediators of stress responses through ABA signaling pathways. Taken together, these results suggested that MeTCPs play potential regulatory roles by modulating phytohormone signaling in plant development or in the responses to stresses. Therefore, it will be particular important to further investigate the potential function of MeTCP genes in hormone signaling in the future. p g g g Previous reports have shown that plant TCP genes are involved in plant growth and development, as well as abiotic stress responses under normal and stressed growth conditions22. In cassava, our data showed that over half of the MeTCP genes were significantly upregulated under cold and drought condition. Among them, 4 genes were up-regulated, and 8 genes were down-regulated by both cold and drought stress. Moreover, combined analysis of expression correlation and promoter content has revealed that most of these MeTCP genes exhibited differential expression in response to more than one stress treatments, suggesting the wide involvement of MeTCP genes in environmental adaptation. Previous reports have shown that knockdown of miR319-dependent TCPs (by con- stitutive miR319 overexpression) increases drought and salinity stress tolerance in bentgrass20. Our data showed that MeTCP3a and MeTCP4, targets of miRNA319, had altered expression patterns under cold, drought and salt stress, suggesting that these genes might play important roles under abiotic stress conditions in cassava. These data indicated that MeTCP might function in resistance to abiotic stresses in cassava.i g In conclusion, we identified 36 TCP transcription factor genes from cassava. Phylogenetic analysis of cassava, Arabidopsis, and rice indicated that these MeTCP genes could be divided into 8 groups, which is supported by fur- ther conserved protein motif, and gene structure analyses. Although nearly all the MeTCP genes were expressed in the examined tissues, some genes were up-regulated in one or several specific organs. mRNA accumulation was altered by a variety of hormone treatments (ABA, IAA, GA, JA, ZT and 6-BA), environmental conditions (drought, high salinity, and low temperature). These results suggested that MeTCP family proteins play critical SCIENTIfIC REPOrTS \| 7: 10016 \| DOI:10.1038/s41598-017-09398-5 10 www.nature.com/scientificreports/ roles in maintaining cassava normal growth under normal or stress conditions through complicated mechanisms. Thus, additional studies on the detailed functions of each gene are warranted in cassava. References TCP transcription factors: architectures of plant form. 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Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre- ative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not per- mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2017 SCIENTIfIC REPOrTS \| 7: 10016 \| DOI:10.1038/s41598-017-09398-5 13
https://openalex.org/W4394819687	https://www.cambridge.org/core/services/aop-cambridge-core/content/view/3DF01666555CE7984FBDB05F49719A46/S0263574724000523a.pdf/div-class-title-frequency-dependent-span-class-inlineformula-span-class-alternatives-img-class-inline-graphic-mathjax-alternative-mathjax-alt-graphic-mathjax-off-data-mimesubtype-png-data-type-src-staticdomain-binary-version-id-urn-cambridge-org-id-binary-20240413180854316-0498-s0263574724000523-s0263574724000523-inline1-png-span-class-mathjax-tex-wrapper-data-mathjax-type-texmath-span-class-tex-math-mathjax-tex-math-mathjax-on-textrm-h-infty-span-span-span-span-control-for-wind-disturbance-rejection-of-a-fully-actuated-uav-div.pdf	English	null	Frequency-dependent control for wind disturbance rejection of a fully actuated UAV	Robotica	2,024	cc-by	8,278	C⃝The 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. https://doi.org/10.1017/S0263574724000523 Published online by Cambridge University Press Abstract In this paper, an H∞dynamic output feedback controller is experimentally implemented for the position regulation of a fully actuated tilted-rotor octocopter unmanned aerial vehicle (UAV) to improve wind disturbance rejection during station-keeping. To apply the lateral forces, besides the standard tilt-to-translate (attitude-thrust) movement, tilted- rotor UAVs can generate vectored (horizontal) thrust. Vectored-thrust is high-bandwidth but saturation-constrained, while attitude-thrust generates larger forces with lower bandwidth. For the ﬁrst time, this paper emphasizes the frequency-dependent allocation of weighting matrices in H∞control design based on the physical capabilities of the fully actuated UAV (vectored-thrust and attitude-thrust). A dynamic model of the tilted-rotor octocopter, including aerodynamic eﬀects and rotor dynamics, is presented to design the controller. The proposed H∞controller solves the frequency-dependent actuator allocation problem by augmenting the dynamic model with weighting transfer functions. This novel frequency-dependent allocation utilizes the attitude-thrust for low-frequency disturbances and vectored-thrust for high-frequency disturbances, which exploits the maximum potential of the fully actuated UAV. Several wind tunnel experiments are conducted to validate the model and wind disturbance rejection performance, and the results are compared to the baseline PX4 Autopilot controller on both the tilted-rotor and a planar octocopter. The H∞controller is shown to reduce station-keeping error by up to 50% for an actuator usage 25% higher in free-ﬂight tests. Frequency-dependent H∞control for wind disturbance rejection of a fully actuated UAV Jérémie X. J. Bannwarth, Shahab Kazemi and Karl Stol Department of Mechanical and Mechatronics Engineering, University of Auckland, Auckland, New Zealand Corresponding author: Shahab Kazemi; Email: shahab.kazemi@auckland.ac.nz Received: 5 October 2023; Accepted: 25 March 2024; First published online: 15 April 2024 Keywords: Multirotor UAV; disturbance rejection; H∞control Robotica (2024), 42, pp. 1781–1795 doi:10.1017/S0263574724000523 Robotica (2024), 42, pp. 1781–1795 doi:10.1017/S0263574724000523 RESEARCH ARTICLE 1. Introduction Performing outdoors is one of the most essential multirotor unmanned aerial vehicle (UAV) applica- tions [1, 2]. However, wind disturbances can adversely aﬀect the performance of UAVs and destabilize them. Many studies have focused on active disturbance rejection with automatic control in multirotor UAVs [3, 4]. H∞control is a well-known method for its high disturbance rejection and stability robust- ness, which has been widely used to address diﬀerent UAV problems [5–7]. The combination of model predictive control and H∞was designed for the wind disturbance rejection of quadrotors in simulation [8, 9]. In another simulation study, Massé et al. [10] incorporated a thorough aerodynamics model in their plant model and compared the control performances of linear quadratic regulation (LQR) and structured H∞synthesis. Structured H∞design was shown to provide a more reliable framework in wind distur- bance rejection compared to LQR. In an experimental study, Dai et al., presented acceleration feedback (AF) enhanced H∞control to deal with the wind disturbance on an underactuated hexacopter [11]. The experimental results suggested the superiority of AF-enhanced H∞over H∞control in eﬀectiveness and robustness against wind disturbance. Disturbance rejection through control is inherently limited by the bandwidth of the actuators. Planar UAVs (with all the rotors parallel) are underactuated which further limits the bandwidth of the actuation along translational axes [12]. Planar UAVs must change their attitude to produce lateral thrust, which is not desirable during station-keeping (attitude-thrust). Meanwhile, vectored-thrust (horizontal thrust) 1782 Jérémie X. J. Bannwarth et al. UAVs, as fully actuated systems, can produce thrust in the body xy-plane without generating moments (as opposed to planar rotors). One way to achieve vectored-thrust is to attach one or more rotors on servomotors to actively adjust their tilt angle [13–16]. This technique increases the number of moving parts and thus decreases reliability. Also, the servomotor bandwidth is a limiting factor, particularly under the inﬂuence of large gyroscopic moments. Another way to design fully actuator UAV is to have (omnidirectional) ﬁxed rotors by arranging them at extreme angles against each other [17, 18]. The downsides of this method are an increase in mass and reduction in hover eﬃciency. Finally, UAVs with passively tilted rotors [19–26] can be used to achieve vectored-thrust. Their rotors are tilted around diﬀering axes, increasing the controllable degrees of freedom of the system. 1. Introduction In this study, a passive tilted-rotor octocopter is presented to simplify the con- trol problem through a speciﬁc rotor tilt pattern and reduce the coupling between thrust generation and attitude control. Current vectored-thrust UAV literature is split between actuator allocation (convert- ing virtual control signals, such as desired body torques and thrusts, to physical actuator commands) and control design. While some studies concentrated on designing actuator allocation schemes [13, 22], some other researchers considered wind disturbance rejection as a control goal and designed a controller accordingly. Many controllers such as adaptive [19], robust chattering-free [21], and integral SMC [24] were designed and simulated for vectored-thrust UAV disturbance rejection, while none of them were implemented experimentally. This paper introduces a novel H∞synthesis tailored for fully actuated UAVs (tilted-rotor octocopter), aiming to enhance wind disturbance rejection. H∞synthesis is used due to its ability to weigh com- peting control goals in the frequency domain. Traditionally, the determination of weighting matrices relied on experimental data [5]. However, this work proposes an experimental implementation of a novel frequency-dependent weighting function allocation, building on the vectored-thrust capability of the fully actuated UAV. Notably, this work incorporates the physical capabilities of the fully actuated UAV into the frequency-dependent allocation. The new controller optimally assigns weights, utilizing attitude-thrust for low-frequency disturbances and vectored-thrust for high-frequency disturbances. This frequency-dependent actuator allocation is desirable because the maximum horizontal thrust that can be produced by changing the attitude of the UAV is signiﬁcantly larger than what is achievable using the vectored-thrust. This recognition is critical, as vectored-thrust is more prone to actuator saturation and better suited for smaller amplitude, higher-frequency commands. By harnessing the UAV’s ability to generate vectored-thrust, the controller achieves a targeted response to wind disturbances with diﬀer- ent frequencies. This allows the total capacity of the fully actuated UAV in wind disturbance rejection while keeping an acceptable performance. In other words, this frequency-dependent weighting is applied to use attitude control to reject low-frequency disturbances and use vectored-thrust for high-frequency disturbances. The rest of the paper is organized as follows. Initially, the tilted-rotor octocopter setup is introduced, and its coordinate frame and physical char- acteristics are presented. A comprehensive model including the eﬀects of wind disturbances and rotor dynamics is proposed in Section 3. Section 4 describes the design of the proposed H∞controller. The ﬂight performance is experimentally examined in a wind tunnel in Section 5. 1. Introduction Finally, conclusions are drawn in Section 6. https://doi.org/10.1017/S0263574724000523 Published online by Cambridge University Press 2. The physical setup and coordinate frame The presented tilted-rotor octocopter is designed and built based on a parameter sweep process to maximize agility for a given endurance and payload [25, 26] (see Fig. 1). The bandwidth from the vectored-thrust design is ten times higher than a comparable tilt-to-translate design. The maximum produced vectored-thrust is 24% of its weight. With reference to Fig. 1b, producing a net force along the x-axis is achieved by increasing the thrust of rotors 3 and 6, while decreasing the thrust of rotors 2 and 7. The moment generated by this operation can then be canceled by increasing the thrust of rotors 1 and 8 and decreasing that of rotors 5 and 4. Table 1 lists the parameters of the selected design. Vectors expressed by W, B, and Mi are expressed in the world, body, and ith-motor frame, respectively. https://doi.org/10.1017/S0263574724000523 Published online by Cambridge University Press 1783 Robotica Figure 1. (a) Photo of the tilted-rotor octocopter that is used in this work. (b) The front and back rotors (1, 4, 5, 8) are tilted about the body x-axis, while the left and right rotors (2, 3, 6, 7) are tilted around the body y-axis. The rotors of the UAV are equally distributed around the center of mass. Figure 1. (a) Photo of the tilted-rotor octocopter that is used in this work. (b) The front and back rotors (1, 4, 5, 8) are tilted about the body x-axis, while the left and right rotors (2, 3, 6, 7) are tilted around the body y-axis. The rotors of the UAV are equally distributed around the center of mass. The world frame and body frame use the typical north-east-down and front-right-down conventions, respectively. The rotors are tilted at an angle ζ. 3. Dynamics Wind introduces uncertainties in the aerodynamic forces acting on the UAV. Modeling these forces accurately is challenging due to the dynamic and unpredictable nature of wind. However, a compre- hensive dynamic model is needed to design the model-based controller. The position of the UAV in the world frame is deﬁned as ξ = [ x y z]T, and its attitude is deﬁned by the Hamilton quaternion q = [ q0 q1 q2 q3 ]T = [ q0 qT 1: 3 ]T [27]. The absolute angular velocity of the craft expressed in the body frame is deﬁned as Bν = [ Bp Bq Br]T. Using Newton–Euler formulas, the acceleration of the UAV is obtained as ¨ξ = 1 mt G + W B R BT + BFaero , (1) (1) where WG = [ 0 0 9.81mt ]T is the weight vector, BT is the total rotor thrust, W B R is the rotation matrix from the body frame to world frame, and BFaero represents the aerodynamic force acting on the frame. Likewise, the angular acceleration of the UAV is where WG = [ 0 0 9.81mt ]T is the weight vector, BT is the total rotor thrust, W B R is the rotation matrix from the body frame to world frame, and BFaero represents the aerodynamic force acting on the frame. Likewise, the angular acceleration of the UAV is B ˙ν = I−1 B Bτ −Bν × IB Bν + BMaero , (2) (2) where IB = diag(Ixx, Iyy, Izz) represents the mass moment of inertia of the frame, Bτ is the total rotor torque, and BMaero is the aerodynamic moment acting on the frame. The frame aerodynamic forces and moments are deﬁned as shed online by Cambridge University Press https://doi.org/10.1017/S0263574724000523 Published online by Cambridge University Press https://doi.org/10.1017/S0263574724000523 Published online by Cambridge University Press 1784 Jérémie X. J. Bannwarth et al. Table 1. Parameters of tilted-rotor octocopter. https://doi.org/10.1017/S0263574724000523 Published online by Cambridge University Press 3. Dynamics Parameter Description Unit Value N Number of rotors – 8 DUAV Rotor-to-rotor diameter m 0.500 Dprop Propeller diameter m 0.1524 ζ Rotor tilt angle degree 31 mt Total UAV mass kg 1.7 Ixx, Iyy x -/y axis MMoI kg m2 0.0240 Izz z-axis MMoI kg m2 0.1051 IR,zz Motor z-axis MMoI kg m2 3.290 × 10−6 cτ Rotor drag coeﬃcient N m s2/rad2 1.041 × 10−8 KE Motor back-EMF constant V s/rad 0.004126 I0 Motor no-load current A 0.6 R Motor resistance 0.117 Table 1. Parameters of tilted-rotor octocopter. BFaero = −1 2ρairAUAVU2 app,f Cx,f αf cosβf Cx,f αf sinβf Cz,f αf T BMaero = 1 2ρairAUAVDUAVCM,f αf U2 app,f −sinβf cosβf 0 T , (3) where αf and βf are the inﬂow angle and sideslip angle of the apparent wind vector (Fig. 2a), AUAV = πD2 UAV 4 is the area of the UAV, and Cx,f(αf), Cz,f(αf), and CM,f(αf) are dimensionless aerodynamic coeﬃcients deﬁned in ref. [28]. In the body frame, the apparent wind vector is expressed as BUapp,f = −B WR(˙ξ + WU), where WU = −[ Ux Uy Uz ]T is the wind velocity vector. y The total force BT = N i=1 BTi, and torque Bτ = N i=1 (Bτ i −Bν(Mi BRMiIR Miωi)), are generated by all the rotors, where Miωi = [ 0 0 ωi ]T represents the angular speed of the ith-rotor, and MiIR = diag(0, 0, IR,zz) is the combined mass moment of inertia of the motor and rotor. The forces and torques acting on a single rotor are BTi = Mi BR Ti,xycosβi Ti,xysinβi Ti,z T , Bτ i = Mi BR ⎡ ⎢⎣ Mi,xysinβi Mi,xycosβi (−1)i+1 bω2 i + IR,zz ˙ωi ⎤ ⎥⎦+ BLi×BTi, (4) (4) where βi and αi represent the sideslip angle and inﬂow angle of the wind on the ith-rotor (see Fig. 2b). The (−1)i+1 term accounts for the rotor direction. The aerodynamic forces and moments acting on each rotor are deﬁned as Ti,xy = −1 2ρairApropDpropCx,i (αi, λi) ωiUapp,i Ti,z = −1 2ρairApropDprop DpropCz,1,iω2 i + Cz,2,i (αi) ωiUapp,i , Mi,xy = −1 2ρairApropD2 propCM,i (αi, λi) ωiUapp,i, (5) (5) where Aprop = πD2prop 4 is the area covered by the propeller. Cx,i(αi, λi), Cz,1,i, Cz,2,i(αi), and CM,i(αi, λi) are dimensionless aerodynamic coeﬃcients deﬁned in ref. [6]. 3. Dynamics Due to the oﬀset between the rotors and the center of mass of the UAV, the apparent wind velocity at each rotor is aﬀected by the rotational speed of the UAV and is given by where Aprop = πD2prop 4 is the area covered by the propeller. Cx,i(αi, λi), Cz,1,i, Cz,2,i(αi), and CM,i(αi, λi) are dimensionless aerodynamic coeﬃcients deﬁned in ref. [6]. Due to the oﬀset between the rotors and the center of mass of the UAV, the apparent wind velocity at each rotor is aﬀected by the rotational speed of the UAV and is given by https://doi.org/10.1017/S0263574724000523 Published online by Cambridge University Press KE is the back-EMF constant, KT = KE is the motor torque constant, and cτ is the rotor torque aerodynamic constant. Finally, Vi is the input voltage to the motor. The PWM to motor voltage mapping for the motors is obtained by mounting a single motor on a RCBenchmark Series 1580 Thrust Stand. The relationship between the normalized PWM signal sent to a motor (χi ∈[0, 1]), and the input voltage is Vi = 6.982χi + 2.351. https://doi.org/10.1017/S0263574724000523 Published online by Cambridge University Press Robotica 1785 1785 Robotica ure 2. Aerodynamic forces and moments acting on octocopter frame and the ith-rotor. (a) Side view he frame when the sideslip angle βf = 0. (b) Side view of the rotor when the sideslip angle βi = 0. Figure 2. Aerodynamic forces and moments acting on octocopter frame and the ith-rotor. (a) Side view of the frame when the sideslip angle βf = 0. (b) Side view of the rotor when the sideslip angle βi = 0. Figure 2. Aerodynamic forces and moments acting on octocopter frame and the ith-rotor. (a) Side view of the frame when the sideslip angle βf = 0. (b) Side view of the rotor when the sideslip angle βi = 0. MiUapp,i = BMiR BUapp,f + Bν × BLi , (6) (6) here BLi represents a position vector from the center of the UAV to the ith-rotor and is de vector from the center of the UAV to the ith-rotor and is deﬁned as BLi = 1 2DUAV cosγi sinγi 0T . (7) (7) Note that in Eq. (7), γi = 45i −22.5◦is the angle from the positive body x-axis to the arm holding rotor i and is measured positive counterclockwise around the body z-axis (see Fig. 1b). The angular acceleration of the ith-rotor is given by Note that in Eq. (7), γi = 45i −22.5◦is the angle from the positive body x-axis to the arm holding rotor i and is measured positive counterclockwise around the body z-axis (see Fig. 1b). The angular acceleration of the ith-rotor is given by ˙ωi = 1 IR,zz KT R (Vi −I0R −KEωi) −cτω2 i , (8) (8) where I0 is the motor idle current and R is the motor resistance [25]. KE is the back-EMF constant, KT = KE is the motor torque constant, and cτ is the rotor torque aerodynamic constant. Finally, Vi is the input voltage to the motor. The PWM to motor voltage mapping for the motors is obtained by mounting a single motor on a RCBenchmark Series 1580 Thrust Stand. The relationship between the normalized PWM signal sent to a motor (χi ∈[0, 1]), and the input voltage is Vi = 6.982χi + 2.351. where I0 is the motor idle current and R is the motor resistance [25]. https://doi.org/10.1017/S0263574724000523 Published online by Cambridge University Press 4. Control architecture To conduct the wind disturbance rejection while hovering at a ﬁxed location, a cascaded closed-loop controller is designed and implemented for the presented tilted-rotor octocopter. For the implementation board and software, a Pixhawk Cube board running the PX4 Autopilot Firmware is employed. Note that https://doi.org/10.1017/S0263574724000523 Published online by Cambridge University Press Jérémie X. J. Bannwarth et al. 1786 Figure 3. The control implementation architecture. Figure 3. The control implementation architecture. the cascaded structure used in this work is mostly identical to that of the position hold control mode of the baseline (BL) PX4 Autopilot version 1.8.2 [29]. The BL closed-loop control architecture is split into inner and outer loops. The inner loop controls the desired UAV attitude (orientation) and comprises of a globally stable nonlinear quaternion-based angular controller [30], and PID control for angular rate. Meanwhile, for the outer loop, the position controller consists of a proportional gain acting on the position error and a PID velocity controller for each axis. In this work, for the outer loop, a novel H∞dynamic output feedback controller is designed to improve the wind disturbance rejection performance. Fig. 3 illustrates the closed-loop cascaded architecture. As only performance in front-facing wind is investigated, the desired yaw angle ψdes = 0◦for the remainder of this work. The output of the H∞block is converted to a desired attitude, qdes, and the normalized thrust BTdes = [ BTdes,x BTdes,y BTdes,z ]T. The attitude controller outputs desired normalized roll, pitch, and yaw torques, in the form of BCη = [ Cφ Cθ Cψ ]T. The actuator allocation is performed by the motor mixer. Note that the motor mixer accepts the desired thrust, BTdes, and torque commands, BCη, in the body frame, whereas the position controller operates in the world frame. To deal with this, two virtual acceleration signals in the world frame are introduced; the virtual attitude-thrust acceleration aA,des ∈R3 and the virtual vectored-thrust (horizontal) acceleration aH,des ∈R3. Initially, aH,des is rotated to the body frame, yielding BaH,des = WBRaH,des = BTdes,x BTdes,y BaH,des,z T . (9) (9) The ﬁrst two elements in the x and y-axis can be produced by the tilted rotors. Yet, BaH,des,z is an unintended side eﬀect of the vectored-thrust scheme when the UAV is not level and must be compensated using attitude control. This element is isolated, rotated back to the world frame, and added to the desired attitude acceleration. https://doi.org/10.1017/S0263574724000523 Published online by Cambridge University Press 4. Control architecture (11) are zero in the BL PX4 motor mixer matrix. https://doi.org/10.1017/S0263574724000523 Published online by Cambridge University Press 4. Control architecture Consequently, the total attitude acceleration ades is deﬁned as The ﬁrst two elements in the x and y-axis can be produced by the tilted rotors. Yet, BaH,des,z is an unintended side eﬀect of the vectored-thrust scheme when the UAV is not level and must be compensated using attitude control. This element is isolated, rotated back to the world frame, and added to the desired attitude acceleration. Consequently, the total attitude acceleration ades is deﬁned as ades = aA,des + W B R ⎡ ⎢⎣ 0 0 BaH,des,z ⎤ ⎥⎦. (10) (10) The PX4 Autopilot thrust-to-quaternion algorithm converts the total attitude acceleration setpoint to the desired quaternion, qdes, and vertical thrust, BTdes,z, that can be fed to the motor mixer (see Fig. 3). The desired vertical thrust is simply the magnitude of the desired attitude acceleration, BTdes,z = −∥ades∥2. In the next step, the motor mixer maps the desired thrust,BTdes, and the torque command, BCη, into a vector of PWM signals (χ) to be sent to the motors (see Fig. 3). Details of how the motor mixer is constructed can be found in ref. [26]. The motor mixer is designed to accept vectored-thrust commands. The motor mixer depends on the angle between the rotor arms, γ = 45◦, and the tilt angle of the rotors, ζ, such that https://doi.org/10.1017/S0263574724000523 Published online by Cambridge University Pre 1787 Robotica Figure 4. H∞closed-loop control schematic with weighting matrices. Figure 4. H∞closed-loop control schematic with weighting matrices. χ = ⎡ ⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣ −Sγ/2 Cγ/2 1 a −b 1 −Cγ/2 Sγ/2 −1 −b a 1 −Cγ/2 −Sγ/2 1 b a 1 −Sγ/2 −Cγ/2 −1 −a −b 1 Sγ/2 −Cγ/2 1 −a b 1 Cγ/2 −Sγ/2 −1 b −a 1 Cγ/2 Sγ/2 1 −b −a 1 Sγ/2 Cγ/2 −1 a b 1 ⎤ ⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦ BCη BTdes , (11) (11) where Sγ/2 = sin (γ/2), Cγ/2 = cos (γ/2), a = 2 tan(γ/2) tan (ζ) , and b = 2 tan ζ. For comparison, the elements of the fourth and ﬁfth column of the motor mixer matrix in Eq. (11) are zero in the BL PX4 motor mixer matrix. where Sγ/2 = sin (γ/2), Cγ/2 = cos (γ/2), a = 2 tan(γ/2) tan (ζ) , and b = 2 tan ζ. For comparison, the elements of the fourth and ﬁfth column of the motor mixer matrix in Eq. 4.1. Linearized plant Synthesizing the H∞controller requires a linearized model of the plant. The attitude is expressed as η = [ φ θ ψ ]T, where φ, θ, and ψ are the roll, pitch, and yaw angles of the octocopter, respectively. The motor mixer and attitude controller are included in the plant model. The input vector of the plant is deﬁned as u = [ aT A,des aT H,des ]T, and the wind velocity (wd = WU) is the disturbance input (see Fig. 4). The state vector is x = [ ξ T ˙ξ T ηT BνT ωT xT PID ]T (27 states), where ω = [ ω1 ω2 · · · ω8 ]T is the rotors’ angular speed vector and xPID is a vector containing the internal states of the angular rate loop. The pitch and roll rate controllers use PID controllers with a second-order derivative ﬁlter (three states each). The yaw rate controller uses a PI controller and only has one state. The attitude controller already stabilizes the internal attitude and angular velocity states. Therefore, the output vector only contains the integral of position, position, and velocity of the UAV, y = [ ξ T ξ T ˙ξ T ]T. The system is trimmed for the wind velocity U* = [ 5.6 0 0 ]T m/s. This velocity corresponds to approximately 50% of the feasible velocity range of the boundary layer wind tunnel used. In the trimming algorithm, position and rate states are constrained to zero. Also, the vectored-thrust is not used at steady state, which simpliﬁes the trimmed input to u* = [ aT A,des 0 ]T. As expected, the trimmed pitch angle, θ = −8.25◦, is negative, corresponding to the UAV pitching into the wind to counteract its drag force. The model is then linearized around the calculated operating point (resulting from the trim process) using MATLAB’s linearize function. The linearized model now needs to be augmented by weighing matrices before controller synthesis. The disturbance rejection, regulation performance, and actuator usage are competing and need to be weighted. Therefore, the linearized model also needs to be augmented by weighting matrices. The linearized plant and H∞controller dynamics to be designed are denoted as the transfer function matrices G(s) and K(s), respectively. Wu(s), Wd(s), Wo(s), and Ws(s) represent the actuator, wind disturbance, oi.org/10.1017/S0263574724000523 Published online by Cambridge University Press 1788 Jérémie X. J. Bannwarth et al. Figure 5. 4.1. Linearized plant The output and sensor noise weighting matrices are arbitrarily chosen as where I is the identity matrix. The output and sensor noise weighting matrices are arbitrarily chosen as Wo = 1 2I, Ws = 1 20I, I ∈R9×9. (14) (14) Finally, the Laplace domain weighing matrices and linearized plant model are combined. Finally, the Laplace domain weighing matrices and linearized plant model are combined. 4.1. Linearized plant Fitting ﬁrst-order transfer function to x-axis wind spectrum. Figure 5. Fitting ﬁrst-order transfer function to x-axis wind spectrum. output, and sensor noise weighting matrices, respectively (see Fig. 4). The regulated output vector, z = [ zT out zact T ]T, and the disturbance input vector, w = [ wT sens wd T ]T, are deﬁned. The actuator weighting matrix is deﬁned as Wu(s) = diag(Wu,A(s), Wu,A(s), Wu,A(s), Wu,H(s), Wu,H(s)), where Wu,A (s) = 50.1 s + ωco s + 10ωco , Wu,H (s) = 8.91 s + ωco s + 0.1ωco , (12) (12) where ωco is the desired cross-over frequency (the frequency at which the vectored-thrust should take over from the attitude control). The controller should use the attitude controller for low-frequency disturbances and vectored-thrust for high-frequency disturbances. This frequency-dependent actuator allocation is desirable because the maximum attitude-thrust of the UAV is signiﬁcantly larger than vectored-thrust. Consequently, vectored-thrust is more likely to saturate the actuators and is better suited to smaller amplitude, higher-frequency commands. The ﬁrst three diagonal elements of Wu(s) corre- spond to the desired virtual attitude-thrust, and the last two correspond to the desired vectored-thrust. The virtual attitude-thrust is penalized at high frequencies, while vectored-thrust is penalized at low fre- quencies. The locations of zeros and poles are chosen based on a desired cross-over frequency, ωco = π rad/s. This frequency is chosen to be below the cutoﬀfrequencies of aA,des,x and aA,des,y, which are 8 rad/s. The DC gains of the attitude and vectored-thrust transfer functions are chosen to be 14 dB and 19 dB, respectively, by the trial-and-error process to prevent actuator saturation. The wind disturbance weighting matrix, Wd, can be deemed as a function that shapes white noise into the expected spectrum of the wind. Fig. 5 shows the x-axis wind spectrum of the wind tunnel and a ﬁrst- order transfer function ﬁtted to it. The high-frequency roll-oﬀof the spectrum is -28.5 dB/dec, which cannot be replicated using a transfer function. An asymptotic approximation is used to ﬁnd the corner frequency of the spectrum (24.1 rad/s). Wd is given the same corner frequency, but its magnitude is set to 15 dB through trial-and-error to improve disturbance rejection while preventing actuator saturation, yielding Wd (s) = 5.62 24.1 s + 24.1I,I ∈R3×3, (13) (13) where I is the identity matrix. https://doi.org/10.1017/S0263574724000523 Published online by Cambridge University Press 4.2. H∞output feedback position controller In the previous section, after trimming the plant, ﬁnding the operating point, and linearizing the plant around the operating point, the model was augmented with weighting matrices. According to Fig. 4, the https://doi.org/10.1017/S0263574724000523 Published online by Cambridge University Press https://doi.org/10.1017/S0263574724000523 Published online by Cambridge University Press 1789 Robotica 1789 Robotica state space dynamics of the controller, K(s) = (AK, BK, CK, DK), are ˙xK = AKxK + BKy, u = CKxK + DKy, (15) state space dynamics of the controller, K(s) = (AK, BK, CK, DK), are ˙xK = AKxK + BKy, u = CKxK + DKy, (15) (15) where xK is the internal state vector of the controller, and AK, BK, CK, and DK are constant matrices. This controller is an output feedback controller because it acts on the measured output, y, rather than on the system state, x, that is described in Section 4.1. where xK is the internal state vector of the controller, and AK, BK, CK, and DK are constant matrices. This controller is an output feedback controller because it acts on the measured output, y, rather than on the system state, x, that is described in Section 4.1. H∞techniques often use a high number of states, which can be undesirable and computationally expensive for real-time applications. Typical H∞synthesis algorithms, such as MATLAB’s inbuilt hin- fsyn, create full-order controllers with the same number of states as the plant. The octocopter plant in this work will result in a 38th-order controller. Combined with the controller’s nine inputs and ﬁve out- puts, a discrete implementation will require more than two thousand multiplication operations at each time step. This complexity causes a substantial computational burden for the onboard ﬂight controller, Pixhawk Cube. The open-source HIFOO 2.0 toolbox (MATLAB) [31] solves this issue by allowing for the creation of reduced order controllers using an optimization-based algorithm. Therefore, HIFOO 2.0 synthesizes the controller from the linearized model described in Section 4.1. The reduced order con- troller (order of 1) is created to keep the performance like the full-order controller while signiﬁcantly reducing the computation cost. Note that implementing the resulting controller on the original system requires accounting for the trimmed input, u* (see Section 4.1). AK = −11.0, (16) CK = −2.82 2.55 −0.956 5.99 0.0202 T , BK = [ 0.847 −2.39 . . . −0.239 −0.0859 ]1×9, DK = ⎡ ⎢⎢⎣ −0.0616 · · · −0.0147 ... ... ... https://doi.org/10.1017/S0263574724000523 Published online by Cambridge University Press 4.2. H∞output feedback position controller 0.0155 · · · 0.0111 ⎤ ⎥⎥⎦ 5×9 . AK = −11.0, (16) CK = −2.82 2.55 −0.956 5.99 0.0202 T , BK = [ 0.847 −2.39 . . . −0.239 −0.0859 ]1×9, DK = ⎡ ⎢⎢⎣ −0.0616 · · · −0.0147 ... ... ... 0.0155 · · · 0.0111 ⎤ ⎥⎥⎦ 5×9 . (16) For the attitude control part, by choosing the Lyapunov candidate function as V(z, q) = qT 1: 3q1: 3 + (q0 −1)2, where z ∈{ z1 z2 } corresponds to the upper and lower hemisphere of the space of unit quater- nions, we can show that V(z, q) > 0 and ˙V(z, q) < 0 (see detailed proof of stability in ref. [28]). This shows that the attitude controller stabilizes the internal attitude and angular velocity states. For the ana- lytical proof of stability of the position control part, since Wu(s), Wd(s), Wo(s), and Ws(s) are positive deﬁnite, we can use the standard linear matrix inequality positive deﬁnite solution (See Appendix B of [11] for the detailed proof of stability). Meanwhile, from Eq. (16) for the newly designed position controller, the controller has a single pole at -11 rad/s. Fig. 6 shows the pole-zero map of the closed-loop system, including the linearized model and the controller. It can be seen that all poles are in the open left halfplane, proving the stability of the linear closed-loop position control system. Note that the other poles to the far left have been removed to improve visibility. Fig. 7 shows the Bode magnitude plot of the transfer function from the wind disturbance input, Ux(s), to the attitude and vectored-thrust accelerations. The magnitude responses of these transfer func- tions indicate which actuator is used to compensate for wind disturbances. The magnitude response of vectored-thrust control is lower than that of attitude control at all frequencies due to the higher DC gain of the weighting function used. Nevertheless, the relative magnitudes of the transfer functions are not con- stant; at low frequencies, the attitude control response is 25 dB higher than the vectored-thrust response, whereas the diﬀerence narrows down to 10.1 dB as ω →∞. These magnitude responses demonstrate the desired frequency-dependent actuator allocation as the vectored-thrust is not used at low frequen- cies to prevent saturation and is used increasingly at higher frequencies to improve disturbance rejection. 1790 Jérémie X. J. Bannwarth et al. Figure 6. 4.2. H∞output feedback position controller Upper half of the pole-zero map close to the imaginary axis for the linearized plant and closed-loop system. Figure 6. Upper half of the pole-zero map close to the imaginary axis for the linearized plant and closed-loop system. Figure 7. Magnitude response of the transfer functions from the wind disturbance to the desired accelerations. Figure 7. Magnitude response of the transfer functions from the wind disturbance to the desired accelerations. The peaks of both responses are at 2.5 rad/s, which is close to the cross-over frequency of the actuator weighting functions at π rad/s. https://doi.org/10.1017/S0263574724000523 Published online by Cambridge University Press 5.1. Free-ﬂight station-keeping performance UAVs encounter a range of disturbances beyond wind, including atmospheric factors like turbulence and thermal updrafts, environmental elements, obstacles in the ﬂight path, potential sensor inaccuracies and noise, communication disruptions, electromagnetic interference, and the risk of system faults. Physical interaction is also another considerable source of disturbance, in which the UAV is interacting through a tool with the surrounding environment. In this paper, however, we assumed all other factors negligible compared to the wind and turbulence disturbances. Also, the UAV is not in contact with its surroundings (free ﬂight). To validate the performance of the H∞controller for wind disturbance rejection, and conducting a comparison, a comprehensive experiment is designed. Three cases are considered: the tilted-rotor octo- copter with the H∞controller, the tilted-rotor octocopter with the BL controller, and a planar version of the octocopter with the BL controller. The UAV is ﬂown inside the wind tunnel in a test section outﬁtted with motion capture cameras to provide position feedback (see Fig. 8a). A disturbance-generating grid is mounted ahead of the ﬂight location to generate turbulence intensities of approximately 10%. Note that the octocopter is powered by two 40A power supplies through an approximately 3 m long tether, as shown in Fig. 8a. The power supplies are set to 13V to account for a measured 2V drop across the tether. This tethered solution is chosen over traditional batteries as it allows faster testing 1791 Robotica Figure 8. Wind disturbance rejection experiment. (a) Octocopter station-keeping in the boundary layer wind tunnel, University of Auckland. (b) RMS error of the position norm. (c) RMS rotation error. (d) Mean RMS of the motor PWM signals. Figure 8. Wind disturbance rejection experiment. (a) Octocopter station-keeping in the boundary layer wind tunnel, University of Auckland. (b) RMS error of the position norm. (c) RMS rotation error. (d) Mean RMS of the motor PWM signals. by removing the need to swap and charge batteries. Furthermore, the constant power supply voltage provides consistency between and during tests. Three wind speeds of Umean = {0, 5.6, 12.8} m/s are investigated. Each ﬂight starts with the UAV sitting on the ﬂoor, midway between the wind tunnel walls and 3.8 m away from the turbulence grid. The operator then switches the UAV to position hold mode to track a position setpoint at the center of the wind tunnel. https://doi.org/10.1017/S0263574724000523 Published online by Cambridge University Press 5.1. Free-ﬂight station-keeping performance The second phase of the experiment involves setting the wind tunnel to the desired speed setting and waiting for the transients to decay before recording the response of the UAV. Each ﬂight yields 100 s of usable data after removing all transient behaviors. Through 72 experimental tests, it was demonstrated that despite increasing the mean wind speed from 5.6 m/s to 12.8 m/s and also adding 10% turbulence intensity, the hovering RMS position error norm remained smaller than 5 cm (Fig. 8b). RMS principal axis rotation error also remained smaller than 1.5◦(Fig. 8c). This shows that the presented linearized model-based controller (linearized based on 5.6 m/s wind with no turbulence) is valid for the actual nonlinear system operating under diﬀerent conditions. These results are also promising compared to another experimental study on H∞control (with a hexacopter roughly the same size of the octocopter used in this study) [11], where the RMS position error was calculated between 10 to 15 cm. Note that in ref. [11], the wind disturbance was generated by a simple fan, without turbulence (with the average wind speed of 7.5 m/s). Meanwhile, this study generated its wind disturbance in the wind tunnel, with 10% turbulence. Fig. 8(b-d) shows the main performance metrics obtained from the experimental data. The presented H∞controller signiﬁcantly outperforms both BL cases in position error (see Fig. 8b). At the maximum wind speed of 12.8 m/s, the H∞controller yields an RMS position error norm (σξ) 51% lower than the BL tilted case and 38% lower than the BL planar case. At 0 and 5.6 m/s, there is a signiﬁcant overlap between the recorded data points for the two BL controllers. However, overlap at 12.8 m/s is minimal, with the BL planar case resulting in a 21% lower RMS error norm than the BL tilted case. The BL planar setup has a higher thrust ceiling, allowing it to outperform the BL tilted conﬁguration slightly. The RMS principal axis rotation error (σ), denoting the shortest rotation between the measured and desired orientations, shows considerable overlap of the three data sets at non-zero wind speeds (see 1792 Jérémie X. J. Bannwarth et al. gure 9. Tilted-rotor octocopter response to a step change in desired vectored-thrust acceleration altitude hold mode. (a) Desired normalized horizontal accelerations, (b) Attitude, (c) Translational celeration, (d) and motor PWM signals. Figure 9. https://doi.org/10.1017/S0263574724000523 Published online by Cambridge University Press 5.1. Free-ﬂight station-keeping performance Tilted-rotor octocopter response to a step change in desired vectored-thrust acceleration in altitude hold mode. (a) Desired normalized horizontal accelerations, (b) Attitude, (c) Translational acceleration, (d) and motor PWM signals. Fig. 8c). Therefore, the conﬁguration does not have a signiﬁcant eﬀect on the attitude control perfor- mance at those wind speeds. However, at zero wind speed, the BL planar case yields an RMS rotation error over twice as large as the other controllers. This diﬀerence is mainly due to the low yaw control authority of the planar arrangement, which results in higher errors. These increased errors are not seen at higher wind speeds, which could be due to the aerodynamic loadings acting on the UAV, but further experimentation is required to identify the source of this behavior. Wind resistance increases energy consumption as the UAV works harder to overcome the wind dis- turbances. Having an energy-eﬃcient control strategy to optimize UAV ﬂight endurance and battery life in the presence of wind is important. The mean RMS of the motor commands (σ χ) is similar for both tilted cases at zero wind speed. However, the actuator usage of the H∞controller is 23% and 25% higher than the BL tilted case at 5.6 and 12.8 m/s, respectively (Fig. 8d). This means the H∞controller yields signiﬁcantly improved station-keeping performance compared to the BL tilted conﬁguration for only a marginal increase in actuator usage. The BL controller on the planar case requires signiﬁcantly more 1793 Robotica Robotica actuator usage across all wind speeds, which is again due to the lack of yaw authority. Resolving this issue would involve replacing the motors and propellers with larger ones. actuator usage across all wind speeds, which is again due to the lack of yaw authority. Resolving this issue would involve replacing the motors and propellers with larger ones. 6. Conclusion A robust H∞output feedback controller was designed and implemented within a cascaded control structure to regulate the position of a tilted-rotor octocopter under wind disturbances while hovering at a point. The tilted pattern of the octocopter allows for high-bandwidth, yet saturation-constrained vectored-thrust, which is not feasible on typical tilt-to-translate planar UAVs. The problem of frequency- dependent actuator allocation, which is largely ignored in current vectored-thrust UAV literature, is solved by augmenting the plant model with weighting transfer functions. Reduced order H∞synthesis is conducted on a linearized version of the augmented model to yield robust performance. 72 free-ﬂight wind tunnel tests were conducted at wind speeds of 0, 5.6, and 12.8 m/s to compare the H∞controller’s performance to that of the BL controller on both a tilted and planar octocopter. The H∞controller sig- niﬁcantly reduced the RMS position error for a slight increase in actuator usage. The H∞RMS position error decreased by 51% compared to the BL tilted case and 38% to the BL planar case. Conversely, its actuator usage is 25% higher than the BL tilted case. In free-ﬂight experiments, the octocopter pro- duced a forward acceleration of 2.4 m/s2 when subjected to a vectored-thrust step while regulating its pitch angle to less than 0.25◦, successfully demonstrating the eﬀectiveness of the new motor mixer. Author contribution. JXB: writing initial draft, methodology, experiments, and data analysis. SK: writing, review, and revision. KS: supervision, review. Financial support. The research reported in this article was conducted as part of “Enabling unmanned aerial vehicles (UAVs) to use tools in complex dynamic environments UOCX2104,” which is funded by the New Zealand Ministry of Business, Innovation and Employment. Competing interests. 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Overton, “Multiobjective robust control with HIFOO 2.0,” IFAC Proceed Vol 42(6), 144–149 (2009). doi: 10.3182/20090616-3-il-2002.00025. https://doi.org/10.1017/S0263574724000523 Published online by Cambridge University Press
https://openalex.org/W4322769913	https://www.cambridge.org/core/services/aop-cambridge-core/content/view/A19DD216DB6471722AC147A48B1A3933/S0034412523000124a.pdf/div-class-title-how-to-believe-in-immortality-div.pdf	English	null	How to believe in immortality	Religious studies	2,023	cc-by	11,197	© The Author(s), 2023. 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. Religious Studies (2024), 60, S117–S130 doi:10.1017/S0034412523000124 Religious Studies (2024), 60, S117–S130 doi:10.1017/S0034412523000124 Religious Studies (2024), 60, S117–S130 doi:10.1017/S0034412523000124 Abstract All the cards seem to be stacked against belief in immortality. Nonetheless, the resources of particu- lar religious traditions may avail where generic philosophical solutions fall short. With attention to the boredom and narcissism critiques, intimations of deathlessness in Śāntideva’s radical altruism, and recent Christian debates on the soul and the intermediate state, I propose two criteria for a coherent religion-specific belief in immortality: (1) the belief is supported by a fully realized reli- gious tradition, (2) the belief satisfies the demand for self-transcendence as well as for self-preser- vation. Where self-transcendence and self-preservation are kept in balance, and where the whole idea rests upon the lattice-work of a fully realized religious tradition, immortality is a fitting object of belief. Moreover, such belief is compatible with considerable speculative freedom concerning matter and spirit, body and soul, and personal identity over time. Keywords: Immortality; soul; intermediate state; Śāntideva Carol Zaleski Carol Zaleski Department of Religion, Smith College, Northampton, MA, USA Email: czaleski@smith.edu Department of Religion, Smith College, Northampton, MA, USA Email: czaleski@smith.edu Department of Religion, Smith College, Northampton, MA, USA Email: czaleski@smith.edu (Received 27 June 2022; revised 10 January 2023; accepted 11 January 2023; first published online 2 March 2023) https://doi.org/10.1017/S0034412523000124 Published online by Cambridge University Press Introduction Many of us have intuitions about death and afterlife that are difficult to articulate, let alone prove, yet are deeply intertwined with our affections and imagination. Think of how inexplicable death seems when it claims a person we’ve known and loved. Or think back to early childhood, to our first impressions of wonder and amazement at the world. Could it be, as Wordsworth has it, that ‘heaven lay about us in our infancy’? Or consider the present moment of consciousness: it is fleeting almost to the point of vanishing, yet when we turn our attention inward, it feels boundless, endless, as big as the universe. Or consider how reason, having emerged from long epochs of evolutionary trial and error, turns around to address us with a voice of its own, pointing towards ideal real- ities which sail above this world of time and change. Consider further the persistent desire for immortality, the common belief in immortality, the moral demand for immor- tality to set right the injustices of this world. Long before we formulate arguments for belief, intuitions like these are already at work. Our very language predisposes us to imagine life beyond death. The person passes on, we say, the breath departs. It’s almost irresistible to imagine that when the breath departs it must go somewhere, carrying ves- tiges of the personality it once sustained. Yet a thought being irresistible doesn’t make it true. We may justly be suspicious of beliefs that got here before we did, so to speak, and are waiting to seduce us. Now a https://doi.org/10.1017/S0034412523000124 Published online by Cambridge University Press Carol Zaleski S118 contrary intuition may present itself. We don’t like to be seduced; we resolve not to be duped by our childish longings. We take a cold, hard look at the facts, and it seems that all the cards are stacked against immortality: the obvious facts of death and decompos- ition, the failure of paranormal research programmes, the moral reasons often adduced for considering immortality an unworthy object of desire, as well as a host of objections to common ways of thinking about matter and spirit, body and soul, and personal identity over time. If, having faced these objections, we still experi- ence intimations of immortality, we’re at an impasse. Philosophy delights in such dilemmas, but philosophy on its own may not have the power to settle this question for us. Introduction Nonetheless, I suggest that – as with the problem of evil – the resources of concrete religious traditions may avail where generic philosophical solutions fall short. Two very different cases, one Buddhist and the other Christian, will serve to illustrate my point. With these two instances before us, I’ll propose two criteria for a coherent religion-specific belief in immortality: (1) the belief is supported by a fully realized reli- gious tradition, rather than by isolated arguments alone, and (2) the belief satisfies the demand for self-transcendence as well as for self-preservation, avoiding exaggeration on either side. Exaggerated notions of self-preservation (preservation, that is, of one’s ordinary ego- istic self) end in tedium, for one is bound eventually to tire of one’s attachments and self-projects. Exaggerated notions of self-transcendence, on the other hand, may give rise to various forms of alienation, whether by severing the connection between one’s mundane life and one’s ultimate transcendent state or by severing the bond between the living and the dead. The corrective for an exaggerated notion of self-transcendence may be found, in part, in practical and ritual observances that reinforce the symbiotic relationship between the living and the dead: care for the dead implies that they are in a position to be helped and promotes a relationship of kin- ship; appeal to the blessed dead implies that they are in a position to be helpful and promotes a relationship of patronage. As I will suggest, immortality without relationship is sterile. If the opposite of relation- ship is confinement or estrangement, then there is a place for relationship in all great soteriological schemes, including śramaṇic teachings such as Jainism, which envisages lib- erated souls as independent monads. Though dwelling (according to Jaina belief) in self- sufficient solitude, each liberated soul is a plenitude of infinite knowledge, bliss, and power without trace of estrangement; and the path towards this inconceivable blessed- ness goes by way of veneration of spiritual exemplars and zealous care for all life forms. g y y p p Where self-transcendence and self-preservation are kept in balance, and where the whole idea rests upon the lattice-work of a fully realized religious tradition, immortality is a fitting object of belief. Moreover, as the examples discussed below will show, such belief can be compatible with considerable speculative freedom in our thinking about matter and spirit, body and soul, and personal identity over time. https://doi.org/10.1017/S0034412523000124 Published online by Cambridge University Press Introduction While Christian thinkers have proposed what they regard as necessary reasons to affirm the existence, uniqueness, and perfection of God, there are other core beliefs, such as those pertaining to motives for particular divine actions in history, which cannot be settled by rational demonstration or by appeal to authoritative texts. Such beliefs are typically defended by citing reasons of fittingness (rationes convenientiae), which are com- pelling even though not probative in force. By such means, Christian thinkers have sought to account for why God, who could have used other means, preferred the Incarnation as the means of human redemption.1 Not just appropriateness but admirability and conform- ity to truths already in possession enter into what is meant by reasons of fittingness. As such, the argument ex convenientia amounts to a vote of confidence in the rational- ity, coherence, and beauty of an entire religious scheme. Such practices of informal rea- soning are broadly characteristic of religious thinking about questions regarded as neither definitively revealed nor capable of being settled in a strictly demonstrative fashion. y p g y Is immortality such a question? I will suggest that in certain cases it can be. y q gg I should clarify, first of all, that I am concerned with immortality in the transcendent sense – as a condition which rises above or passes beyond the circle of birth and death. Transcendent immortality is admittedly difficult to describe; to count as immortality it must involve some kind of personal continuity or persistence, but to count as transcend- ent immortality it must also involve a profound transformation (a self-transcendence which may, from our present vantage point, look like a self-death). There are many and various ways of picturing what it would be like to persist and yet be radically trans- formed. Images of the afterlife are famously riddled with inconsistencies. But this much can be said: the inescapable facts of birth and death, mutability, decay, and decomposition cannot rule out the possibility of a transcendent immortality, for they are confined to the hither side of the boundary between this life and the next. Nor can mediumistic commu- nications, near-death experiences, or reincarnation memories establish its reality. Such phenomena also belong to the hither side, and are liable to being explained naturalistic- ally. Introduction Some working definitions are in order. By fully realized religious tradition I have in mind a religious tradition that exhibits staying power and aptitude for development. Following Paul Griffiths’s (1999, 3, 7) characterization of a religion as ‘principally an account’ which exhibits ‘comprehensiveness, unsurpassability, and centrality’, I treat as ‘fully rea- lized’ any religious tradition that has the resources to sustain and develop such an account. This would include resources such as sacred texts and foundational narratives (literate or non-literate); guidance from authorities, prophets, or paradigmatic figures (living or dead); practices like memorization, recitation, exegesis, and commentary; rules of life applicable to different walks of life; liturgical, spiritual, devotional, ascetic, penitential, and artistic disciplines. The precise weight given to particular resources S119 Religious Studies Religious Studies will vary, and the lack of one or more of these resources need not be disqualifying. Nor does it matter that individuals are rarely unflagging in their commitment or articulate in defending it; for the religious account is in the custody of the tradition as a whole, and as such remains recoverable for as long as the tradition survives. To speak of tradition may also raise questions. As the word suggests, a tradition is some- thing handed on. Better still, it is the activity of handing on and of receiving what is given. What a tradition is not is a static thing easily marked off from its cultural rivals. The reli- gious traditions of the world are internally diverse, permeable, and subject to historical contingencies. Nonetheless, a sensitive interpretation of religion will avoid approaches that emphasize multivocality and the play of power interests to an extent that under- mines the very idea of a coherent religious Gestalt. Fully realized religious traditions flour- ish by keeping faith with what is handed on, even as they grow, adapt, and change. y g y g g With this understanding of ‘fully realized religious tradition’ in mind, my aim is to defend the fittingness of one’s given belief in immortality when nurtured by such a trad- ition. Here fittingness has a special sense which I take from the Latin convenientia. In the background is Cicero’s use of convenientia to render the Platonic and Stoic ὁμολογία (agreement, harmony, fittingness, decorum, conformity to nature). In the foreground, for this article, is the argumentum ex convenientia, a form of reasoning that has proved indispensable for theology, famously within Christian scholasticism. https://doi.org/10.1017/S0034412523000124 Published online by Cambridge University Press Tedium and narcissism In his famous essay, ‘The Makropulos Case: Reflections on the Tedium of Immortality’, Bernard Williams (1973) argued that, while long life is a good thing, immortality is undesirable. Given endless time we would eventually exhaust the core projects and inter- ests that made us wish for prolonged life in the first place, and so would sink into tedium. Williams is not alone in this view, of course. Schopenhauer, Mark Twain, Paul Tillich, and Tolkien’s Elves all tell us that endless longevity is undesirable; and most twenty-first-century philosophers agree. Yet in recent years, John Martin Fischer (2019) has been arguing, against Williams and others he refers to as ‘immortality curmudgeons’, that an endless life spent pursuing a variety of interests, including the repetition of intrin- sically worthwhile activities, is a reasonable and attractive picture of human immortality. My own view, however, is that Bernard Williams has embedded in his argument the key for dismantling it. That key is his choice of the word tedium, instead of the more common- place boredom, in his essay’s subtitle. Boredom is a word of modern coinage and shallow associations, while tedium (or its synonym acedia) has a long history in classical and post- classical moral psychology, and in the literature of late antique Christian monasticism, where it is classified among the eight temptations (logismoi) with which the demons assail contemplatives.2 p For Williams, tedium occurs because there is a natural limit to our human capacity to take an interest, pay attention, lay plans, and fulfil them. For ascetic and monastic writers, however, tedium is not a natural limitation, but a vice. John Cassian (1900), writing in the fifth century, gives us a typical account of a Christian monk in the grip of tedium: ‘he looks about anxiously this way and that, and sighs that none of the brethren come to see him, and often goes in and out of his cell, and frequently gazes up at the sun, as if it was too slow in setting, and so a kind of unreasonable confusion of mind takes posses- sion of him like some foul darkness, and makes him idle and useless for every spiritual work’.3 An analogous syndrome – thīna-middha (’sloth-and-torpor’) – appears in the Abhidhamma Piṭaka and in later Buddhist literature among the unwholesome mental fac- tors that endanger spiritual practice. Introduction But if the case for a transcendent immortality neither stands nor falls on empirical evidence, it does remain vulnerable to other kinds of criticism, including the charge that immortality is an unworthy object of desire. For example, a Buddhist may be expected to discourage the desire for immortality if immortality is construed as the craving for an eternal independent self, while a Carol Zaleski S120 Christian may be expected to discourage the desire for immortality if construed in a way that makes bodily resurrection superfluous. There are also general objections to the desir- ability of immortality: an endless life is bound to end in boredom, we are told; moreover, it is narcissistic to want such a life for oneself. I will begin by responding to these general objections – from boredom and from narcissism – before turning to specifically Buddhist and Christian considerations. https://doi.org/10.1017/S0034412523000124 Published online by Cambridge University Press Tedium and narcissism The Mahāyāna teacher Śāntideva (to be discussed below) warns monks against the idle habit of ‘breaking up clods of earth, ripping grass, and drawing lines in the earth without any purpose’ (Śāntideva (1996), 5.46, p. 38). One hears, in these small details, the voice of seasoned elders. Monastics and ascetics have a special intimacy with tedium; I take them to be experts on the subject. h d h f d f f f The good news is that if tedium is a vice or, if you prefer, an impairment of attention and affections, it may admit of a cure. If tedium is a vice rather than a natural limitation, it follows that in principle human beings have the potential to be healed and transformed into a condition which would make immortality a worthy object of desire. We know from experience that we have it within us to be utterly absorbed in an object we find desirable or beautiful or otherwise worthy of attention. We also know how quickly we exhaust our powers of attention. But perhaps we were not meant to be so restless, so easily bored; per- haps we were meant to have inexhaustible reserves of attention, admiration, and love. As we are now, we aren’t fit for immortality, but perhaps we may be made fit, and in that remaking become more, rather than less, like ourselves. https://doi.org/10.1017/S0034412523000124 Published online by Cambridge University Press S121 Religious Studies Religious Studies That we aren’t very good at imagining this transformed way of being doesn’t mean that it is wholly alien to our nature. Monks and nuns, ascetics and mystics, and ordinary pious folks have already set their sights on this transformation and are preparing, here and now, by prayer, meditation, spiritual exercises, and works of love, to receive it. So much for the boredom critique. But the narcissism critique should still give us pause. Picture a certain kind of seeker obsessively combing the annals of psychical research in search of evidence for personal survival of bodily death. The spectacle is unattractive, to say the least. Now contrast this with the selfless equanimity with which so many celebrated non-believers have faced the prospect of their own death. Think of David Hume dying (according to the testimony of his friends) with his patience, wisdom, kindliness, and humour intact. Tedium and narcissism Or think of Derek Parfit, making the cheerful dis- covery that as soon as he abandoned the idea of personal identity, the ‘glass tunnel’ he had been living in disappeared and he was cured of self-centeredness as well as fear of death (Parfit (1984), 281; cf. Idem (1971)). My colleague Jay Garfield has developed this theme of moral awakening through giving up the idea of a substantial self in many of his writings, including his recent book Losing Ourselves (2022). Such examples ought to make one wonder: isn’t it nobler to face death squarely, not demanding the assurance of a persistent ego-self? It’s a good question which, if not fatal to immortality belief, can perform the service of cleansing that belief of its narcissistic contaminants. For an exaggerated preoccupation with personal immortality is disqualifying for any immortality worthy of the name. In seeking ego-centred self-preservation at all costs, the narcissist paradoxically annihilates the self by refusing the gift of true blessedness. This is the point of the second criterion I propose: a fitting belief in immortality will hold fast to the middle path, satisfying the demand for self-transcendence as well as for self- preservation, avoiding extremes. https://doi.org/10.1017/S0034412523000124 Published online by Cambridge University Press Śā ntideva and intimations of deathlessness To explore the possibility that a sceptical challenge to immortality belief may purify rather than abrogate such belief, thus rendering it ‘fitting’ within a religious context, I turn to my first illustration, from the Bodhicaryāvatāra of the eighth-century Indian Buddhist scholar-poet Śāntideva. The Bodhicaryāvatāra is Śāntideva’s summons and guide to the bodhisattva way of life, and to the bodhicitta, the altruistic resolve to attain awakening (bodhi) for the benefit of all beings, which is at its heart. Ś g g Admittedly, Śāntideva may seem an odd figure to recruit for an article on how to believe in immortality. Formed by the Mahāyāna sūtras and the Madhyamaka (‘Middle Way’) school, Śāntideva is adept at hammering down would-be absolutes wherever they pop up. If we are to think with Śāntideva, we have to imagine that everything and every- one, from gods to garden snails, is impermanent, dependently arising, empty of self, intrinsic nature, or ‘own-being’(svabhāva) – and that we harm ourselves and others by grasping for a permanent hold where none is to be found. Clinging to unreal mental con- structions, making ourselves and others miserable with ego-making thoughts, sowing deeds (karma) that for good or ill must bear fruit in future lives, we are captive to the cycle of birth and death. Reborn as a hell being, an animal, or a hungry ghost, I’ll pay the consequences for my evil deeds – and will be hard pressed to amass the merit I need to improve future lives. Even in paradise there is no immortality to be found. Reborn as a god, after long eons of bliss I will exhaust my merit and fall into a lower birth. The situation is urgent, Śāntideva says: just now as I am enjoying the exceedingly rare privilege of a human birth, neither lulled by the pleasantness of paradise nor crushed by the miseries of lower births, I should resolve without a moment’s delay to become awa- kened for the benefit of all sentient beings. https://doi.org/10.1017/S0034412523000124 Published online by Cambridge University Press S122 Carol Zaleski Carol Zaleski One point needs clarification at the outset. I take rebirth to be a background assump- tion for Śāntideva as for traditional Buddhists generally, though scholars and practi- tioners offer differing assessments of its centrality to the Dharma. Śā ntideva and intimations of deathlessness For the purpose of this article it matters little if the standard arguments for rebirth – whether offered by Dharmakīrti in the late sixth century or by investigators of reincarnation memories in our own time – are persuasive or not. For it is not within the ordinary course of rebirth that one should look for immortality. On a traditional Buddhist account, every birth, how- ever pleasant, is stalked by death, and fear of death only tightens death’s grip upon us.4 What intrigues me, rather, are the intimations of deathlessness to be found in accounts of Buddhist awakening – the suggestion that deathlessness is somehow a feature of awaken- ing, whether beyond or (as in Mahāyāna traditions) transcendently within cyclic exist- ence. For Śāntideva, deathlessness is tasted by the bodhisattva who no longer pines for that storied ‘other shore’ beyond saṃsāra, but who resolves, rather, to awaken for the benefit of other beings. g How to generate this altruistic resolve? Whether because there is more than one hand in its composition, or because its author is a virtuoso of upāya (skill in means), the Bodhicaryāvatāra dispenses an assortment of instructions ranging from the folk-proverbial to the technically philosophical, suitable for every stage of the journey. If I am a beginner on the path, I should heed Śāntideva’s warnings about the torments of hell. If assailed by carnal temptations, I should employ time-honoured ascetical techniques – neutralizing lust, for example, by meditating on the beloved’s entrails. As a moral incentive, I should reflect on the equality of all sentient beings – for it is a truism that everyone wishes to be happy. Suffering is simply bad, and should therefore be removed wholesale without pref- erential treatment (Bodhicaryāvatāra 8.103). Finally I should realize that there is no other way for me to attain true happiness than to donate myself to others, dedicating to their well-being whatever merit I accumulate through spiritual practice. Echoing the Mahāyāna liturgy called the ‘supreme worship’ (anuttara-pūja), the Bodhicaryāvatāra abounds in per- formative utterances: May the virtue that I have acquired by doing all this relieve every suffering of sen- tient beings. May I be the medicine and the physician for the sick. May I be their nurse until their illness never recurs . . . https://doi.org/10.1017/S0034412523000124 Published online by Cambridge University Press Śā ntideva and intimations of deathlessness For the sake of accomplishing the welfare of all sentient beings, I freely give up my body, enjoyments, and all my virtues of the three times. (Śāntideva (1997), 3.6, 3.7, 3.10, p. 34) Here is altruism to the nth power, enacted with language that is at once prayerful and heroically sacrificial. Here is altruism to the nth power, enacted with language that is at once prayerful and heroically sacrificial. In the ninth chapter on the Perfection of Wisdom, Śāntideva moves from ethics to metaphysics, drawing upon the Abhidharma analysis of existence as a causal stream of impersonal, ownerless dharmas while rejecting the view of dharmas as atom-like things, honouring Cittamātra teachings on equality of self and other while rejecting the view of consciousness as something independently real. He is free to be eclectic metaphysically because he holds fast to the Madhyamaka understanding of emptiness as his touchstone; with his core commitments in place, there is room for considerable speculative freedom. Having acknowledged the difference between conventional and ultimate truth, Śāntideva uses conventional words about death and rebirth to set free those caught in language’s nets. While he devotes himself to the flourishing of sentient beings, he har- bours no doubts as to their ultimately illusory character.5 One might expect, therefore, that he would be as eager as a Hume or a Parfit to wean us away from immortality lan- guage. Nonetheless, I cannot read the Bodhicaryāvatāra without noticing imagery evocative of immortality thinking. https://doi.org/10.1017/S0034412523000124 Published online by Cambridge University Press S123 Religious Studies Religious Studies Such intimations are present from the very beginning of Śāntideva’s guide. The text begins with an extraordinary promise: the instant you set forth on the bodhisattva way of life, the base metal of your ordinary body will be transmuted into the precious gem that is the body of a Buddha. The bodhicitta is like an alchemical elixir, Śāntideva tells us: ‘the elixir of life, produced to vanquish death in the world’ (Śāntideva (1997), 3.28, p. 36). p The compound noun translated here as ‘elixir of life’ is an elaboration of amṛta, a remarkable word of ancient Indic ancestry which, like the Greek words ambrosia and nec- tar, means both deathlessness and the elixir that conquers death.6 In Buddhist literature, we hear the Buddha praised as amṛta, his Dharma as amṛta, Nirvana as amṛta, mindfulness as amṛta. In the Perfection of Wisdom sūtras, emptiness is amṛta. Śā ntideva and intimations of deathlessness Amitabha, portrayed as the Buddha of Infinite Life (Amitāyus), holds a vase of amṛta. The lay Buddhist master Vimalakīrti, in the sūtra that bears his name, treats his guests to a magical feast of amṛta. Among Mādhyamikas, both Bhāviveka and his critic Candrakīrti employ the expres- sion tattvāmṛta to refer to the way things really are, undistorted by conceptualizations.7 Amṛta may be taken to be metaphorical in these Buddhist cases, but if so it is a particu- larly strong metaphor, steeped in myth, ritual, alchemy, and medicine, and assimilated in meditative and visionary experience. If only in this strong metaphorical sense, the Bodhicaryāvatāra conveys intimations of immortality. The bodhisattva’s ‘perfection of generosity’ (dānapāramitā) imparts a gleam of deathlessness to this death-stalked world: ‘As long as space abides and as long as the world abides, so long may I abide, destroying the sufferings of the world’ is Śāntideva’s prayerful resolve (Śāntideva (1996), 10.55, p. 143). He does not seek to escape from contingency into some absolute realm. Keeping to the Middle Way, he avoids the extremes of annihilationism and eternalism. He finds freedom from contingency within contingency, nirvana within this dreamlike show of a world. Call it deathlessness rather than immortality if that makes it less likely to be misunderstood. Call it deathlessness within life, rather than after life, if you prefer. On the other hand, if we widen our view of the text to encompass its non-monastic as well as monastic audience, if we think of what it must be like to hear Śāntideva’s words spoken by revered teachers, to hear them again during instructions for the dying and funeral rites for the dead, to have them echo in memory during private moments of grief, then the intimations of deathlessness acquire a powerful resonance. For ordinary folk hard-pressed by life, the bodhisattva path may be a far-off dream, but the good news is that there are magnanimous beings dispensing well-being out of their inexhaust- ible treasury of merit. Knowing this, Śāntideva dedicates his merit not only for the ultim- ate awakening of all sentient beings, but also for the fulfilment of their mundane desires: ‘May all beings have immeasurable life. May they always live happily. May the very word “death” perish’ (Śāntideva (1996), 10.33, p. 141). Such intimations of deathlessness are muted, however, if we turn to secular analogues. https://doi.org/10.1017/S0034412523000124 Published online by Cambridge University Press Śā ntideva and intimations of deathlessness Mark Johnston (2012), in his remarkable book Surviving Death, traces a movement from anatta to agape, that is, from no-self to self-sacrificing love. He suggests, in a humanistic vein, that immortality can be ours provided we deny the self and join the ‘onward rush of humankind’ (an expression Johnston takes from John Stuart Mill). For Johnston, the good are so thoroughly identified with others that they live on, as it were, in multiple embodi- ments. It’s a beautiful thought, but it makes no provision for ongoing communion between the living and the dead. Similarly for Derek Parfit: when the glass tunnel of per- sonal identity dissolves, other people come closer, but they are other people only of the sort that a naturalistic account could permit. For Śāntideva, in contrast, the other people who come closer constitute a vast company of buddhas, bodhisattvas, gods, and other dazzling beings in addition to the ordinary folk Carol Zaleski S124 one meets every day. It’s not only the living who have a moral claim upon one; as in most traditional cultures, the dead also have a moral claim. To think otherwise would be to make nonsense of pious practices regarding the deceased, and Śāntideva has no wish to make nonsense of pious practices. But here is the Buddhist distinctive: the bodhisattva’s response to this moral claim is unconditional; it extends to countless multitudes; and it is heightened by the conviction that its beneficiaries are dependently originated, lacking a self, empty of own-being. To accept the moral claim of others on these terms is to taste the ambrosia of deathlessness, purified of narcissistic craving for personal survival. Such is Śāntideva’s altruistic intimation of deathlessness. For Śāntideva, this altruistic intimation of deathlessness goes hand in hand with the no-self teaching, set against a background of religious practices that link the living and the dead. But for other Indic philosophical and religious traditions (such as Hindu or Jain) which proclaim the reality of a transcendent, changeless, blissful ātman, there are analogous moral fruits. The glass tunnel must dissolve for the ātmavādin, too, for the only way to realize my true self is to disidentify from my ego-self. Śā ntideva and intimations of deathlessness The spiritual disciplines that support self-realization, thus understood, aim at loosening the ego’s grip by subduing the passions, retraining the attention, warming the heart with devotion, and balancing ecstatic states with prudence about the needs of the body and sensitivity to the needs of others. So it is that the overt affirmations of immortality that figure so powerfully in many South Asian traditions, as well as the subtle intimations of deathlessness one finds in Śāntideva, rest upon the full length and breadth of their respective teachings and prac- tices. Isolated arguments for immortality are vulnerable to being discredited; but where an intuition of immortality rests upon the lattice-work of a fully realized religious tradition, its claims become stronger and its moral fruits more wholesome. So far I have suggested that immortality is a fitting object of belief provided it is sup- ported by a fully realized religious tradition which, keeping to the Middle Way, balances self-preservation with self-transcendence and preserves the relationship between the liv- ing and the dead. For the remainder of this article, to pursue the same suggestion from a quite different angle, I turn to a question about the immortality of the soul as it pertains to the two-stage afterlife characteristic of classical Judaism, Christianity, and Islam. https://doi.org/10.1017/S0034412523000124 Published online by Cambridge University Press Immortality of the soul and the intermediate state For Jews, Christians, and Muslims, the traditional belief is that the dead will be raised at the end of time as complete embodied, perfected individuals.8 But between death and res- urrection there is a puzzling interregnum. Do the dead utterly blink out of existence, only to be reconstituted by resurrection? Are they unconscious, in suspended animation, or asleep? If the dead persist as separated souls without sense organs, what kind of experi- ence, if any, are they capable of enjoying? If the blessed are admitted even now to the rewards of paradise, does that make the future resurrection superfluous? Which of the standard models of personal identity and selfhood is best suited to make sense of the two- stage afterlife traditionally professed by Jews, Christians, and Muslims? One popular option is dualism. Soul–body dualism of a Platonic or Cartesian kind does a good job of securing the self’s persistence even in a disembodied state, and has the vir- tue of simplicity, as Richard Swinburne likes to point out. Yet our biological evolution, our kinship to other animals, and the intricate entanglement of mind and brain make dual- ism – even if it should turn out to be the best candidate – difficult to theorize and defend. More attractive to many theists today are hybrid models that stress the integrity of the human organism, regarding body and soul as naturally intertwined. On the view called hylomorphism, the human soul is the substantial form (morphē) which communicates S125 Religious Studies life and structure to the human body. Once separated by death, the body loses its human signature and becomes a mere corpse, while the soul is deprived of its normal body- constituting role. Hence, on a hylomorphic account, a complete and flourishing human life beyond death would require bodily resurrection. Each of these models – dualistic and hylomorphic, with their many variations – faces a particular challenge. The dualist’s challenge is to explain why resurrection of the body is necessary in addition to immortality of the soul; the hylomorphist’s challenge is to account for the continuity of the human person during the interim period between death and resurrection. Yet with good will and skilful reasoning, these challenges can be met, though in different eras the climate of opinion will favour one model over the other for a time. Immortality of the soul and the intermediate state An interesting case in point is the current debate concerning ‘survivalist’ versus ‘cor- ruptionist’ interpretations of Thomas Aquinas’s teaching on the status of the separated soul.9 The nomenclature is somewhat misleading: no one denies that, for Thomas, the separated soul (whose incorruptibility is knowable by reason) survives throughout the intermediate period from death until bodily resurrection. But the corruptionist reading is that the human person does not survive the loss of the body at death. Thomas’s obser- vation (2012), anima mea non est ego (‘my soul is not I’), is frequently quoted in this regard. The survivalist reading, however, interprets Thomas as allowing for the idea that human personhood persists throughout the intermediate period. For believers well grounded in their tradition, the existence of such rival views of the soul isn’t in itself a great problem. Scholars may disagree about what Thomas Aquinas meant, but no one could imagine him endorsing a view of the soul which makes nonsense of such pious practices as praying to the saints or praying for the souls in purgatory. Aquinas remains a Catholic on any sound reading. The real worry arises when a disagree- ment over the metaphysics of the human person shakes the foundation of core religious practices and commitments. The remainder of this article will consider just such a prac- tical case: a twentieth-century theological contretemps concerning immortality of the soul. Here’s how the issue was framed by the eminent Lutheran theologian Oscar Cullmann, in his 1955 Ingersoll Lecture on Human Immortality at Harvard: y g p g y Here’s how the issue was framed by the eminent Lutheran theologian Oscar Cullm in his 1955 Ingersoll Lecture on Human Immortality at Harvard: If today one asks an average Christian, no matter whether Protestant or Catholic . . . what the New Testament teaches about the destiny of the individual human being after death, in almost every case one will receive the answer: ‘The immortality of the soul’. In this form, this opinion is one of the greatest misunderstandings of Christianity there can be. (Cullmann (1965), 9) Cullmann was not the first advocate of this view, nor was he alone in promoting it. Many exegetes and theologians were urging Christians to close their ears to the siren song of immortality of the soul, deeming it a Greek import unfaithful to the holism of biblical anthropology, unmindful of the centrality of Christ’s redeeming work. https://doi.org/10.1017/S0034412523000124 Published online by Cambridge University Press Immortality of the soul and the intermediate state Resurrection should be the exclusive focus of Christian hope. And in that case, the argument was, it is better to think of the intermediate period as unconscious sleep or temporary extinction, and thus conveniently set aside the church-dividing doctrine of purgatory.10 p g y By the 1960s, a dehellenizing campaign, as Joseph Ratzinger called it (1988), was well advanced among Catholics as well as Protestants, with a number of biblical theologians arguing that one should think of life after death as entry into God’s eternity, rather than as a temporal interlude in which separated souls hang around waiting to receive their bodies. A novel theory called ‘resurrection in death’ was proposed as a way to https://doi.org/10.1017/S0034412523000124 Published online by Cambridge University Press Carol Zaleski S126 overcome body–soul dualism and eliminate the redundancies of the traditional two-stage eschatology. All this might have been a tempest in an exegetical teapot if it weren’t for the fact that the dehellenizing campaign contributed to a general climate of embarrassment about soul language – an embarrassment that spread beyond the guild of professional theologians to affect the congregation. Some pastors began to wonder if it was improper to speak of going to heaven when you die, improper to comfort mourners with assurances about the souls of the departed, improper, even, to address prayers to the saints. Why pray for the dead, or to the dead, if there is no conscious life on the receiving end? , , g There were other practical consequences. Catholic liturgists seeking to implement the Vatican II mandate to reform the traditional (1962) Roman Missal trimmed back the use of the word anima (soul), deleting it even from Masses for the Dead.11 This was part of a gen- eral movement to restrain what were deemed excessively individualistic and otherworldly tendencies in public worship. Moreover, in places where anima was retained in the 1970 Roman Missal, English-language translators favoured prosaic alternatives. Immortality of the soul and the intermediate state The Communion Rite prayer, ‘Lord, I am not worthy that you should enter under my roof, but only say the word and my soul shall be healed’ (Domine non sum dignus ut intres sub tectum meum; sed tantum dic verbo et sanabitur anima mea), echoing the prayer of the cen- turion for his paralysed servant (Matthew 8:8), became by the translator’s licence the abstract utterance ‘Lord, I am not worthy to receive you, but only say the word and I shall be healed.’ The assumption was that the pronoun I is more holistic than the noun soul. But the effect was paradoxically disembodying, for the word soul is far richer than the pronoun I in sensory and spiritual associations. It was entirely unnecessary, too: on a traditional Christian account the soul indwells the body not as a mere part like an appendix, but as a part that unifies the whole, is the deepest dimension of the whole, and represents the whole by synecdoche. That’s about as holistic as any anthropological term could be. Moreover, as they await the general resurrection, the souls of the blessed already enjoy a corporate existence, so to speak, as part of the heavenly society in which they abide as members of Christ’s body, in fellowship with all the saints, at once at rest and active in contemplation and charity. y Eventually the dehellenization campaign subsided. In 2011, the word soul was restored to English-language liturgical texts, reflecting principles set down earlier by a ‘Letter on Certain Questions concerning Eschatology’ published by the Sacred Congregation for the Doctrine of the Faith (1979), and a subsequent intervention by the International Theological Commission (1992). Against the view that would oppose resurrection of the whole person to immortality of the soul, the Letter affirmed: that a spiritual element survives and subsists after death, an element endowed with consciousness and will, so that the ‘human self’ subsists. To designate this element, the Church uses the word ‘soul’, the accepted term in the usage of Scripture and Tradition. Although not unaware that this term has various meanings in the Bible, the Church thinks that there is no valid reason for rejecting it; moreover, she con- siders that the use of some word as a vehicle is absolutely indispensable in order to support the faith of Christians. Immortality of the soul and the intermediate state And, most importantly: ‘The Church excludes every way of thinking or speaking that would render meaningless or unintelligible her prayers, her funeral rites and the religious acts offered for the dead. All these are, in their substance, loci theologici.’ The Letter sets forth a principle of restraint, not to make nonsense of pious practices that link the living and the dead; and it joins this principle of restraint to a principle of speculative freedom, not demanding uniformity in matters of metaphysics. One may be a Platonist or an https://doi.org/10.1017/S0034412523000124 Published online by Cambridge University Press S127 Religious Studies Religious Studies Aristotelian, a survivalist or a corruptionist, provided the non-negotiables are honoured. Śāntideva, as I’ve suggested, observed a comparable restraint and enjoyed a comparable speculative freedom. For Jews, Christians, and Muslims alike, there is one word in the lexicon of theological anthropology which overshadows the words soul and body and renders disputes about them less worrisome: that word is creature. As a creature, the human being receives each moment of existence as a sheer gift. This is a key point that regulates metaphysical speculation: the fact that we are made is more important than what we are made of. We could be as immaterial as angels and God could destroy us if it were just for God to do so. We could be as material as mayflies and God could preserve us. Immortalism fails when it becomes forgetful of creaturehood. Mortalism fails when it severs the relationship between this life and the next, between the living and the dead, between the human person and God. By the same token, personal identity in the afterlife becomes conceivable by dint of relation to one’s maker. As the infant’s development of a unified personal identity is fos- tered by the face-to-face vision of the mother and thwarted by its deprivation, so personal identity in the future life may be fostered by the face-to-face vision of God, the beatific vision.12 In principle, if I am Jewish, Christian, or Muslim, I believe in immortality because I believe in God, not because I have prior or independent proof of immortality. I need not be discouraged by the inconclusive character of isolated arguments for life after death. Immortality of the soul and the intermediate state As long as I understand immortality in a way that harmonizes with the total pat- tern of creation, revelation, and redemption to which I subscribe, it is rational for me to believe in it. And from this it follows that I may judge models of body, soul, and self on their fittingness in relation to what I believe about God and in relation to what I do as a member of the spiritual community in which I am enfolded. Analogously, if I am Buddhist, I believe in amṛta – deathlessness – because I honour my interdependence with all sentient beings and entrust myself to the protection of fully awakened beings of invincible com- passion. To satisfy mere curiosity about the afterlife is not conducive to my awakening, but deathlessness becomes intelligible as a Mahāyāna Buddhist theme when it is seen in light of the total Gestalt of the bodhisattva way of life. g y There is admittedly a certain circularity to the claim that belief in immortality may be justified by internal considerations of harmony or fittingness. Yet I hope to have shown that belief in immortality is not always an automatic concomitant of a religious outlook. In the Buddhist and Christian cases examined here, there are tensions to resolve and work to be done in order to harmonize belief in immortality with the total pattern of religious belief. How to believe in immortality? Immerse yourself in an immortality-bearing tradition which offers you something even greater than your own immortality to believe in and to love, and a thousand things besides your own immortality in which to take an interest. It should present a comprehensive vision of reality. It should appeal to your sense of what is good, beautiful, and true; engage your reason and your affections; integrate what you know while surpassing what you know; challenge as well as console; and direct you towards the fulfilment that is proper to your nature. As the examples I’ve discussed illustrate, fully realized religious traditions have the resources to nurture as well as to restrain the impulse to believe in immortality. Coherent, religion-specific immortality beliefs balance self-transcendence and self- preservation, honour the connection between the living and the dead, and tolerate con- siderable speculative freedom provided the core beliefs of the tradition are in place. https://doi.org/10.1017/S0034412523000124 Published online by Cambridge University Press Notes Śāntideva’s opinion on the harmful character of death is discussed by Garfield (2010/2011); also in ‘Seeing 3. Cassian s account is modelled on that of Evagrius of Pontos, Praktikos 12 Acedia ; see Sinkewicz (2006), 99. 4. Śāntideva’s opinion on the harmful character of death is discussed by Garfield (2010/2011); also in ‘Seeing sentient beings: Śāntideva’s moral phenomenology’ in Gold and Duckworth (2019, 200). In other contexts, fear of death or of rebirth in lower realms is a salutary means to spur spiritual and moral exertion. 4. Śāntideva’s opinion on the harmful character of death is discussed by Garfield (2010/2011); also in ‘Seeing sentient beings: Śāntideva’s moral phenomenology’ in Gold and Duckworth (2019, 200). In other contexts, fear of death or of rebirth in lower realms is a salutary means to spur spiritual and moral exertion. y p p 5. Scholars disagree about the precise relationship between his moral teaching of radical unselfishness and his metaphysical teaching of no-self, but there’s no doubt that both are non-negotiable commitments for Śāntideva. See Garfield et al. (2016), Harris (2011), Siderits (2000), Williams (1998). y p p 5. Scholars disagree about the precise relationship between his moral teaching of radical unselfishness and his metaphysical teaching of no-self, but there’s no doubt that both are non-negotiable commitments for Śāntideva. See Garfield et al. (2016), Harris (2011), Siderits (2000), Williams (1998). 6. The Sanskrit is jagan-mṛtyu-vināśāya, from jagat (the universe, all beings), mṛtyu (death), and vināśāya (annihi- lation). The Tibetan counterpart to this verse, as rendered by the Padmakara Translation Group, is more colour- ful: ‘This is the supreme draft of immortality \| That slays the Lord of Death, the slaughterer of beings’ (3.29, 53). For amṛta in the Vedas see Olivelle (2011). For amṛta in the purāṇas and in South Asian iconography, see Narayanan (2014). 6. The Sanskrit is jagan-mṛtyu-vināśāya, from jagat (the universe, all beings), mṛtyu (death), and vināśāya (annihi- lation). The Tibetan counterpart to this verse, as rendered by the Padmakara Translation Group, is more colour- ful: ‘This is the supreme draft of immortality \| That slays the Lord of Death, the slaughterer of beings’ (3.29, 53). For amṛta in the Vedas see Olivelle (2011). For amṛta in the purāṇas and in South Asian iconography, see Narayanan (2014). 7. See Eckel (2008), on this expression in Bhāviveka’s The Heart of the Middle Way (Madhyamakahṛdaya) verse 5.1, pp. 213–214, n. 2. Notes Eckel cites an interesting discussion by Lamotte (1976) ‘Perfumed amṛta and the sacred meal’, 307–314, on differences between Buddhist and Hindu treatments of this theme. Candrakīrti (1979, para. 372, p. 182). p 8. For resurrection in biblical and classical Judaism, see Levenson (2006); for Islamic traditions, see Lange (2015) and Smith and Haddad (1981). p 8. For resurrection in biblical and classical Judaism, see Levenson (2006); for Islamic traditions, see Lange (2015) and Smith and Haddad (1981). 9. For an influential survivalist interpretation of Aquinas, see Stump (2012). For an influential corruptionist inter- pretation, see Toner (2009 and 2010). See also the following for the ongoing debate: Oderberg (2012); Nevitt (2014), (2016); Spencer (2014); Brower (2017), 279–310; De Haan and Dahm (2019); Skrzypek (2020); Rooney (2021); . 10. Discussed in Zaleski Ingersoll Lecture (2004) and Yates (2017) (with a detailed defence of the traditional two- stage eschatology). For an ecumenical rapprochement between Lutheran and Catholic theologians on questions of the intermediate state, including purgatory, see The Hope of Eternal Life (2011). 9. For an influential survivalist interpretation of Aquinas, see Stump (2012). For an influential corruptionist inter- pretation, see Toner (2009 and 2010). See also the following for the ongoing debate: Oderberg (2012); Nevitt (2014), (2016); Spencer (2014); Brower (2017), 279–310; De Haan and Dahm (2019); Skrzypek (2020); Rooney (2021); . (2016); Spencer (2014); Brower (2017), 279 310; De Haan and Dahm (2019); Skrzypek (2020); Rooney (2021); . 10. Discussed in Zaleski Ingersoll Lecture (2004) and Yates (2017) (with a detailed defence of the traditional two- stage eschatology). For an ecumenical rapprochement between Lutheran and Catholic theologians on questions of the intermediate state, including purgatory, see The Hope of Eternal Life (2011). 11. For documentation of some of these changes, see Hazell (2021). 12. Christians haven’t always agreed about whether the souls of the blessed will see God immediately upon death. For Catholics, the question was settled in the affirmative by Pope Benedict XII in the 1336 Constitution Benedictus Deus and seconded by the bull Laetentur coeli at the 1439 Council of Florence. Immortality of the soul and the intermediate state Narcissism is not an issue – for on any well-developed religious conception, the price of immortality is death to self; the promise of immortality is relationship unbroken by death; and the perfection of generosity is to draw others into this deathless relationship. S128 Carol Zaleski Conflict of interest. None. Notes 1. Thomas Aquinas made liberal use of this strategy. For a twentieth-century example, see Bernard Lonergan (2011). 2. Boredom is too recent for Samuel Johnson to have included it in his 1755 Dictionary of the English Language, while an entry for tedious defines the word as ‘wearisome by continuance’. See ‘tedious, adj.’. A Dictionary of the English Language, by Samuel Johnson. 1755. https://johnsonsdictionaryonline.com/1755/tedious_adj (Accessed 8 June 2022). l h f ll i See also the following: g ‘acedia, n.’. (no date) OED Online. Oxford University Press. http://www.oed.com/view/Entry/1335#eid331335 (A d J ) edia, n.’. (no date) OED Online. Oxford University Press. http://www.oed.com/view/Entry/1335#eid33133512 ccessed 3 June 2022). acedia, n. . (no date) OED Online. Oxford University Press. http://www.oed.com/view/Entry/1335#eid33133512 (Accessed 3 June 2022). ‘accidie, n.’. (no date) OED Online. Oxford University Press. http://www.oed.com/view/Entry/1068? (Accessed 3 June 2022). ‘accidie, n.’. (no date) OED Online. Oxford University Press. http://www.oed.com/view/Entry/1068? (Accessed 3 June 2022). ‘accidie, n.’. (no date) OED Online. Oxford University Press. http://www.oed.com/view/Entry/1068? (Accessed 3 June 2022). A Latin Dictionary, Lewis CT and Short C (eds), taedĭum (no date). https://www.perseus.tufts.edu/hopper/text? doc=Perseus:text:1999.04.0059:entry=taedium (Accessed 3 June 2022). A Latin Dictionary, Lewis CT and Short C (eds), taedĭum (no date). https://www.perseus.tufts.edu/hopper/text? A Latin Dictionary, Lewis CT and Short C (eds), taedĭum (no date). https://www.perseus.tufts.edu/hopper/tex doc=Perseus:text:1999.04.0059:entry=taedium (Accessed 3 June 2022). y 3. Cassian’s account is modelled on that of Evagrius of Pontos, Praktikos 12 ‘Acedia’; see Sinkewicz (2006), 99. 4. Śāntideva’s opinion on the harmful character of death is discussed by Garfield (2010/2011); also in ‘Seeing sentient beings: Śāntideva’s moral phenomenology’ in Gold and Duckworth (2019, 200). In other contexts, fear f d h f b h l l l l d l Cassian’s account is modelled on that of Evagrius of Pontos, Praktikos 12 ‘Acedia’; see Sinkewicz (2006), 99 Śā tid ’ i i th h f l h t f d th i di d b G fi ld (2010/2011) l i ‘S i 3. Cassian’s account is modelled on that of Evagrius of Pontos, Praktikos 12 ‘Acedia’; see Sinkewicz (2006), 99. 4. Śāntideva’s opinion on the harmful character of death is discussed by Garfield (2010/2011); also in ‘Seeing sentient beings: Śāntideva’s moral phenomenology’ in Gold and Duckworth (2019 200) In other contexts fear 3. Cassian’s account is modelled on that of Evagrius of Pontos, Praktikos 12 ‘Acedia’; see Sinkewicz (2006), 99. 4. p g y g Cassian J (1900) Institutes 10, 2 in The Twelve Books of John Cassian on the Institutes of the Coenobia, and the Remedies for the Eight Principal Faults, A Select Library of Nicene and post-Nicene Fathers of the Christian Church, second series, Schaff P and Wace H (eds), vol. 11. 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https://openalex.org/W4306796041	https://www.jmir.org/2022/10/e36767/PDF	English	null	The Effectiveness of Patient Training in Inflammatory Bowel Disease Knowledge via Instagram: Randomized Controlled Trial	JMIR. Journal of medical internet research/Journal of medical internet research	2,022	cc-by	10,727	JOURNAL OF MEDICAL INTERNET RESEARCH JOURNAL OF MEDICAL INTERNET RESEARCH Blunck et al Original Paper The Effectiveness of Patient Training in Inflammatory Bowel Disease Knowledge via Instagram: Randomized Controlled Trial Dominik Blunck1, MSc; Lena Kastner1, MSc; Michael Nissen2, MSc; Jacqueline Winkler3, MSc 1Department of Health Management, Institute of Management, Friedrich-Alexander-Universität Erlangen-Nürnberg, Nuremberg, Germany 2Machine Learning and Data Analytics Lab, Department Artificial Intelligence in Biomedical Engineering, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany 3Bristol-Myers Squibb GmbH & Co KGaA, Munich, Germany *these authors contributed equally Corresponding Author: Dominik Blunck, MSc Department of Health Management Institute of Management Friedrich-Alexander-Universität Erlangen-Nürnberg Lange Gasse 20 Nuremberg, 90403 Germany Phone: 49 911 5302 96394 Email: dominik.blunck@fau.de Corresponding Author: Dominik Blunck, MSc Department of Health Management Institute of Management Friedrich-Alexander-Universität Erlangen-Nürnberg Lange Gasse 20 Nuremberg, 90403 Germany Phone: 49 911 5302 96394 Email: dominik.blunck@fau.de Abstract Background: Patients’ knowledge was found to be a key contributor to the success of therapy. Many efforts have been made to educate patients in their disease. However, research found that many patients still lack knowledge regarding their disease. Integrating patient education into social media platforms can bring materials closer to recipients. Objective: The aim of this study is to test the effectiveness of patient education via Instagram. Methods: A randomized controlled trial was conducted to test the effectiveness of patient education via Instagram among patients with inflammatory bowel disease. Participants were recruited online from the open Instagram page of a patient organization. The intervention group was educated via Instagram for 5 weeks by the research team; the control group did not receive any educational intervention. The knowledge about their disease was measured pre- and postintervention using the Inflammatory Bowel Disease Knowledge questionnaire. Data were analyzed by comparing mean knowledge scores and by regression analysis. The trial was purely web based. Results: In total, 49 participants filled out both questionnaires. The intervention group included 25 participants, and the control group included 24 participants. The preintervention knowledge level of the intervention group was reflected as a score of 18.67 out of 24 points; this improved by 3 points to 21.67 postintervention. The postintervention difference between the control and intervention groups was 3.59 points and was statistically significant (t32.88=–4.56, 95% CI 1.98-5.19; P<.001). Results of the regression analysis, accounting for preintervention knowledge and group heterogeneity, indicated an increase of 3.33 points that was explained by the intervention (P<.001). Conclusions: Patient education via Instagram is an effective way to increase disease-related knowledge. Future studies are needed to assess the effects in other conditions and to compare different means of patient education. Trial Registration: German Clinical Trials Register DRKS00022935; https://tinyurl.com/bed4bzvh (J Med Internet Res 2022;24(10):e36767) doi: 10.2196/36767 J Med Internet Res 2022 \| vol. 24 \| iss. 10 \| e36767 \| p. 1 (page number not for citation purposes) social media; Instagram; patient training; patient education; disease-related knowledge; RCT; randomized controlled trial; Germany; inflammatory bowel disease; IBD-KNOW Inflammatory Bowel Disease Inflammatory bowel disease (IBD) is a group of chronic inflammatory diseases of the gastrointestinal tract. IBD can be divided into Crohn disease, ulcerative colitis, and other diseases that present with different gastrointestinal symptoms, such as diarrhea [1]. The global prevalence of IBD is approximately 3.9 million females and 3.0 million males, with a worldwide accelerating incidence [2,3]. The economic burden of IBD is highly relevant. Annual costs per patient were shown to be 3-fold in IBD patients compared to patients without IBD [4]. A systematic review estimated the mean annual health care cost of IBD patients in North America to be over US $13,000 [5]. Although the disease is not yet fully understood [6], there exist different pharmaceutical and nonpharmaceutical interventions. For pharmaceutical interventions, aminosalicylates, corticosteroids, antibiotics, immunomodulative treatments, and different biologic treatments are used, depending on the clinical stage of IBD [7-10]. Nonpharmaceutical interventions are surgery—for example, for patients who are refractory to treatment—and other interventions, such as diets [7]. Because of a greater likelihood of depression or anxiety, resulting in lower quality of life, psychotherapy is a common therapeutic approach as well [7,11-13]. In the case of IBD, different methods to increase disease-related knowledge have been studied. One study compared a telemedicine intervention (ie, SMS text messaging) with standard care (ie, educational materials at clinical appointments) to increase disease-related knowledge in IBD. On a 24-point scale, telemedicine increased the baseline value of 12.6 by 2.4 points, whereas standard care only yielded 1.8 points [36]. In a study where patients received a CD-ROM for self-paced autodidactic learning, participants were able to increase their knowledge from 12.2 points on a 30-point scale to 19.9 points, an increase of 7.6 points. After 9 months of follow-up, the knowledge increase was still 5.3 points higher than at baseline [37]. Another study compared a 12-hour structured education program with standard care (ie, teaching by physician during regular visits). On a 24-point scale, the intervention group’s disease-related knowledge increased by 7.71 points immediately after the intervention and 7.94 points after 8 weeks compared to baseline. The control group’s disease-related knowledge increased by 3.55 points immediately after standard care and 4.05 points after 8 weeks compared to baseline [38]. In another study, IBD patients were educated through counseling, pill cards, and educational material. In that study, knowledge increased from 8.15 points to 11.65 points [23]. KEYWORDS social media; Instagram; patient training; patient education; disease-related knowledge; RCT; randomized controlled trial; Germany; inflammatory bowel disease; IBD-KNOW J Med Internet Res 2022 \| vol. 24 \| iss. 10 \| e36767 \| p. 1 (page number not for citation purposes) https://www.jmir.org/2022/10/e36767 XSL•FO RenderX JOURNAL OF MEDICAL INTERNET RESEARCH Blunck et al requires taking different pharmaceuticals as well as lifestyle and nutrition changes. Hence, the literature implies a need to enhance knowledge of IBD and related therapies for better adherence. In one study by Elkjaer et al [26], patients with IBD who participated in dedicated educational programs showed better compliance and adherence, higher disease-related knowledge, better quality of life, and better coping with relapsing, leading to a mean relapse duration of 18 days compared to 77 days in the control group. Shared decision-making improves clinical outcomes because therapy plans are aligned with patients’ values, lifestyles, and expectations [27-29]. In IBD, shared decision-making is a relevant factor regarding medication therapy [30]. For shared decision-making, however, equitable collaboration between patients and physicians is required. Therefore, high levels of disease-related knowledge are necessary to enable a common understanding of the underlying problems and therapy options [29,31]. Additionally, the majority of patients with IBD also want to be actively involved in the decision-making process, as surveys have shown [32-34], which might be due to high levels of uncertainty associated with IBD [35]. Thus, one important antecedent of shared decision-making is informing patients. Inflammatory Bowel Disease Studies show that IBD patients benefit from higher disease-related knowledge, which has positive effects on the clinical outcomes of their overall therapy [14,15]. Not only in IBD, but also in other, especially chronic, conditions, higher levels of knowledge of the respective condition are related to better outcomes [16,17]. Besides the clinical importance, improving patients’ disease-related knowledge is also economically important. A study by Colombara et al [18] found that an increase of 5 points in patients’ disease-related knowledge on a 24-point scale could decrease costs in the first year after diagnosis by over €1000. https://www.jmir.org/2022/10/e36767 J Med Internet Res 2022 \| vol. 24 \| iss. 10 \| e36767 \| p. 2 (page number not for citation purposes) Aim The purpose of this study is, thus, to explore whether patient education via Instagram stories is an effective method for educating and informing adult patients with IBD, as compared to patients receiving no intervention, by conducting a randomized controlled trial (RCT). Disease-Related Knowledge Besides the sole presentation of pictures or videos in the story, creators can also integrate different interactive functionalities, such as quizzes. A recent study evaluated the use of social media platforms and showed that 59% of Instagram users visited Instagram at least daily, and more than one-third of the users visited the app several times a day [42]. Therefore, it seems like a reasonable approach for integrating patient education into everyday life. Recruitment and Randomization For recruitment, we were supported by CHRONISCH GLÜCKLICH e.V., a German patient organization for IBD. The organization owns and operates an Instagram page that had 2332 followers (87.5% female) at the start of recruitment. Comparable pages have similar demographics. They announced the study in their publicly available “Instagram Stories” and called for participation. Participants were included if they met the following inclusion criteria: (1) were older than 18 years of age, (2) had an Instagram account, and (3) were able to fill out a questionnaire. After a recruitment period of 2 weeks, we assigned the participants to either the intervention group or the control group with the help of the online program Research Randomizer [47]. Dropout Effects According to the intention-to-treat concept, we included all data from all patients in our analysis, whether or not they followed the study protocol [48]. To ensure robustness of our results, we conducted all analyses without dropouts. To better understand dropout effects, we investigated group differences between included participants and those who dropped out with respect to current age, age at diagnosis, sex, diagnosis, and prestudy disease-related knowledge. Previous studies of social media–based interventions showed overall good results in improving clinical outcomes and patients’ disease-related knowledge about different conditions, for example, diabetes [43]. A review article by Grajales et al [44] reported various approaches for applying social media to health care and patient education. For example, several apps in Facebook are described as well as weblogs. Another paper studied the effect of participation in social health networks on patient activation. Patients with a chronic condition participated in a dedicated social network where they could find medical advice from experts as well as the opportunity to connect with other patients. Higher frequency and duration of usage of this network was associated with higher patient activation, and patients felt more empowered [45]. Intervention The intervention group received access to a nonpublic Instagram account, which posted educational material to the story function one to three times per week from June 29, 2020, to July 31, 2020. Furthermore, the stories were saved using the highlights function to be watched later. The posted educational material was either informational or interactive (Figure 1). All educational content was publicly available information about IBD and was reviewed by a physician before being posted by the research team. For interactive purposes, quizzes, for example, were included in the educational stories. Furthermore, participants were not forced or controlled to watch the Instagram stories; they solely received access and followed the account. If participants provided feedback or made requests during the study, such as comments on a story, this was incorporated into successive stories over the 5-week period (ie, higher contrast). Disease-Related Knowledge In this section we describe (1) why higher disease-related knowledge might positively affect clinical outcomes, (2) how other studies approached increasing disease-related knowledge in IBD, (3) how we propose to integrate patient education into patients’ daily lives via social media, and (4) how others did so for other indications. Higher disease-related knowledge has a positive effect on clinical outcomes because it improves adherence and enables shared decision-making, which ultimately leads to better clinical outcomes. Adherence to the treatment plan is a major success factor in therapy. However, in chronic diseases in particular, studies found that medication adherence often is insufficient [19,20]. Higher levels of patient knowledge showed improved adherence in different conditions, for example, because of higher motivation or dispelled misbeliefs [21,22]. Several studies found an improvement in adherence among patients with IBD through different educational interventions and, subsequently, higher rates of knowledge of IBD [23,24]. Bucci et al [25] investigated the factors that predict adherence among Italian patients with IBD and described the complex treatment plan for IBD, which Although different approaches for informing patients have already been studied, they might lack sustainable integration into patients’ daily lives. For example, Yin et al [39] argued that most of the educational apps they identified in a scoping review did not proactively inform patients, and patients instead had to access the app by themselves manually; this could be why they were poorly embedded into patients’ daily routines. In contrast, social media is discussed as a way to potentially overcome this problem, as many patients already use it and it comes with high interactivity [40]. Therefore, we suggest distributing information via Instagram. Instagram is a widely used social media platform with 1 billion https://www.jmir.org/2022/10/e36767 J Med Internet Res 2022 \| vol. 24 \| iss. 10 \| e36767 \| p. 2 (page number not for citation purposes) J Med Internet Res 2022 \| vol. 24 \| iss. 10 \| e36767 \| p. 2 (page number not for citation purposes) XSL•FO RenderX XSL•FO RenderX JOURNAL OF MEDICAL INTERNET RESEARCH Blunck et al (Consolidated Standards of Reporting Trials of Electronic and Mobile Health Applications and Online Telehealth) guideline [46]. The intervention group received disease-related education for 5 weeks, and the control group did not receive any educational treatment. Outcomes were assessed before and after the intervention. users worldwide [41]. J Med Internet Res 2022 \| vol. 24 \| iss. 10 \| e36767 \| p. 3 (page number not for citation purposes) Disease-Related Knowledge In most cases, Instagram is accessed via its corresponding smartphone app, which is used to view and share pictures or videos. Users can view pictures and videos in two ways: either via their timeline or the so-called story function. Media in the timeline is presented once to the user by the Instagram algorithm but is constantly available. Furthermore, the algorithm orders content as a result of user-based analyses. The story function is found in the top section of the Instagram home screen. Content creators can share short video clips or pictures in the story function, which are then presented to the creator’s followers. The order of the stories presented to a user also depends on user-based analyses. Instagram stories are available for 24 hours; however, creators can save their stories using the so-called “Story Highlights” feature, which makes stories constantly available. Buttons to view different categories of highlights are available on every user profile. Besides the sole presentation of pictures or videos in the story, creators can also integrate different interactive functionalities, such as quizzes. A recent study evaluated the use of social media platforms and showed that 59% of Instagram users visited Instagram at least daily, and more than one-third of the users visited the app several times a day [42]. Therefore, it seems like a reasonable approach for integrating patient education into everyday life. users worldwide [41]. In most cases, Instagram is accessed via its corresponding smartphone app, which is used to view and share pictures or videos. Users can view pictures and videos in two ways: either via their timeline or the so-called story function. Media in the timeline is presented once to the user by the Instagram algorithm but is constantly available. Furthermore, the algorithm orders content as a result of user-based analyses. The story function is found in the top section of the Instagram home screen. Content creators can share short video clips or pictures in the story function, which are then presented to the creator’s followers. The order of the stories presented to a user also depends on user-based analyses. Instagram stories are available for 24 hours; however, creators can save their stories using the so-called “Story Highlights” feature, which makes stories constantly available. Buttons to view different categories of highlights are available on every user profile. Overview The study’s primary outcome was patients’ knowledge about IBD. The outcome was measured at baseline (ie, preintervention) and 1 week after the last story was published (ie, postintervention). We measured patients’ knowledge by self-assessment using an online questionnaire. There exist different validated questionnaires to measure patients’ knowledge about IBD, such as the Crohn's and Colitis Knowledge score [49] and the Inflammatory Bowel Disease Knowledge (IBD-KNOW) questionnaire [50]. We chose the IBD-KNOW questionnaire because it is newer and includes a broader field of disease and therapy-related knowledge, such as biologics. We measured the patients’ knowledge about IBD by using the validated IBD-KNOW questionnaire. For this purpose, we translated the original English-language questionnaire into German (Multimedia Appendix 1). This translated version was reviewed by a physician. The questionnaire consists of 24 items, asking questions about IBD facts with response options of “true,” “false,” and “I don’t know.” The number of correct answers—“I don’t know” is not counted as correct—represents the respondent’s level of knowledge about IBD and, hence, the score ranges from 0 to 24 points. The online questionnaire was evaluated by application of the Checklist for Reporting Results of Internet E-Surveys (CHERRIES) [51]. We analyzed the study’s data in three ways. Firstly, we descriptively analyzed the study participants’ characteristics. Secondly, we conducted inferential statistics to display group and time differences in level of knowledge. Thirdly, we conducted a regression analysis. All statistical analyses were performed with R statistical software (version 4.0.0; R Foundation for Statistical Computing) [54,55]. We used the following R packages: pwr for power calculation [53], ggplot2 for data visualization [56], car for calculating variance inflation factors [57], and dplyr and tidyr for data management [58,59]. P values of less than .05 were considered statistically significant. Regression Analysis To further analyze the effects, account for group heterogeneity, and ensure robustness of our results, we estimated an ordinary least squares (OLS) regression model of patients’ knowledge with a difference-in-differences approach (ie, lm()-function in R). The dependent variable in the regression model was the IBD-KNOW score. The independent variables included a group dummy variable, a time dummy variable, and an interaction term of group and time. The group dummy value was 1 for the treatment group and 0 for the control group; the time dummy value was 1 for the postintervention questionnaire and 0 for the preintervention questionnaire. The covariates were chosen to control for further effects that are associated with learning. Hence, we controlled for sex (dummy variable, female = 1), age in years, the duration in years that the patient has lived with their IBD diagnosis at the time of the study (ie, current age – age at diagnosis), and diagnosis (dummy variable for Crohn disease) [60,61]. This is reflected in the following equation: Besides the 24 IBD-specific questions, we included several sociodemographic and disease-related variables in the questionnaire, which were included as control variables in the regression analyses. Inferential Statistics To analyze group differences regarding categorical variables, we used the chi-square test. For continuous variables, we conducted the Welch t test. J Med Internet Res 2022 \| vol. 24 \| iss. 10 \| e36767 \| p. 4 (page number not for citation purposes) https://www.jmir.org/2022/10/e36767 Statistical Analysis Overview Outcome Measure Design This study was conducted as a 2-arm, parallel-group, purely web-based RCT, following the CONSORT-EHEALTH J Med Internet Res 2022 \| vol. 24 \| iss. 10 \| e36767 \| p. 3 (page number not for citation purposes) https://www.jmir.org/2022/10/e36767 https://www.jmir.org/2022/10/e36767 JOURNAL OF MEDICAL INTERNET RESEARCH Blunck et al Figure 1. Example screenshots of educational material [content in German]. Figure 1. Example screenshots of educational material [content in German]. Sample Size To identify the required sample size, we performed a power analysis. An improvement of 3 points in the IBD-KNOW score has been previously regarded as clinically important [36,52]. At an SD of 4.7 [15] and to detect group differences of at least 3 points on the IBD-KNOW scale, with power greater than 0.8 and α<.05, a sample size of 40 participants per group was required [53]. We anticipated a dropout rate of 20%, giving a total planned sample size of 100 participants. https://www.jmir.org/2022/10/e36767 J Med Internet Res 2022 \| vol. 24 \| iss. 10 \| e36767 \| p. 4 (page number not for citation purposes) XSL•FO RenderX JOURNAL OF MEDICAL INTERNET RESEARCH Blunck et al We did not find significant group differences between the included participants and the dropout group with respect to age at diagnosis (P=.34), sex (P=.37), type of diagnosis (P=.93), and prestudy IBD knowledge (P=.17). A difference in age between the dropout group and the included participants was found (P=.04), with the dropouts being 3 years older on average. This difference in age did not yield a difference regarding the length of IBD history, which is the difference between current age and age at diagnosis (P=.27). y = β0 + β1dSex + β2dDiagnosis + β3Age + β4Duration + β5dTime + β6dGroup + β7(dTime × dGroup) + e In the regression analysis, we followed the intention-to-treat approach by including all dropouts in the analysis. However, we estimated further models with dropouts excluded to ensure robustness of the results. Multicollinearity was checked by calculating variance inflation factors. Values greater than 5 were considered to indicate multicollinearity [62]. Ethics Approval Without excluding dropouts (ie, intention-to-treat approach), preintervention knowledge in the control group was reflected by a mean of 17.73 (SD 3.72) points, and preintervention knowledge in the intervention group was reflected by a mean of 18.33 (SD 3.13) points; the difference was not statistically significant (t76.47=–0.79, 95% CI –2.11 to 0.91; P=.43). When dropouts were excluded, preintervention knowledge in the whole sample was reflected by a mean of 18.47 (SD 3.40) points. With dropouts excluded, preintervention knowledge in the control group was reflected by a mean of 18.28 (SD 3.76) points, and preintervention knowledge in the intervention group was reflected by a mean of 18.67 (SD 3.05) points. The difference between the control and intervention groups before the intervention was not statistically significant (t45.73=–0.40, 95% CI –2.35 to 1.58; P=.69). Postintervention knowledge was reflected by a mean of 18.08 (SD 3.60) points in the control group and 21.67 (SD 1.55) points in the intervention group. This difference of 3.59 points was statistically significant (t32.88=–4.56, 95% CI –5.19 to –1.98; power=0.99; P<.001). The pre- and postintervention knowledge levels by the control and intervention groups are displayed in Figure 3. This study was prospectively approved by the Ethics Committee of Friedrich-Alexander-Universität Erlangen-Nürnberg (reference No. 202_20 B) and retrospectively registered in the German Clinical Trials Register (DRKS00022935). All participants declared informed consent before the study after receiving patient information and the data privacy declaration. Figure 2. Flowchart of participants. https://www.jmir.org/2022/10/e36767 J Med Internet Res 2022 \| vol. 24 \| iss. 10 \| e36767 \| p. 6 (page number not for citation purposes) Results Out of 83 initial participants, 40 (48%) were assigned to the control group and 43 (52%) were assigned to the treatment group. In total, 15 participants from the control group and 19 from the intervention group were lost to follow-up because they did not fill out both questionnaires and were, thus, regarded as dropouts. This left a total of 49 participants—25 (51%) in the control group and 24 (49%) in the intervention group—who were analyzed (Figure 2). However, all outcome analyses are reported with and without dropouts in this section. The characteristics of the intervention and control group participants are displayed in Table 1; we did not find statistically significant differences between the control and intervention groups. Figure 2. Flowchart of participants. J Med Internet Res 2022 \| vol. 24 \| iss. 10 \| e36767 \| p. 5 https://www.jmir.org/2022/10/e36767 (page number not for citation purposes) •FO J Med Internet Res 2022 \| vol. 24 \| iss. 10 \| e36767 \| p. 5 (page number not for citation purposes) https://www.jmir.org/2022/10/e36767 https://www.jmir.org/2022/10/e36767 JOURNAL OF MEDICAL INTERNET RESEARCH Table 1. Characteristics of the study participants. P value χ2a (df) t testa (df) Full sample (N=49) Intervention group (n=24) Control group (n=25) Characteristics .55 N/Ab –0.60 (45.53) 26.41 (6.22) 26.96 (6.69) 25.88 (5.82) Age (years), mean (SD) .47 N/A 0.73 (43.91) 20.65 (7.24) 19.88 (8.07) 21.40 (6.42) Age at diagnosis (years), mean (SD) Sex, n (%) .49 0.5 (1) N/A 47 (96) 24 (100) 23 (92) Female — —c N/A 2 (4) 0 (0) 2 (8) Male Type of diagnosis, n (%) .64 0.2 (1) N/A 30 (61) 16 (67) 14 (56) Crohn disease — — N/A 19 (39) 8 (33) 11 (44) Ulcerative colitis Knowledge about IBDd, IBD-KNOWe score, mean (SD) .69 N/A –0.40 (45.73) 18.47 (3.40) 18.67 (3.05) 18.28 (3.76) Preintervention <.001 N/A –4.56 (32.88) 19.84 (3.31) 21.67 (1.55) 18.08 (3.60) Postintervention aThe t test (2-tailed) and chi-square test were used to measure the difference between the control and intervention groups. Table 1. Characteristics of the study participants. aThe t test (2-tailed) and chi-square test were used to measure the difference between the control and intervention groups. bN/A: not applicable; this test was not applied to this variable ( ) q w w v g p bN/A: not applicable; this test was not applied to this variable. Results 6 (page number not for citation purposes) XSL•FO RenderX JOURNAL OF MEDICAL INTERNET RESEARCH Blunck et al Results of the robustness test, including the participants who dropped out, confirmed our results: estimate of time × treatment = 3.21 (P=.01); adjusted R2=0.17; F7,90=3.83 (P=.001). The results can be found in Table S1 in Multimedia Appendix 3. variance in patients’ knowledge is explained by the control variables. After adding the independent variables, the R2 of model 2 shows that 18% of the variance of patients’knowledge is explained by the variables. The adjusted R2, which considers the number of control variables, in model 2 indicates that 13% of the variance is explained by model 2, a gain of 15 percentage points (pp) compared to model 1. The statistically significant F test values in model 2 indicate an overall significant model [61]. Qualitative feedback from participants was incorporated during the study. For example, participants noted that some story slides were difficult to read, as IBD can affect patients’ eyes. Therefore, story slides were designed in high contrast after this feedback. Furthermore, we received a lot of positive feedback. Participants regarded the interventions as useful and meaningful. They also noted that they learned a lot—especially newly diagnosed participants—and stated that these interventions should be much more common. Variance inflation factors were all well below the cutoff of 5, with a maximum in model 1 of 1.06 in age and a maximum in model 2 of 2.57 in the interaction term; this was expected, as the interaction was a linear combination of two other variables. Given these results, we do not consider multicollinearity to be a major problem in our analysis. Table 2. Difference-in-differences regression of knowledge about inflammatory bowel disease. Results cThe chi-square value and its related P value for a group are reported in the top row for that group. dIBD: inflammatory bowel disease. BD-KNOW: Inflammatory Bowel Disease Knowledge; scores range from 0 to 24 points. d postintervention knowledge by control and intervention groups. IBD-KNOW: Inflammatory Bowel Disease Knowledge. Figure 3. Levels of pre- and postintervention knowledge by control and intervention groups. IBD-KNOW: Inflammatory Bowel Disease Knowledge. Figure 3. Levels of pre- and postintervention knowledge by control and intervention groups. IBD-KNOW: Inflammatory Bowel Disease Knowledg The results of the OLS regression analysis are displayed in Table 2. Model 1 shows the baseline effect of the selected control variables on patients’ knowledge scores. Model 2 adds the time and group dummy variables, as well as the interaction term for these variables. The variable of interest is the interaction term, as it describes the main treatment effect. Patients in the treatment group increased their knowledge score by 3.07 point all other things being equal, compared to the control grou (P=.001; see Multimedia Appendix 2 for a visualization of th treatment effect). R2 represents the proportion of variance in the depende variable that is explained by the model In model 1 1% of th treatment group increased their knowledge score by 3.07 points, all other things being equal, compared to the control group (P=.001; see Multimedia Appendix 2 for a visualization of the treatment effect). treatment group increased their knowledge score by 3.07 points, all other things being equal, compared to the control group (P=.001; see Multimedia Appendix 2 for a visualization of the treatment effect). The results of the OLS regression analysis are displayed in Table 2. Model 1 shows the baseline effect of the selected control variables on patients’ knowledge scores. Model 2 adds the time and group dummy variables, as well as the interaction term for these variables. The variable of interest is the interaction term, as it describes the main treatment effect. Patients in the R2 represents the proportion of variance in the dependent variable that is explained by the model. In model 1, 1% of the https://www.jmir.org/2022/10/e36767 J Med Internet Res 2022 \| vol. 24 \| iss. 10 \| e36767 \| p. Results Model 2 Model 1 Variables and measures P value Value P value Value Control variables, estimated β coefficient (SE) <.001 19.72 (1.77) <.001 19.82 (1.87) Constant .23 –1.45 (1.19) .60 –0.68 (1.29) Female .79 0.16 (0.59) .59 0.34 (0.63) Crohn diseasea — Reference —b Reference Ulcerative colitisa .53 –0.03 (0.05) .49 –0.03 (0.05) Age .95 0.00 (0.05) .51 0.04 (0.06) Duration Independent variables, estimated β coefficient (SE) .69 0.33 (0.83) N/A N/Ac Timea .38 0.64 (0.72) N/A N/A Interventiona .001 3.07 (1.17) N/A N/A Time × intervention — 132 — 132 Observations, n — 0.18 — 0.01 R 2 — 0.17 N/A N/A Delta R2 — 0.13 — –0.02 Adjusted R2 — 0.15 N/A N/A Delta adjusted R2 <.001 3.889 (7, 124) .86 0.320 (4, 127) F test (df) a Table 2. Difference-in-differences regression of knowledge about inflammatory bowel disease. bNot calculated. knowledge. Therefore, this study provides evidence for the effectiveness of patient education via Instagram. https://www.jmir.org/2022/10/e36767 J Med Internet Res 2022 \| vol. 24 \| iss. 10 \| e36767 \| p. 7 (page number not for citation purposes) Principal Findings With a mean of 76.95% correct answers (mean score of 18.47 out of 24), our sample showed an already-high mean knowledge level at baseline, compared to other studies in this area. For example, Abutaleb et al [36] found 52.50% correct answers during the preintervention stage. Others found mean baseline knowledge levels of 26.67% (8/30) [63], 33.33% (8/24) [18], 40.67% (12.2/30) [37], 40.79% (9.79/24) and 48.25% (11.58/24) To answer the research question of whether educating adult patients with IBD via Instagram is effective, we conducted an RCT in a sample of 49 participants. After 5 weeks of training via Instagram stories, the intervention group yielded statistically significant and relatively higher levels of disease-related https://www.jmir.org/2022/10/e36767 J Med Internet Res 2022 \| vol. 24 \| iss. 10 \| e36767 \| p. 7 (page number not for citation purposes) XSL•FO RenderX XSL•FO RenderX Blunck et al JOURNAL OF MEDICAL INTERNET RESEARCH Blunck et al [38], and 62.90% (18.87/30) [64]. Along with the relatively high baseline knowledge level, our study showed an increase in mean disease-related knowledge by 12.50 pp. Other studies achieved increases of 10 pp with telemedicine and 7.5 pp with standard interventions [36], 25.33 pp with a CD-ROM program [37], and 32.13 pp with a formal education program and 14.79 pp with a standard intervention [38]. Hence, the knowledge increase presented in our study is on the lower bound compared to other interventions. However, the study designs are not comparable without restrictions, for example, because of different intensity and frequency of interventions. Furthermore, higher baseline values come with less improvement from educational interventions [36], which is reasonable due to a saturation effect and a natural upper limit of the knowledge scale. content, high levels of monitoring and interaction, or the use of Instagram ads to increase visibility. However, the latter mechanism, in particular, might bias results in the study setting and would be more suitable in a regular care setting. A difference in this study compared to previous studies is that participants in this study did not participate in dedicated trainings. This means that patients only received access to the Instagram account and were responsible for watching or actively participating. In classical patient educational interventions [38], patients actively participate in a training session, a physician visit, or similar. Principal Findings As it is not feasible in a regular care setting to ensure continued training via dedicated trainings, we contributed by providing a solution that is integrated into patients’ daily routines, without a cost to health care providers, and that can be used on a long-term and continued basis. Once educational material is designed and conceptualized, it could be used and reused in a large patient population. Compared to other, previously mentioned, ways of increasing patients’ disease-related knowledge, our approach is easy to implement, comes with good scalability, integrates educational content into patients’ lives, and addresses young people in particular. Furthermore, the proposed approach allows possibilities for patient organizations to closer engage with patients. Another application of educational social media interventions is the education of patients’ friends and family members. As those people are often affected or involved in the care of patients with chronic conditions, higher disease-related knowledge among friends and family members could also increase their understanding of patients’ situations and therapies, which subsequently would support patients. Furthermore, we contributed by providing a German translation of the IBD-KNOW questionnaire. The dropout rate in this study was 41% (34/83) and was, thus, relatively high compared to other studies; for example, one study found 25% loss to follow-up after 6 months and 26% loss to follow-up after 12 months [36], whereas another study found 16% dropout immediately after the intervention and 22% loss to follow-up after 8 weeks [38]. We believe that the high dropout rate in our study may be due to the fact that, in order to prevent forced results, we did not send reminders to the participants to complete the questionnaires. Although the dropout group did not differ from the included participants regarding parameters such as length of IBD history or prestudy knowledge, dropouts were significantly older than included participants. A reason for this observation might be that older patients might have lower computer literacy and, thus, were more likely to drop out. Hence, future studies could address this issue in further elaborating the interplay of age and learning via social media in patients with IBD. The unexpectedly high dropout rate ultimately led to a relatively low number of participants. This was not in line with the assumptions used for the power analysis. Principal Findings Future studies should take measures to either (1) expect a higher dropout rate and recruit a larger number of participants or (2) decrease the overall dropout rate. The latter may be achieved by using reminders or incentives. We did not take these measures in our study in order to reduce bias. Limitations Our study comes with several limitations. First, patient recruitment took place via the Instagram pages of a German patient organization. This might bias and underestimate results for the total relevant population because we assumed that the patient organization’s Instagram page was being followed by an already-interested audience. For example, studies found that patients who are members of a patient organizations yield higher knowledge scores than patients who are not [50]. Therefore, the knowledge levels of this respective sample might already be above average. On the other hand, however, one could argue that the sample of patients could be more highly motivated and have a higher willingness to learn due to their higher level of interest, which counteracts this effect. Additionally, the study setting may have led to another selection bias because young and computer-literate people, in particular, are Instagram users, which limits generalizability. Another limitation might arise from dropouts. As 34 participants were lost to follow-up, our overall findings might be biased if the dropout probability was associated with the knowledge score, specifically with learning. Due to the unexpectedly high dropout rate, the sample size of our study was relatively small. Inclusion of larger study populations might be beneficial in gaining a better understanding of our findings. Finally, we found a high proportion of women among the followers of the organization specific to patients with IBD on Instagram. This may suggest that men generally have different coping strategies for dealing with IBD than women. J Med Internet Res 2022 \| vol. 24 \| iss. 10 \| e36767 \| p. 8 (page number not for citation purposes) https://www.jmir.org/2022/10/e36767 Future Research This study recommends different questions for future research. First, patient education via Instagram or other social media should be directly compared with other means of patient education, in order to compare effectiveness in a head-to-head comparison. Second, the effectiveness of Instagram patient education should be tested in other chronic conditions as well. Third, the economic effects of patient education via Instagram—or social media in general—should be explored. Integration into patients’ daily routines might reduce costs for transportation to a training facility or physician. Additionally, patient education via social media, such as Instagram, is easy to scale and increases accessibility, which leads to lower costs at training facilities or for physicians. Fourth, before rolling out Instagram patient education in regular settings, quality requirements should be defined to enable systematic dissemination and prevent communication of misleading or false information to patients. To assert the sustainability of the effect of education via Instagram, further studies with a longer follow-up period are needed. However, the real-life setting of the proposed educational mode has a continuous character. This means that patients have continuous access to the educational material instead, for example, of a one-time visit at a seminar, which rather reduces the need for follow-up studies. Furthermore, previous studies found that the knowledge increase gained by patients with IBD stays relatively constant over time [37,38]. Additionally, we only considered German patients, which might reduce the generalizability of our results. Studies show that knowledge levels differ between countries [65]. Future studies should, therefore, focus on multicenter study designs or evaluate results across countries. Conclusions To test the effectiveness of patient training via Instagram, we conducted an RCT with 49 patients with IBD. The intervention group received access to an Instagram account, which posted educational material over 5 weeks. The outcome—patients’ knowledge about IBD—was measured at the pre- and postintervention stages using a questionnaire whose response scores ranged from 0 to 24 points. The intervention group yielded 3.59 more points than the control group, on average, after the intervention (P<.001), with no significant differences before the intervention. Therefore, we conclude that Instagram is an effective tool for educating patients and demonstrates large potential for future support of chronic conditions. The interest in the educational material in our study might be higher than in a real-life setting because of a trial effect. Patients might be interested more or might learn more because they know they are part of a study [66] and not blinded. Therefore, the effect might be overestimated. To validate the effectiveness of patient education via Instagram or other social media channels, further research (eg, observational studies) is needed. Acknowledgments We would like to thank CHRONISCH GLÜCKLICH e.V. for supporting the recruitment of participants. We acknowledge financial support by Deutsche Forschungsgemeinschaft and Friedrich-Alexander-Universität Erlangen-Nürnberg within the funding program Open Access Publication Funding. Conflicts of Interest JW worked during her master’s course—where this paper was initiated as a student project—for Roche Pharma AG as a patient partnership manager in the field of gastroimmunology. Roche did not provide any funding for the study and had no influence in initiating, planning, designing, and conducting the study; collection, analysis, or interpretation of data; the writing of the manuscript; or the decision to publish the paper. Multimedia Appendix 1 Inflammatory Bowel Disease Knowledge questionnair [DOCX File , 18 KB-Multimedia Appendix 1] Inflammatory Bowel Disease Knowledge questionnaire. [DOCX File , 18 KB-Multimedia Appendix 1] JOURNAL OF MEDICAL INTERNET RESEARCH Additionally, participants in this study were almost exclusively female. As the proportion of women among all patients with IBD is much lower [2], the generalizability of this study to the whole IBD population may be limited. However, the high proportion of women in our study is due to the demographic composition of Instagram followers of the patient organization with which we collaborated for recruitment. Authors' Contributions JW was responsible for conceptualization of the study, methodology, investigation, and writing, reviewing, and editing the paper. DB was responsible for conceptualization of the study; methodology; formal analysis; writing the original draft; reviewing and editing subsequent drafts; visualization; supervision; and project administration. LK and MN were responsible for writing the original draft and for reviewing and editing subsequent drafts. Blunck et al Blunck et al https://www.jmir.org/2022/10/e36767 Contribution To our knowledge, this was the first study to analyze the effect of patient training via Instagram on patients’ disease-related knowledge. One main contribution of our study is evidence for the effectiveness of patient education via Instagram. Future work in this area should focus on disseminating educational content in regular care. One major challenge for this could be quality assurance because everybody could publish apparent educational content without expert review. If health care providers actively use social media platforms in the future, a high level of quality in educational material could be ensured. Another challenge might be the long-term motivation of users. Potential ways to reduce retention issues are high-quality https://www.jmir.org/2022/10/e36767 XSL•FO RenderX XSL•FO RenderX Blunck et al JOURNAL OF MEDICAL INTERNET RESEARCH References Lancet Gastroenterol Hepatol 2020 Jan;5(1):17-30 [FREE Full text] [doi: 10.1016/S2468-1253(19)30333-4] [Medline: 31648971] 3. Liu S, Zhao W, Lan P, Mou X. The microbiome in inflammatory bowel diseases: From pathogenesis to therapy. Protein Cell 2021 May;12(5):331-345 [FREE Full text] [doi: 10.1007/s13238-020-00745-3] [Medline: 32601832] y 4. Park KT, Ehrlich OG, Allen JI, Meadows P, Szigethy EM, Henrichsen K, et al. 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J Med Internet Res 2022 \| vol. 24 \| iss. 10 \| e36767 \| p. 9 (page number not for citation purposes) J Med Internet Res 2022 \| vol. 24 \| iss. 10 \| e36767 \| p. 9 (page number not for citation purposes) J Med Internet Res 2022 \| vol. 24 \| iss. 10 \| e36767 \| p. 9 (page number not for citation purposes) https://www.jmir.org/2022/10/e36767 https://www.jmir.org/2022/10/e36767 XSL•FO RenderX JOURNAL OF MEDICAL INTERNET RESEARCH Blunck et al [PNG File , 6 KB-Multimedia Appendix 2] Multimedia Appendix 3 Difference-in-differences regression results (robustness check). [DOCX File , 17 KB-Multimedia Appendix 3] Multimedia Appendix 4 CONSORT-eHEALTH checklist (V 1.6.1). [PDF File (Adobe PDF File), 1184 KB-Multimedia Appendix 4] References 1. Guan Q. A comprehensive review and update on the pathogenesis of inflammatory bowel disease. J Immunol Res 2019;2019:7247238 [FREE Full text] [doi: 10.1155/2019/7247238] [Medline: 31886308] 2. GBD 2017 Inflammatory Bowel Disease Collaborators. 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[doi: 10.1136/bmj.h4672] [Medline: 26341898] J Med Internet Res 2022 \| vol. 24 \| iss. 10 \| e36767 \| p. 12 (page number not for citation purposes) https://www.jmir.org/2022/10/e36767 XSL•FO RenderX XSL•FO RenderX JOURNAL OF MEDICAL INTERNET RESEARCH Blunck et al J Med Internet Res 2022 \| vol. 24 \| iss. 10 \| e36767 \| p. 13 (page number not for citation purposes) Abbreviations CHERRIES: Checklist for Reporting Results of Internet E-Surveys CONSORT-EHEALTH: Consolidated Standards of Reporting Trials of Electronic and Mobile Health Applications and Online Telehealth IBD: inflammatory bowel disease IBD-KNOW: Inflammatory Bowel Disease Knowledge OLS: ordinary least squares pp: percentage points RCT: randomized controlled trial Edited by G Eysenbach; submitted 16.02.22; peer-reviewed by S Esworthy, FH Leung; comments to author 29.07.22; revised version received 12.09.22; accepted 19.09.22; published 19.10.22 Please cite as: Blunck D, Kastner L, Nissen M, Winkler J The Effectiveness of Patient Training in Inflammatory Bowel Disease Knowledge via Instagram: Randomized Controlled Trial J Med Internet Res 2022;24(10):e36767 URL: https://www.jmir.org/2022/10/e36767 doi: 10.2196/36767 PMID: Please cite as: Blunck D, Kastner L, Nissen M, Winkler J The Effectiveness of Patient Training in Inflammatory Bowel Disease Knowledge via Instagram: Randomized Controlled Trial J Med Internet Res 2022;24(10):e36767 URL: https://www.jmir.org/2022/10/e36767 doi: 10.2196/36767 PMID: ©Dominik Blunck, Lena Kastner, Michael Nissen, Jacqueline Winkler. Originally published in the Journal of Medical Internet Research (https://www.jmir.org), 19.10.2022. This is an open-access article distributed 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, first published in the Journal of Medical Internet Research, is properly cited. The complete bibliographic information, a link to the original publication on https://www.jmir.org/, as well as this copyright and license information must be included. https://www.jmir.org/2022/10/e36767 XSL•FO RenderX
https://openalex.org/W2067032659	https://www.frontiersin.org/articles/10.3389/fnhum.2014.01004/pdf	English	null	Your body, my body, our coupling moves our bodies	Frontiers in human neuroscience	2,014	cc-by	5,193	COUPLING AND AUTONOMY, THE TWO FACES OF COORDINATION )] Rhythmic coordination of movements emerges from the dynamics of interac- tion between multiple component pro- cesses (Kelso, 1995; see also Van Orden et al., 2003); those interactions bring forth a dynamical landscape that pre- orients behavior and that can be mod- ulated by intention (Kelso, 2002). For instance, rhythmic coordination of two limbs is driven by the dynamics of their relation, rather than by their sole intrin- sic properties (Kelso, 1984). Coordination of one limb to an external pacer is gov- erned by their relational dynamics as well (Kelso, 1981). Such dynamical interactions lead to coordination of activity across mul- tiple scales of brain and behavior (Ihlen and Vereijken, 2010; Kelso et al., 2013). Human subjects can embody both pluri- frequential rhythms (Toiviainen et al., 2010) and the complexity of their ﬂuc- tuations (Rankin et al., 2009; Marmelat et al., 2014). Thanks to the intrinsic com- plexity that underlies behavioral coordi- nation, coupling allows coordination with the environment across scales (Laroche et al., 2014). If relational dynamics play a role in rhythmic coordination they may also play a key role in social inter- action. Indeed, interacting with others is organized rhythmically and at multi- ple timescales since infancy (Gratier and Apter-Danon, 2009). The development Coordination is related to two comple- mentary aspects: autonomy and inter- active coupling (Clayton et al., 2004). Autonomy refers to the intrinsic laws of organization of a living system (Varela, 1979). In turn, such laws deﬁne the envi- ronment to which a living system can couple. Autonomy thus provides a system with a dynamical background in the con- text of which sensory perturbations occur; it provides a perspective that shapes the lived world. In turn, as a result of their modulation, internal dynamics carry the imprint of the system’s own environment. In other words, internal dynamics depend on agent∼world relational dynamics (i.e., they depend on the way the relation between agent and world evolves). In short, complex systems and their environ- ment co-determine each other. Patterns of coordination thus emerge dynami- cally from agent∼world coupling and are therefore both autonomous and rela- tional. By emergence, we mean that despite the fact that coordination is a by-product of intra-individual processes (Ross and Balasubramaniam, 2014), it is not reducible to them. Edited by: Gregory Bryant, University of California, Los Angeles, USA Keywords: coupling, coordination, interpersonal, dynamical systems, human interaction of interacting have been studied in ani- mals and humans (Strogatz, 2003), rang- ing from ﬂock behavior (Okubo, 1986) to language (Dale et al., 2013; Manson et al., 2013)]. intertwined in coordination phenomena, and outline existing methods to address those issues. Sensitivity to temporal contingencies appears early in life and plays a key role in the ontogeny of socio-cognitive abili- ties in humans (Nadel et al., 1999; Gratier and Apter-Danon, 2009). The tendency for rhythmic coordination, sometimes referred to as “entrainment,” requires sensory-motor coupling (Phillips-Silver et al., 2010). In most of the ﬁelds of cognitive science, action-perception and agent-world coupling views are replacing the classical stimulus-response dichotomy (Marsh et al., 2009; Silberstein and Chemero, 2012; Schilbach et al., 2013; Novembre and Keller, 2014). Such con- ceptual frameworks are well suited to study coordination phenomena as they emphasize the dynamical nature of cog- nition (Varela et al., 1993; Kelso, 1995; Buzsáki and Draguhn, 2004; Lehmann and Schönwiesner, 2014). Moreover, they leave room for the balance of autonomy, a cen- tral feature of complex biological systems, and interactive coupling, through which such systems relate to—and make sense of—their environment (Di Paolo, 2005; Barandiaran et al., 2009; Buhrmann et al., 2013). A naturalistic study of autonomy and coupling requires both embracing ecological situations and considering ﬁrst- person perspective. Furthermore, many social coordination phenomena cannot be observed in the laboratory without the interaction of at least two subjects. We propose to consider linking ﬁrst- and third-person measures, and even relate them across multiple interacting individu- als. We will discuss how these concepts are OPINION ARTICLE published: December 2014 doi: 10.3389/fnhum.2014.01004 Your body, my body, our coupling moves our bodies Guillaume Dumas 1,2,3, Julien Laroche 4 and Alexandre Lehmann 5,6,7 1 Institut Pasteur, Human Genetics and Cognitive Functions Unit, Paris, France 2 CNRS UMR3571 Genes, Synapses and Cognition, Institut Pasteur, Paris, France 3 Human Genetics and Cognitive Functions, University Paris Diderot, Sorbonne Paris Cité, Paris, France 4 Akoustic Arts, Paris, France 5 Department of Otolaryngology - Head and Neck Surgery, Faculty of Medicine, McGill University, Montreal, QC, Canada 6 Centre for Research on Brain, Language and Music, Montreal, QC, Canada 7 International Laboratory for Brain, Music and Sound Research, Montreal, QC, Canada Correspondence: guillaume.dumas@pasteur.fr Edited by: Jessica Phillips-Silver, Georgetown University Medical Center, USA Reviewed by: Gregory Bryant, University of California, Los Angeles, USA Keywords: coupling, coordination, interpersonal, dynamical systems, human interaction HUMAN NEUROSCIENCE 16 OPINION ARTICLE published: December 2014 doi: 10.3389/fnhum.2014.01004 Your body, my body, our coupling moves our bodies Guillaume Dumas 1,2,3, Julien Laroche 4 and Alexandre Lehmann 5,6,7 1 Institut Pasteur, Human Genetics and Cognitive Functions Unit, Paris, France 2 CNRS UMR3571 Genes, Synapses and Cognition, Institut Pasteur, Paris, France 3 Human Genetics and Cognitive Functions, University Paris Diderot, Sorbonne Paris Cité, Paris, France 4 Akoustic Arts, Paris, France 5 Department of Otolaryngology - Head and Neck Surgery, Faculty of Medicine, McGill University, Montreal, QC, Canada 6 Centre for Research on Brain, Language and Music, Montreal, QC, Canada 7 International Laboratory for Brain, Music and Sound Research, Montreal, QC, Canada Correspondence: guillaume.dumas@pasteur.fr Edited by: Jessica Phillips-Silver, Georgetown University Medical Center, USA Reviewed by: Gregory Bryant, University of California, Los Angeles, USA Keywords: coupling, coordination, interpersonal, dynamical systems, human interaction HUMAN NEUROSCIENCE 16 Guillaume Dumas 1,2,3*, Julien Laroche 4 and Alexandre Lehmann 5,6,7 1 Institut Pasteur, Human Genetics and Cognitive Functions Unit, Paris, France Frontiers in Human Neuroscience COUPLING AND AUTONOMY, THE TWO FACES OF COORDINATION Indeed, mutual interac- tion allows for more accurate/stable coordination than unilateral situations where only one partner is responsive to the other (Cummins, 2009; Konvalinka et al., 2010; Noy et al., 2011). Dynamics that are properly collective thus arise in mutual interaction, and they can attract and coordinate individual behaviors (De Jaegher and Di Paolo, 2007; Auvray et al., 2009; Lenay and Stewart, 2012; Laroche and Kaddouch, 2014). Such dynamics are therefore irreducible to purely intra- individual processes. Interacting subjects can yet rely on those relational dynamics; they can jointly regulate them. Overall, neither the dynamics of the interaction process nor their co-regulation can be observed when subjects are isolated from each other. It is thus important to study the dynamical properties of the interaction process itself in order to understand how we co-regulate them. More individually- centered processes of coordination might derive from recurrent social interactions. Recent methodological and technological advances make this change of paradigm possible. Over the last decade, social neuro- science took an interactive turn. Two-body and second-person neuroscience have especially been supporting the use of eco- logical paradigms for understanding the neural underpinning of social interac- tion (Schilbach et al., 2013). This ongoing interactive turn relies on the develop- ment of new methods. Hyperscanning, for instance, allows recording the brain activ- ity of multiple individuals engaged in an interaction. This approach already demon- strated differential effects of social context (e.g., induced/spontaneous, see Dumas et al., 2012a) and role (e.g., leader/follower, see Dumas et al., 2012a; Sänger et al., 2013; Konvalinka et al., 2014) during interpersonal coordination. Music is an ideal ecological context for the study of coordination and has been used in the burgeoning ﬁeld of social interaction neuroscience. The work of Lindenberger and colleagues has for instance revealed the inter-individual brain dynamics of joint improvisation (Müller et al., 2013) and how the global system should be described through both intra- and inter- individual processes (Sänger et al., 2012). An open question is how much does the observed inter-brain relationships rely on shared biological structure (Dumas et al., 2012b), task and environment (Burgess, 2013), or even cultural back- ground (Vogeley and Roepstorff, 2009; Kitayama and Park, 2010). For instance, heart rate coordination can be induced by a common task (e.g., singing the same song in Vickhoff et al., 2013) or socially modulated coupling (Konvalinka et al., 2011). COUPLING AND AUTONOMY, THE TWO FACES OF COORDINATION In this opin- ion, we emphasize the inter-individual dimension of coordination, especially in the case of the human speciﬁc activi- ties of music and dance [numerous ways December 2014 \| Volume 8 \| Article 1004 \| 1 Frontiers in Human Neuroscience Frontiers in Human Neuroscience www.frontiersin.org www.frontiersin.org Your body, my body, our coupling moves our bodies Dumas et al. (Toiviainen et al., 2010). Low-cost motion capture devices such as wireless accelerom- eters from video-game devices have been used to ecologically investigate inter- personal coordination between listeners dancing to music (De Bruyn et al., 2009). Recent analytical tools can deal with non-linear dynamics of movements and interpersonal coordination across multiple timescales [e.g., windowed-cross correlation (Boker et al., 2002); cross- wavelet transform (Issartel et al., 2007); frame-differencing methods (Paxton and Dale, 2013); cross-recurrence quantiﬁ- cation analysis (Coco and Dale, 2014; Demos et al., 2014); detrended cross- correlation analysis (Hennig, 2014); mul- tifractal detrended ﬂuctuation analysis (Bedia et al., 2014)]. Such behavioral mea- sures can discriminate between roles (i.e., leader/follower, Sacheli et al., 2013), indi- vidual strategies of regulation of coupling (Fairhurst et al., 2014), types of personal- ity (Schmidt et al., 1994) or can identify signatures of social disorders (Varlet et al., 2014). With tools grasping the complex- ity of movements, more ecological exper- iments are within reach. as EEG recordings (Debener et al., 2012; De Vos et al., 2014), allowing to exper- iment in contexts more ecological than the laboratory (e.g., a concert venue). As smartphones get powerful enough to pro- cess brain signals in real-time, conduct- ing in-ﬁeld or at-home EEG protocols is becoming feasible (Stopczynski et al., 2014). Because those systems are low-cost and yet can provide research-grade quality signals (Badcock et al., 2013), they can eas- ily scale up to record multiple participants. of our capacity to coordinate rhythmi- cally might lie in the dynamics of social interactions, rather than in purely intra- individual processes (Dumas, 2011): while we obviously need intra-individual capac- ities (e.g., vision) to handle social interac- tion, the development of our coordination capacities is shaped by those interactions as well (De Jaegher et al., 2010; Dumas et al., 2014b; Froese et al., 2014b) Two-body approaches—at least two participants interacting in real-time— demonstrate that interactive contexts bring forth different qualities of coordi- nation, in comparison with perceptive contexts in which participants’ behav- iors do not have any impact on their environment. COUPLING AND AUTONOMY, THE TWO FACES OF COORDINATION Neurocomputational modeling has already helped to measure the potential contribution of similarity at both anatom- ical and dynamical levels to our propensity to coordinate with others (Dumas et al., 2012b). Such empirically grounded Several innovations in brain-imaging methods can be readily applied to the study of rhythmic coordination in music and dance contexts. Through a careful design of control conditions, the use of ecological musical stimuli is possible in fMRI (Blood and Zatorre, 2001) as well as in EEG with the Steady-States Evoked Potential (SS-EP) technique. Traditional event-related potential approaches require numerous stimuli repetitions; hence stim- uli durations are usually kept to a minimum. With the SS-EP technique, a continuous stimulus such as music that has a periodic structure (or is frequency- tagged) can be presented and requires very few repetitions. It has been used success- fully to demonstrate neural oscillations underlying listening and tapping to syn- thetic beats (Nozaradan et al., 2011, 2012, 2013) and could be extended to study eco- logical musical beats. Adequately studying natural cognition may require the inte- gration of multiple modality and sensing techniques, while participants move freely (Makeig et al., 2009; Gramann et al., 2014). The recent years have seen the develop- ment of wearable devices for electrophys- iological (Codrons et al., 2014), as well Frontiers in Human Neuroscience DECIPHERING FACTORS ENABLING COORDINATION THROUGH AN ECOLOGICAL AND DYNAMICAL APPROACH Recent technological improvements made the continuous tracking of complex move- ments possible. For instance, whole-body motion capture through multi-camera set- tings showed that subjects can coor- dinate their movements simultaneously to multiple timescales of musical events December 2014 \| Volume 8 \| Article 1004 \| 2 Frontiers in Human Neuroscience Frontiers in Human Neuroscience www.frontiersin.org www.frontiersin.org Your body, my body, our coupling moves our bodies Dumas et al. environment and others have a causal role at the intra-individual level and non-additive consequences at the inter- individual level, and (2) deciphering the different factors of coupling across neural, behavioral and cultural scales. A last chal- lenge is to embrace the ongoing change of paradigm that shall bridge the gap between the lived and the observed experience of social coordination in ecological contexts. Taken together, integrating ﬁrst-, second- and third-person perspectives is a required move to accurately study natural human coordination phenomena. models can moreover be combined with experiments through human-machine interaction (Dumas et al., 2014a). This is especially interesting for operationalizing real time and reciprocal social interactions while keeping a rigorous experimental control (e.g., parametrically manipulate coupling). Burgess, A. P. (2013). On the interpretation of syn- chronization in EEG hyperscanning studies: a cau- tionary note. Front. Hum. Neurosci. 7:881. doi: 10.3389/fnhum.2013.00881 Buzsáki, G., and Draguhn, A. (2004). Neuronal oscillations in cortical networks. Science 304, 1926–1929. doi: 10.1126/science.1099745 Chapin, H., Jantzen, K., Kelso, J. S., Steinberg, F., and Large, E. (2010). Dynamic emotional and neu- ral responses to music depend on performance expression and listener experience. PLoS ONE 5:e13812. doi: 10.1371/journal.pone.0013812 p g) While at the objective level, many approaches have been proposed to relate intra- and inter-individual dynamics (Hasson et al., 2012; Konvalinka and Roepstorff, 2012; Dumas et al., 2014b), the link between the third (objective) and ﬁrst person (subjective) accounts remains unclear. How can interaction help escap- ing this dichotomy? We argue that closing this gap requires a joint study of intrinsic and relational dynamics. Introspection in experimental psychology has been heavily criticized in the past decades, but more rigorous approaches are now being designed to study subjective experience while overcoming previous limitations (Bockelman et al., 2013; Petitmengin and Lachaux, 2013). Recent work has managed to question the lived experience of the intersubjective dimension of coordination (Froese et al., 2014a). 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December 2014 \| Volume 8 \| Article 1004 \| 5 Frontiers in Human Neuroscience CONCLUSION This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduc- tion is permitted which does not comply with these terms. Schmidt, R. C., Christianson, N., Carello, C., and Baron, R. (1994). Effects of social and physical variables on between-person visual coordination. Ecol. Psychol. 6, 159–183. doi: 10.1207/s15326969 eco0603_1 Varela, F. J., Rosch, E., and Thompson, E. (1993). The Embodied Mind: Cognitive Science and Human Experience. Cambridge, MA: MIT press. Varlet, M., Marin, L., Capdevielle, D., Del-Monte, J., Schmidt, R. C., Salesse, R. N., et al. (2014). 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https://openalex.org/W4221073878	https://bit.mephi.ru/index.php/bit/article/download/1409/1271	Russian	null	Algorithms of network data analysis in the disclosure of a tax crime scheme	Bezopasnostʹ informacionnyh tehnologij	2,022	cc-by	6,585	р Ду , р ф , р руф АЛГОРИТМЫ СЕТЕВОГО АНАЛИЗА ДАННЫХ В РАСКРЫТИИ СХЕМЫ НАЛОГОВОГО ПРЕСТУПЛЕНИЯ Сергей В. Дуга1, Виктория В. Ефимова2, Андрей И. Труфанов3 1Судебно-экспертный центр Следственного комитета Российской Федерации, Строителей ул., 8, корпус 2, Москва, 119313, Россия 2Следственное управление Следственного комитета Российской Федерации по Иркутской области, Володарского ул., 11, Иркутск, 664011, Россия 3Иркутский национальный исследовательский технический университет, Лермонтова ул., 83, Иркутск, 664074, Россия 1e-mail: siber@list.ru, https://orcid.org/0000-0002-5894-9855 2e-mail: efimova.vika1977@mail.ru, https://orcid.org/0000-0003-3990-1917 3e-mail: troufan@gmail.com, https://orcid.org/0000-0002-6967-3495 Сергей В. Дуга1, Виктория В. Ефимова2, Андрей И. Труфанов3 1Судебно-экспертный центр Следственного комитета Российской Федерации, Строителей ул., 8, корпус 2, Москва, 119313, Россия 2Следственное управление Следственного комитета Российской Федерации по Иркутской области, Володарского ул., 11, Иркутск, 664011, Россия 3Иркутский национальный исследовательский технический университет, Лермонтова ул., 83, Иркутск, 664074, Россия 1e-mail: siber@list.ru, https://orcid.org/0000-0002-5894-9855 2e-mail: efimova.vika1977@mail.ru, https://orcid.org/0000-0003-3990-1917 3e-mail: troufan@gmail.com, https://orcid.org/0000-0002-6967-3495 Сергей В. Дуга, Виктория В. Ефимова, Андрей И. Труфанов Сергей В. Дуга, Виктория В. Ефимова, Андрей И. Труфанов АЛГОРИТМЫ СЕТЕВОГО АНАЛИЗА ДАННЫХ В РАСКРЫТИИ СХЕМЫ НАЛОГОВОГО ПРЕСТУПЛЕНИЯ DOI: http://dx.doi.org/10.26583/bit.2022.1.08 Аннотация. В статье рассматривается возможность применения средств сетевого (графового) анализа при раскрытии схемы налогового преступления и формировании стратегии его расследования. Предложена модель данных, позволяющая построить сетевую топологию преступления основываясь как на непосредственных материалах о событии преступления, так и на дополнительной информации, полученной путем доступа к базам данных правоохранительных и контрольных органов. Рассмотрены алгоритмы сетевого анализа, способствующие выявлению новых сведений о преступной схеме. Данные сетевые алгоритмы позволяют обнаружить скрытые и неочевидные связи между фигурантами дела, выявить иерархическую структуру их отношений, что способствует установлению ключевых участников преступной схемы. На примерах различных уголовных дел налоговых преступлений показано, что применение сетевого анализа данных позволяет сформировать схему преступления, дать правильную криминалистическую характеристику преступления, определить круг его субъектов, предложить следователю криминалистическую методику расследования данного вида преступлений (алгоритм расследования) и типовые следственные версии, что в совокупности способствует организации расследования должным образом. слова: сетевой анализ, налоговые преступления, схема налогового преступления, сетевого анализа, расследование преступлений. Ключевые слова: сетевой анализ, налоговые преступления, схема налогового преступления, алгоритмы сетевого анализа, расследование преступлений. Для цитирования: ДУГА, Сергей В.; ЕФИМОВА, Виктория В.; ТРУФАНОВ, Андрей И. АЛГОРИТМЫ СЕТЕВОГО АНАЛИЗА ДАННЫХ В РАСКРЫТИИ СХЕМЫ НАЛОГОВОГО ПРЕСТУПЛЕНИЯ. Безопасность информационных технологий, [S.l.], т. 29, № 1, с. 82–93, 2022. ISSN 2074-7136. URL: https://bit.mephi.ru/index.php/bit/article/view/1409. DOI: http://dx.doi.org/10.26583/bit.2022.1.08. Для цитирования: ДУГА, Сергей В.; ЕФИМОВА, Виктория В.; ТРУФАНОВ, Андрей И. АЛГОРИТМЫ СЕТЕВОГО АНАЛИЗА ДАННЫХ В РАСКРЫТИИ СХЕМЫ НАЛОГОВОГО ПРЕСТУПЛЕНИЯ. Безопасность информационных технологий, [S.l.], т. 29, № 1, с. 82–93, 2022. ISSN 2074-7136. URL: https://bit.mephi.ru/index.php/bit/article/view/1409. DOI: http://dx.doi.org/10.26583/bit.2022.1.08. Sergey V. Duga1, Viktoriya V. Efimova2, Andrey I. Trufanov3 1Forensic Expert Center of the Investigative Committee of the Russian Federation, Stroitelej str., 8, k. 2, Moscow, 119313, Russia 2Investigative Committee of the Russian Federation Irkutsk Region, Volodarskogo str., 11, Irkutsk, 664011, Russia 3Irkutsk National Research Technical University, Lermontova str., 83, Irkutsk, 664074, Russia 1e-mail: siber@list.ru, https://orcid.org/0000-0002-5894-9855 2e-mail: efimova.vika1977@mail.ru, https://orcid.org/0000-0003-3990-1917 3e-mail: troufan@gmail.com, https://orcid.org/0000-0002-6967-3495 DOI: http://dx.doi.org/10.26583/bit.2022.1.08 БЕЗОПАСНОСТЬ ИНФОРМАЦИОННЫХ ТЕХНОЛОГИЙ = IT Security, Том 29, № 1 (2022) 82 Сергей В. Дуга, Виктория В. Ефимова, Андрей И. Труфанов Сер е . Ду , ор . ф о , дре . руф о АЛГОРИТМЫ СЕТЕВОГО АНАЛИЗА ДАННЫХ В РАСКРЫТИИ СХЕМЫ НАЛОГОВОГО ПРЕСТУПЛЕНИЯ р Ду , р ф , р руф АЛГОРИТМЫ СЕТЕВОГО АНАЛИЗА ДАННЫХ В РАСКРЫТИИ СХЕМЫ НАЛОГОВОГО ПРЕСТУПЛЕНИЯ Abstract. The paper considers the possibility of using network (graph) analysis tools in the disclosure of a tax crime scheme and the formation of a strategy for its investigation. АЛГОРИТМЫ СЕТЕВОГО АНАЛИЗА ДАННЫХ В РАСКРЫТИИ СХЕМЫ НАЛОГОВОГО ПРЕСТУПЛЕНИЯ DOI: http://dx.doi.org/10.26583/bit.2022.1.08 A data model is proposed that allows building a network topology of a crime based both on direct material about a crime event and on additional information obtained by accessing the databases of law enforcement and control agencies. The algorithms of network analysis that contribute to the identification of new information about the criminal scheme are also considered. These network algorithms make it possible to detect hidden and non-obvious connections between the defendants in the case, to identify the hierarchical structure of their relationships, which helps to identify the key participants in the criminal scheme. Using the examples of various criminal cases of tax crimes, it is shown that the use of the considered variants of network data analysis allows forming a crime scheme, providing the correct criminalistic characterization of the crime, determining the range of its subjects, offering the investigator a forensic methodology for investigating this type of crime (investigation algorithm) and standard investigative versions, that all together contribute to the proper organization of the investigation. Keywords: mathematical modeling network analysis, tax crimes, tax crime scheme, network analysis algorithms, crime investigation. g g For citation: DUGA, Sergey V.; EFIMOVA, Viktoriya V.; TRUFANOV, Andrey I. Algorithms of network data analysis in the disclosure of a tax crime scheme. IT Security (Russia), [S.l.], v. 29, n. 1, p. 82–93, 2022. ISSN 2074-7136. URL: https://bit.mephi.ru/index.php/bit/article/view/1409. DOI: http://dx.doi.org/10.26583/bit.2022.1.08. Введение Расследование преступления – сложный интеллектуальный процесс, в котором задействованы множество механизмов. Специалист в процессе расследования сталкивается со значительным числом трудностей, главный из которых – ограниченность временных ресурсов для установления всех обстоятельств преступления и завершения расследования. У каждого вида преступлений своя специфика и особенности при выборе стратегии и тактики расследования. Вместе с тем выделяется категория «интеллектуальных» преступлений, раскрытие которых напрямую зависит от правильности сбора первичного аналитического материала и способностей его анализа. В их числе налоговые преступления. Предложенные в научной и практической литературе алгоритмы расследования налоговых преступлений не могут претендовать на всеохватность. Каждое преступление индивидуально, а применяемые криминальные схемы уникальны. Поэтому на первый план выходит способность подхода к сбору и анализу информации, формированию схемы преступления. От правильно построенной схемы налогового преступления зависит оперативность в расследовании и возможность применения превентивных мер к их совершению, обеспечение возмещения ущерба, причиненного государству. В настоящее время на практике следователь является единственным лицом, который фиксирует ход следствия в установленном законом порядке, проводит аналитику собранных доказательств и обеспечивает выявление новых составов преступлений. Многие правоохранительные и контрольные органы имеют специализированные подразделения, включающие группы лиц, занимающихся отдельными направлениями процесса по выявлению фактов уклонения от уплаты налогов, и осуществляющих аналитику. Следователь такой поддержки лишен, так как в структуре Следственного комитета РФ отсутствуют собственные оперативные подразделения, а также аналитические службы, которые могли бы облегчить следователю процесс выявления преступления и формирования доказательств. При этом сбор информации для анализа, позволяющий отнести его результаты к доказательствам, осуществляется следователем в отсутствие каких-либо специфических программных комплексов, в так называемом «ручном» режиме, что существенно растягивает во времени процесс расследования. у р р р р Доказывание начинается со сбора информации для формирования схемы преступления. На данном этапе важнейшее значение приобретает оперативность получения БЕЗОПАСНОСТЬ ИНФОРМАЦИОННЫХ ТЕХНОЛОГИЙ = IT Security, Том 29, № 1 (2022) 83 Сергей В. Дуга, Виктория В. Ефимова, Андрей И. Труфанов Сергей В. Дуга, Виктория В. Ефимова, Андрей И. Труфанов АЛГОРИТМЫ СЕТЕВОГО АНАЛИЗА ДАННЫХ В РАСКРЫТИИ СХЕМЫ НАЛОГОВОГО ПРЕСТУПЛЕНИЯ р Ду , р ф , др руф АЛГОРИТМЫ СЕТЕВОГО АНАЛИЗА ДАННЫХ В РАСКРЫТИИ СХЕМЫ НАЛОГОВОГО ПРЕСТУПЛЕНИЯ значимой информации из различных источников, формирование собственной базы данных, содержащей сведения об участии потенциальных субъектов схемы в преступной деятельности, связь таких субъектов с иными субъектами в схеме. Традиционно, налоговые преступления отличаются длительностью их совершения, системным характером преступных действий и их проработанностью. Сбор аналитического материала для выявления и расследования налогового преступления осуществляется из огромного числа источников, наиболее значимые из них – базы данных налоговых, регистрирующих органов, коммерческих банков, таможенных органов, судов. 1. Обзор работ, близких по тематике 1. Обзор работ, близких по тематике В настоящее время, со стороны исследователей всего мира, наблюдается рост интереса к применению новейших методов анализа данных для борьбы с преступностью в общем контексте [1–4], так и для расследования налоговых преступлений [5–7]. В настоящее время, со стороны исследователей всего мира, наблюдается рост интереса к применению новейших методов анализа данных для борьбы с преступностью в общем контексте [1–4], так и для расследования налоговых преступлений [5–7]. [ ], р р у [ В [8] предложена аналитическая система для выявления влиятельных членов преступной организации. Подход состоит из последовательных этапов: В [8] предложена аналитическая система для выявления влиятельных членов преступной организации. Подход состоит из последовательных этапов: 1) построение сети (сеть создается либо из данных мобильной связи, поддерживаемой преступной организацией, либо из отчетов о преступлениях, содержащих информацию о членах преступной организации), 1) построение сети (сеть создается либо из данных мобильной связи, поддерживаемой преступной организацией, либо из отчетов о преступлениях, содержащих информацию о членах преступной организации), 2) назначение веса каждой связи в сети (вес связи представляет собой количество телефонных звонков/сообщений между двумя преступниками), 3) вычисление кратчайшего пути по степени посредничества (мера, которая отражает значимость узла (вершины) при передаче информации из одной части сети в другую), ру у 4) присвоение оценки каждому узлу в сети на основе концепции зависимости существования. Преступники, представленные узлами (вершинами) высшего ранга, считаются влиятельными членами преступной организации. ру у 4) присвоение оценки каждому узлу в сети на основе концепции зависимости существования. Преступники, представленные узлами (вершинами) высшего ранга, считаются влиятельными членами преступной организации. В [9] представлена аналитическая система «ATTENet», предназначенная для обнаружения и объяснения подозрительных групп уклонения от уплаты налогов на основе аффилированных транзакций. Для решения задачи, во-первых, система создает сеть, которая включает данные о налогах и налогоплательщиках из официальной налоговой базы данных. Затем система объединяет основные характеристики и особенности структуры каждой группы в сети методом «Structure2Vec», после чего, с использованием алгоритма «Random Forest», обнаруживает подозрительные группы. Наконец, для изучения и объяснения результатов, система предоставляет визуализацию с интерактивными инструментами. Введение Получение информации из таких источников в настоящее время занимает значительное время, осуществляется в формате «запрос-ответ» в бумажном виде, имеет ограниченный формат. Впоследствии полученные сведения следователь аккумулирует и обобщает, составляя схему преступления. Применение средств сетевого анализа при раскрытии схемы налогового преступления и формировании стратегии и тактики расследования способно существенно облегчить процесс доказывания, оптимизировать работу следователя и сократить сроки расследования уголовных дел. 2. Используемые источники данных Для наполнения информационной системы сведениями, нами используются различные источники. Кратко перечислим их: БЕЗОПАСНОСТЬ ИНФОРМАЦИОННЫХ ТЕХНОЛОГИЙ = IT Security, Том 29, № 1 (2022) 84 Сергей В. Дуга, Виктория В. Ефимова, Андрей И. Труфанов р Ду , р ф , др руф АЛГОРИТМЫ СЕТЕВОГО АНАЛИЗА ДАННЫХ В РАСКРЫТИИ СХЕМЫ НАЛОГОВОГО ПРЕСТУПЛЕНИЯ − материалы уголовных дел. На текущий момент используется система «Pullenti» [10] для извлечения именованных сущностей и семантического анализа материалов уголовных дел, в частности протоколов допросов. На рис. 1 представлен пример такого анализа. Из первоначального текста извлекаются именованные сущности, а также, в результате семантического анализа, строится сетевая модель; Рис. 1. Пример анализа текста с использованием системы «Pullenti» Fig. 1. An example of text analysis using the “Pullenti” system Рис. 1. Пример анализа текста с использованием системы «Pullenti» Fig. 1. An example of text analysis using the “Pullenti” system − данные из мобильных телефонов. По результатам осмотров мобильных телефонов фигурантов уголовных дел формируются отчеты, которые, в последующем, загружаются в систему. Использование средств коммуникации, таких как телефонные звонки и мессенджеры, оставляют цифровые следы, которые можно использовать для анализа. Это позволяет следователям лучше понимать внутреннюю иерархию преступных организаций, обнаруживая субъектов, которые играют центральную роль и/или обеспечивают связь между подгруппами; − сведения от налоговых органов. Также в систему загружаются сведения из программ учета финансово- хозяйственной деятельности, полученные в ходе производства осмотров электронных носителей информации, данные из единой информационной системы в сфере закупок, результаты арбитражных процессов. р Ду , р ф , др руф АЛГОРИТМЫ СЕТЕВОГО АНАЛИЗА ДАННЫХ В РАСКРЫТИИ СХЕМЫ НАЛОГОВОГО ПРЕСТУПЛЕНИЯ В данном исследовании используется сетевая модель, которую можно представить в виде кортежа (V, E, L(V), T(E), X), представляющая собой неориентированный граф, вершины и ребра которого связаны с одной или несколькими метками, где: V – множество вершин, V – множество вершин, V – множество вершин, E – множество ребер, L(V) – сюръективное отображение (𝑣, l), которое связывает вершины с метками, такое, что каждой вершине 𝑣∈𝑉, соответствует хотя бы одна метка l ∈𝐿, L(V) – сюръективное отображение (𝑣, l), которое связывает вершины с метками, такое, что каждой вершине 𝑣∈𝑉, соответствует хотя бы одна метка l ∈𝐿, T(E) – сюръективное отображение (𝑒, t), которое связывает ребра с их типами, такое, что каждому ребру 𝑒∈𝐸 соответствует хотя бы один тип t ∈𝑇. T(E) – сюръективное отображение (𝑒, t), которое связывает ребра с их типами, такое, что каждому ребру 𝑒∈𝐸 соответствует хотя бы один тип t ∈𝑇. Кроме того, каждая вершина связана с соответствующим вектором признаков. Здесь X является матрицей признаков для графа (X𝑁×𝐷), так, что i-я строка X является вектором признаков для узла 𝑣𝑖(𝑖= 1,2 ….\|V \|). Кроме того, каждая вершина связана с соответствующим вектором признаков. Здесь X является матрицей признаков для графа (X𝑁×𝐷), так, что i-я строка X является вектором признаков для узла 𝑣𝑖(𝑖= 1,2 ….\|V \|). Для дальнейшего численного анализа определим матрицу смежности (𝐴𝑁×𝑁) данного графа, такую что: 3. Модель данных Налоговое преступление, как правило, представляет собой сложную систему – совокупность объектов и субъектов, взаимодействующих друг с другом нетривиальным образом. При расследовании данного вида преступлений важно проводить анализ сети в целом, не сосредоточивая внимание на отдельных субъектах. Такой тип оценки может существенно выиграть от применения сетевого анализа, когда лица рассматриваются как акторы, которые связаны друг с другом в рамках взаимозависимой системы. Чтобы изучить сложные системы в разных дисциплинах и областях, первым шагом является конкретное представление системы с использованием унифицированного математического языка. Кроме того, эти формализмы позволяют конструировать эффективные алгоритмы и могут использоваться для определения структуры, функции и динамики системы. Представляя исследуемые структуры в виде сетей, можно применять различные математические и сетевые методы для количественной оценки и выявления структурных особенностей. БЕЗОПАСНОСТЬ ИНФОРМАЦИОННЫХ ТЕХНОЛОГИЙ = IT Security, Том 29, № 1 (2022) 85 Сергей В. Дуга, Виктория В. Ефимова, Андрей И. Труфанов Сергей В. Дуга, Виктория В. Ефимова, Андрей И. Труфанов 𝐴𝑖𝑗 {1, если существует связь между вершинами 𝑖 и 𝑗 0, в ином случае Тип связи задается вручную, или определяется автоматически на основе анализа телефонных соединений, обмена сообщениями и иных источников. Для иллюстрации изложенного на рис. 2 приведена часть концептуальной модели данных, сформированной в графовой базе данных «Neo4j». Рис. 2. Часть концептуальной модели данных Fig. 2. Part of the conceptual data model Рис. 2. Часть концептуальной модели данных Fig. 2. Part of the conceptual data model ис. 2. Часть концептуальной модели данных Сеть строится на основе данных, получаемых в рамках расследования уголовного дела, предполагая, как ручное внесение данных, так и автоматическое извлечение сведений из различных источников. 𝐴𝑖𝑗 {1, если существует связь между вершинами 𝑖 и 𝑗 0, в ином случае Кроме того, используются двудольные сети для разделения неоднородного графа на однородные подграфы, с целью последующего анализа. В двудольных графах вершины разбиты на два непересекающихся подмножества, так, что связи (ребра) могут возникать только в том случае, если вершины принадлежат разным множествам. Применение двудольных сетей (графов) может использоваться для представления связей субъект- организация, когда один тип представляет исходные узлы (например, люди), а другой представляет группы, к которым принадлежит первый тип узлов (например, организация). Двудольная сеть и ее граф определяется матрицей инцидентности (𝐴𝑁×𝐺). Например, если имеется n субъектов и g организаций: 𝐴𝑖𝑗 {1, если 𝑗 субъект принадлежит к организации 𝑖 0, в ином случае 𝐴𝑖𝑗 {1, если 𝑗 субъект принадлежит к организации 𝑖 0, в ином случае 0, в ином случае Основываясь на предложенных в [11, 12] моделях, узлы используются для представления, таких сущностей как: р у – Субъекты. Физические лица, имеющие отношение к расследуемому уголовному делу. – Субъекты. Физические лица, имеющие отношение к расследуемому уголовному делу. – Организации. Юридические лица, имеющие отношение к расследуемому уголовному делу. – Организации. Юридические лица, имеющие отношение к расследуемому уголовному делу. – События – время, место, способ и другие обстоятельства совершения преступления, обстоятельства, способствовавшие совершению преступления (УПК РФ статья 73), а также иные обстоятельства, имеющие значение для уголовного дела (встречи людей, телефонные звонки, передача данных и пр.). – Объектами могут быть любые предметы, которые служили орудиями, оборудованием или иными средствами совершения преступления, предметы и документы, которые могут служить средствами для обнаружения преступления и установления обстоятельств уголовного дела (УПК РФ статья 81). – Места – место совершения преступления, домашний/рабочий адрес человека, адрес регистрации юридического лица и пр. – Места – место совершения преступления, домашний/рабочий адрес человека, адрес регистрации юридического лица и пр. В зависимости от типов узлов используются различные связи между ними, например, связь между организацией и физическим лицом, а также между организациями, может быть установлена из программ учета финансово-хозяйственной деятельности В зависимости от типов узлов используются различные связи между ними, например, связь между организацией и физическим лицом, а также между организациями, может быть установлена из программ учета финансово-хозяйственной деятельности БЕЗОПАСНОСТЬ ИНФОРМАЦИОННЫХ ТЕХНОЛОГИЙ = IT Security, Том 29, № 1 (2022) 86 Сергей В. Дуга, Виктория В. Ефимова, Андрей И. Труфанов р Ду , р ф , др руф АЛГОРИТМЫ СЕТЕВОГО АНАЛИЗА ДАННЫХ В РАСКРЫТИИ СХЕМЫ НАЛОГОВОГО ПРЕСТУПЛЕНИЯ организаций, сведений, полученных из налогового органа или вручную. В свою очередь, связь между физическими лицами может быть охарактеризована различными типами: родственники, коллеги, частое общение, общаются редко. 5. Прогноз существования связи Данную задачу можно сформулировать следующим образом: Если имеется набор данных в виде сети 𝐺 = (𝑉, 𝐸), где V – множество узлов, E – множество наблюдаемых связей, то задача заключается в том, чтобы предсказать, насколько вероятно существование ненаблюдаемой связи (𝑒𝑖𝑗∉𝐸) между произвольной парой узлов 𝑣𝑖, 𝑣𝑗. Для решения подобных задач используются различные методы [13–15]. Одним из таких методов является метод «Общие соседи» (Common Neighbors) [16]. В основе данного метода лежит предположение, что два узла с большой вероятностью связаны между собой (они будут связаны в ближайшем будущем), если у них много общих соседей: 𝑠𝑐𝑜𝑟𝑒(𝑥, 𝑦) = \|Г(𝑥) ∩Г(𝑦)\|, 𝑠𝑐𝑜𝑟𝑒(𝑥, 𝑦) = \|Г(𝑥) ∩Г(𝑦)\|, е Г(x) –набор узлов, смежных с узлом x, а Г(y) –набор узлов, смежных с узлом y. ( р у у (y р у у y Применение данного метода целесообразно ввиду того, что, зачастую, у следователя есть только частичная информация для анализа. Применение данного метода целесообразно ввиду того, что, зачастую, у следователя есть только частичная информация для анализа. Несмотря на свою простоту, метод хорошо работает в большинстве реальных сетей и превосходит сложные подходы [17]. Например, при расследовании уголовного дела по факту хищения бюджетных средств путем незаконного возмещения НДС с деятельности ООО Б. в особо крупном размере решение о потенциальному круге фигурантов уголовного дела (Р., Ф., С.) сделан следователем с учетом данных о связях лица Р., заявленного в уставных документах в качестве руководителя организации, с иными лицами, хотя и не имеющими официального отношения к Обществу, но фактически являющимися его руководителями. В ходе расследования были получены сведения от налогового органа о лицах, заявленных в качестве работников ООО Б., сведения из базы ЕГРЮ, сведения от кредитных организаций по месту открытия расчетных счетов общества. На основании полученных сведений была построена сетевая модель преступления и применен метод прогноза существования связи, который показал высокую вероятность наличия связи между ООО Б. и физическими лицами Ф. и С. 6. Использование метрики «центральность по собственному вектору» Целью данной метрики является определение наиболее значимых фигурантов уголовного дела путем присваивания веса каждой вершине в сети посредствам использования алгоритмов центральности [18–21]. На основе предложенной модели вычисляются степени значимости (определение важности отдельных узлов в сети) как классическими методами сетевого анализа, так и методами, адаптированными для предметной области (предварительное следствие). Основная цель этой оценки – выявить те субъекты, которые с большей вероятностью будут причастны к деятельности по уклонению от уплаты налогов. Это особенно полезно, когда нет надежной информации, которая позволила бы идентифицировать соответствующих фигурантов. 4. Используемые алгоритмы сетевого анализа Анализ отношений между физическими и юридическими лицами представляет собой выявление и определение степени (важности) личных связей, имущественных прав. Такие связи могут быть как формальными, так и неформальными. Формальные связи легко проверить с помощью соответствующих документов и реестров. Неформальными связи можно определить на основе различных данных, полученных в ходе расследования преступления, например сведения о телефонных соединениях или финансовые операции. Анализ, основанный на различных источниках данных, также позволяет определить роль отдельных узлов во всей сети. В ходе анализа связей необходимо, в частности, выявить следующие признаки аффилированности: В ходе анализа связей необходимо, в частности, выявить следующие признаки аффилированности: − участие одних и тех же лиц в создании, управлении организациями, в том числе через родственников или подконтрольных лиц; − пересечение по работе в одной и той же организации, т.е. либо одновременно являются работниками организации, либо работали в ней в разное время; овпадение адресов государственной регистрации организаций. − совпадение адресов государственной регистрации организаций. БЕЗОПАСНОСТЬ ИНФОРМАЦИОННЫХ ТЕХНОЛОГИЙ = IT Security, Том 29, № 1 (2022) 87 Сергей В. Дуга, Виктория В. Ефимова, Андрей И. Труфанов р Ду , р ф , др руф АЛГОРИТМЫ СЕТЕВОГО АНАЛИЗА ДАННЫХ В РАСКРЫТИИ СХЕМЫ НАЛОГОВОГО ПРЕСТУПЛЕНИЯ ьшее собственное значение матрицы смежности), 𝑥 собственный вектор этой р В рамках выше описанного примера, расследования уголовного дела по факту хищения бюджетных средств путем незаконного возмещения НДС с деятельности ООО Б. в особо крупном размере, решение об организаторе преступления и его исполнителях, сделано следователем с учетом информации о центральности указанных фигур в связи с большим количеством фактов хозяйственной деятельности ООО Б. по сравнению с иными потенциальными субъектами преступления: участие в создании ООО Б. и их аффилированных организаций, использованных в схеме преступления (из информации регистрационных дел налогового органа); представление интересов ООО Б. и аффилированных к нему организаций, участвующих в схеме преступления во взаимодействии с налоговыми органами (информация налогового органа по запросу следственного органа, анализ осмотра документов, изъятых в ходе обысков, выемок); участие в распоряжении денежными средствами ООО Б. и аффилированных к нему организаций, участвующих в схеме преступления (по данным юридических дел кредитных организаций); осуществление деятельности по адресу отправления налоговой отчетности по системе телекоммуникационной связи в налоговых орган (по данным налогового органа по IP-адресам отправки налоговой отчетности указанных организаций); наличие сведений о согласовании текущих вопросов деятельности ООО Б. и аффилированных к нему организаций, участвующих в схеме преступления (информация по результатам осмотров документов учета, изъятых в ходе обыска, выемок). 5. Прогноз существования связи Выявление влиятельных членов преступной организации – одна из важнейших задач, которую берут на себя следователи по уголовным делам. Возможное решение – выявить наиболее значимых субъектов при помощи алгоритма центральности собственного вектора на основе матрицы смежности (Eigenvector Centrality) [22]. Оценка центральности (значимости) вершины пропорциональна сумме оценок всех вершин, соединенных с ней. Таким образом, если вершина соединена со многим значимыми вершинами, она также будет считаться значимой. Математически это можно выразить уравнением 𝐴𝑥= λ𝑥, где А – матрица смежности с собственным значением λ (согласно теореме Перрона-Фробениуса БЕЗОПАСНОСТЬ ИНФОРМАЦИОННЫХ ТЕХНОЛОГИЙ = IT Security, Том 29, № 1 (2022) 88 Сергей В. Дуга, Виктория В. Ефимова, Андрей И. Труфанов р Ду , р ф , др руф АЛГОРИТМЫ СЕТЕВОГО АНАЛИЗА ДАННЫХ В РАСКРЫТИИ СХЕМЫ НАЛОГОВОГО ПРЕСТУПЛЕНИЯ λ – наибольшее собственное значение матрицы смежности), 𝑥 собственный вектор этой матрицы. 7. Поиск изоморфного подграфа где a – множество смежных вершин интересующей организацией, b – множество смежных вершин организации (одной из), полученной на шаге 3 описанного выше алгоритма, c – пересечение этих множеств. Применение данного алгоритма, в ходе расследования уголовного дела по факту уклонения от уплаты налогов с деятельности ООО Ж. в особо крупном размере путем необоснованного применения льготы по уплате НДС, позволило выявить признаки подконтрольности девяти организаций, формально не имеющих отношений к деятельности ООО Ж., однако, фактически с ним связанных. На основании информации, полученной из регистрирующего органа по результатам анализа документов регистрационных дел указанных организаций, установлено, что участниками указанных организаций являются лица, ранее являвшиеся административными работниками ООО Ж.; указанные организации также как и ООО Ж. имеют один адрес государственной регистрации; из информации, полученной из налогового органа установлено, что все организации имеют связь по месту осуществления бухгалтерского учета, формирования и направления налоговой отчетности; идентичными оказались и сведения о месте выхода в сеть по системе электронных расчетов Банк-Клиент всех указанных организаций, включая ООО Ж. Выявленные связи позволили сделать вывод о наличии подозрительной группы, состоящей из десяти субъектов (ООО Ж. и девять выше указанных организаций), использованных в преступной схеме. 7. Поиск изоморфного подграфа Многие реальные случаи уклонения от уплаты налогов реализуются посредством взаимодействия нескольких субъектов. Задача состоит в том, чтобы на основе сетевой онтологии, описывающей сетевые шаблоны подозрительной деятельности, извлекать топологическую информацию из комплексной сети, также известную как подграфы в анализе графов. Использование данного метода позволяет характеризовать, в частности, социальные связи, экономические взаимоотношения. Для примера приведём некоторые критерии аффилированности субъектов предпринимательской деятельности: − участие одних и тех же лиц в создании, управлении организациями, в том числе через родственников или подконтрольных лиц; − участие одних и тех же лиц в создании, управлении организациями, в том числе через родственников или подконтрольных лиц; − большая часть операций по расчетному счету приходится на взаимоотношения с аффилированной организацией; − большая часть операций по расчетному счету приходится на взаимоотношения с аффилированной организацией; один адрес государственной регистрации. − один адрес государственной регистрации. Применения алгоритмов поиска сетевых шаблонов широко используется для выявления подозрительных групп при уклонении от уплаты налогов [23–27]. Применения алгоритмов поиска сетевых шаблонов широко используется для выявления подозрительных групп при уклонении от уплаты налогов [23–27]. На основе используемой онтологии (рис. 3) сформулированы запросы на графовом языке запросов «Cypher». Используя данные запросы, можно, в частности, получить организации, связанные с интересующей организацией опосредованно. Данный алгоритм можно описать следующим образом: 1. указать интересующую организацию; 2. получить организации, связанные с интересующей организацией на удалении например, до трех перемещений (прыжков); 3. для каждой организации, полученной на шаге 2, найти смежные вершины; 4. для каждого множества смежных вершин, полученных на шаге 3, вычислить коэффициент сходства Жаккарда с множеством смежных вершин интересующей организации. БЕЗОПАСНОСТЬ ИНФОРМАЦИОННЫХ ТЕХНОЛОГИЙ = IT Security, Том 29, № 1 (2022) 89 Сергей В. Дуга, Виктория В. Ефимова, Андрей И. Труфанов р Ду р ф р руф АЛГОРИТМЫ СЕТЕВОГО АНАЛИЗА ДАННЫХ В РАСКРЫТИИ СХЕМЫ НАЛОГОВОГО ПРЕСТУПЛЕНИЯ Рис. 3. Часть используемой онтологии Рис. 3. Часть используемой онтологии Рис. 3. Часть используемой онтологии у Fig. 3. Part of the ontology used Fig. 3. Part of the ontology used Fig. 3. Part of the ontology used Коэффициент Жаккара (Jaccard Similarity) [28] в настоящее время является наиболее часто используемой мерой подобия в контексте анализа поведенческих связей, сходства между преступлениями. Привлекательность данного метода, отчасти, заключается в его простоте. Математически его можно выразить уравнением: 𝑐 𝐽= 𝑐 𝑎+𝑏−𝑐, й где a – множество смежных вершин интересующей организацией, b – множество смежных вершин организации (одной из), полученной на шаге 3 описанного выше алгоритма, c – пересечение этих множеств. СПИСОК ЛИТЕРАТУРЫ: 1. Bogahawatte K., Adikari S. 8th International Conference on Computer Science & Education. Intelligent criminal identification system, Colombo, Sri Lanka. 2013, p. 633–638. DOI: http://dx.doi.org/10.1109/ICCSE.2013.6553986. 1. Bogahawatte K., Adikari S. 8th International Conference on Computer Science & Education. Intelligent criminal identification system, Colombo, Sri Lanka. 2013, p. 633–638. DOI: http://dx.doi.org/10.1109/ICCSE.2013.6553986. 2. Hamdy E., Adl A., Hassanien A.E., Hegazy O. and Kim T. -H. Criminal Act Detection and Identification Model, 2015 Seventh International Conference on Advanced Communication and Networking (ACN). 2015, p. 79–83. DOI: http://dx.doi.org/10.1109/ACN.2015.30. 2. Hamdy E., Adl A., Hassanien A.E., Hegazy O. and Kim T. -H. Criminal Act Detection and Identification Model, 2015 Seventh International Conference on Advanced Communication and Networking (ACN). 2015, p. 79–83. DOI: http://dx.doi.org/10.1109/ACN.2015.30. p p g 3. Nath S.V. Crime Pattern Detection Using Data Mining, 2006 IEEE/WIC/ACM International Conference on Web Intelligence and Intelligent Agent Technology Workshops. 2006, p. 41–44. DOI: http://dx.doi.org/10.1109/WI-IATW.2006.55. p p g 3. Nath S.V. Crime Pattern Detection Using Data Mining, 2006 IEEE/WIC/ACM International Conference on Web Intelligence and Intelligent Agent Technology Workshops. 2006, p. 41–44. DOI: http://dx.doi.org/10.1109/WI-IATW.2006.55. p g 4. Saravanan P., Selvaprabu J., Arun Raj L., Abdul Azeez Khan A., Javubar Sathick K. (2021) Survey on Crime Analysis and Prediction Using Data Mining and Machine Learning Techniques. In: Zhou N., Hemamalini S. (eds) Advances in Smart Grid Technology. Lecture Notes in Electrical Engineering, vol 688. Springer, Singapore. DOI: http://dx.doi.org/10.1007/978-981-15-7241-8_31. 5. Jihal H., Ounacer S., Ardchir S., Azouazi M. (2020) Clustering Model of False Positive Elimination in Moroccan Fiscal Fraud Detection. In: Ezziyyani M. (eds) Advanced Intelligent Systems for Sustainable Development (AI2SD’2019). Advances in Intelligent Systems and Computing, vol 1104. Springer, Cham. DOI: http://dx.doi.org/10.1007/978-3-030-36671-1_12. 6. Wu Y., Dong B., Zheng Q., Wei R., Wang Z. and Li X. A Novel Tax Evasion Detection Framework via Fused Transaction Network Representation, 2020 IEEE 44th Annual Computers, Software, and Applications Conference (COMPSAC). 2020, p. 235–244. DOI: http://dx.doi.org/10.1109/COMPSAC48688.2020.00039. ( ) p p g 7. Didimo W., Grilli L., Liotta G., Menconi L., Montecchiani F. and Pagliuca D. Combining Network Visualization and Data Mining for Tax Risk Assessment, in IEEE Access. Vol. 8, p. 16073–16086, 2020. DOI: http://dx.doi.org/10.1109/ACCESS.2020.2967974. p g 8. Taha K., Yoo P.D. Using the Spanning Tree of a Criminal Network for Identifying Its Leaders, in IEEE Transactions on Information Forensics and Security. Vol. 12, no. 2, p. 445–453, 2017. DOI: http://dx.doi.org/10.1109/TIFS.2016.2622226. p g 9. Zheng Q. et al. р Ду , р ф , др руф АЛГОРИТМЫ СЕТЕВОГО АНАЛИЗА ДАННЫХ В РАСКРЫТИИ СХЕМЫ НАЛОГОВОГО ПРЕСТУПЛЕНИЯ р Ду , р ф , др руф АЛГОРИТМЫ СЕТЕВОГО АНАЛИЗА ДАННЫХ В РАСКРЫТИИ СХЕМЫ НАЛОГОВОГО ПРЕСТУПЛЕНИЯ Предложенные алгоритмы позволяют анализировать собранную в рамках расследования информацию, включая сведения электронных баз данных учета налогоплательщика, данные электронных переписок, телефонных переговоров, соединений конкретных лиц, пр., позволяют при наличии межведомственного взаимодействия оперативно получать запрашиваемую информацию у различных источников (налоговые органы, органы внутренних дел, иные контрольные и правоохранительные органы), имеют существенное практическое значение для выявления и расследования налоговых преступлений, поскольку позволяют формировать схему налогового преступления, изменять ее с учетом новой криминалистической информации, обрабатываемой с применением информационных технологий, и на ее основе выдвинуть обоснованную и максимально достоверную в соответствии с установленным набором входных данных следственную версию события преступления. Заключение На примере расследования реальных уголовных дел продемонстрирована эффективность применения используемых алгоритмов в рассматриваемой предметной области. Показано, что использование алгоритмов сетевого анализа, таких как поиск изоморфного подграфа, предсказания связи, а также методов анализ социальных сетей, в частности определение наиболее значимых фигурантов, может способствовать значительному сокращению сроков расследования уголовного дела, выявить неочевидные/скрытые факты. БЕЗОПАСНОСТЬ ИНФОРМАЦИОННЫХ ТЕХНОЛОГИЙ = IT Security, Том 29, № 1 (2022) 90 Сергей В. Дуга, Виктория В. Ефимова, Андрей И. Труфанов р Ду , р ф , др руф АЛГОРИТМЫ СЕТЕВОГО АНАЛИЗА ДАННЫХ В РАСКРЫТИИ СХЕМЫ НАЛОГОВОГО ПРЕСТУПЛЕНИЯ р Ду , р ф , др руф АЛГОРИТМЫ СЕТЕВОГО АНАЛИЗА ДАННЫХ В РАСКРЫТИИ СХЕМЫ НАЛОГОВОГО ПРЕСТУПЛЕНИЯ Bulletin del la Socit Vaudoise des Sciences Naturelles, no. 37, p. 547–579. DOI: http://dx.doi.org/10.5169/seals-266450. р Ду , р ф , др руф АЛГОРИТМЫ СЕТЕВОГО АНАЛИЗА ДАННЫХ В РАСКРЫТИИ СХЕМЫ НАЛОГОВОГО ПРЕСТУПЛЕНИЯ 14. Zhou T., Lü L., Zhang Y.C. Predicting missing links via local information. The European Physic Vol. 71, no. 4, p. 623–630, 2009. DOI: http://dx.doi.org/10.1140/epjb/e2009-00335-8. 15. Barabâsi A.L. et al. Evolution of the social network of scientific collaborations. Physica A: Statistical mechanics and its applications. Vol. 311, no. 3-4, p. 590–614, 2002. DOI: http://dx.doi.org/10.1016/S0378-4371(02)00736-7. 16. Newman M.E.J. Clustering and preferential attachment in growing networks. Physical review. 2001. DOI: http://dx.doi.org/10.1103/PhysRevE.64.025102. 17. Martínez V., Berzal F., Cubero J C. A survey of link prediction in complex networks. ACM computing surveys (CSUR). Vol. 49, no. 4, p. 1–33, 2016. DOI: http://dx.doi.org/10.1145/3012704. 18. Rungsawang A., Manaskasemsak B. An Efficient Partition-Based Parallel PageRank Algorithm. Proceedings of the 11th International Conference Parallel and Distributed Computing, 2004. DOI: http://dx.doi.org/10.1109/ICPADS.2005.85. 19. Brandes U. A faster algorithm for betweenness centrality. Journal of mathematical sociology. Vol. 25, no. 2, p. 163–177, 2001. DOI: http://dx.doi.org/10.1080/0022250X.2001.9990249. Ü p p g 20. Sariyüce A.E., Kaya K., Saule E and Çatalyiirek Ü.V. Incremental algorithms for closeness centrality, 2013 IEEE International Conference on Big Data. 2013, p. 487–492. DOI: http://dx.doi.org/10.1109/BigData.2013.6691611. 21. Bihari A. and Pandia M.K. Eigenvector centrality and its application in research professionals' relationship network, 2015 International Conference on Futuristic Trends on Computational Analysis and Knowledge Management (ABLAZE). 2015, p. 510–514. DOI: http://dx.doi.org/10.1109/ABLAZE.2015.7154915. 22. Li X. et al. Identifying social influence in complex networks: A novel conductance eigenvector centrality model. Neurocomputing. Vol. 210, p. 141–154, 2016. DOI: http://dx.doi.org/10.1016/j.neucom.2015.11.123. 23. Didimo W. et al. Visual querying and analysis of temporal fiscal networks. Information Sciences p. 406–421, 2019. DOI: http://dx.doi.org/10.1016/j.ins.2019.07.097. 24. Ruan J. et al. Identifying suspicious groups of affiliated-transaction-based tax evasion in big data. Information Sciences. Vol. 477, p. 508–532, 2019. DOI: http://dx.doi.org/10.1016/j.ins.2018.11.008. 25. Jihal H., Ounacer S., Ardchir S., Azouazi M. (2020) Clustering Model of False Positive Elimination in Moroccan Fiscal Fraud Detection. In: Ezziyyani M. (eds) Advanced Intelligent Systems for Sustainable Development (AI2SD’2019). Advances in Intelligent Systems and Computing, vol. 1104. Springer, Cham. DOI: https://doi.org/10.1007/978-3-030-36671-1_12. p g 26. Adamov A.Z. IEEE 13th International Conference on Application of Information and Communication Technologies (AICT). 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DOI: http://dx.doi.org/10.5169/seals-266450. Поступила в редакцию – 31 октября 2021 г. Окончательный вариант – 01 марта 2022 г. Received – October 31, 2021. The final version – March 01, 2022. Поступила в редакцию – 31 октября 2021 г. Окончательный вариант – 01 марта 2022 г. Received – October 31, 2021. The final version – March 01, 2022. Поступила в редакцию – 31 октября 2021 г. Окончательный вариант – 01 марта 2022 г. Received – October 31, 2021. The final version – March 01, 2022. REFERENCES: [1] Bogahawatte K., Adikari S. 8th International Conference on Computer Science & Education. Intellige identification system, Colombo, Sri Lanka. 2013, p. 633–638. DOI: http://dx.doi.org/10.1109/ICCSE.201 [1] Bogahawatte K., Adikari S. 8th International Conference on Computer Science & Education. Intelligent criminal identification system, Colombo, Sri Lanka. 2013, p. 633–638. DOI: http://dx.doi.org/10.1109/ICCSE.2013.6553986. [2] Hamdy E., Adl A., Hassanien A.E., Hegazy O. and Kim T. -H. 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Труфанов р Ду , р ф , р руф АЛГОРИТМЫ СЕТЕВОГО АНАЛИЗА ДАННЫХ В РАСКРЫТИИ СХЕМЫ НАЛОГОВОГО ПРЕСТУПЛЕНИЯ 93 БЕЗОПАСНОСТЬ ИНФОРМАЦИОННЫХ ТЕХНОЛОГИЙ = IT Security, Том 29, № 1 (2022)
https://openalex.org/W3033417100	https://ieeexplore.ieee.org/ielx7/6287639/8948470/09109259.pdf	English	null	An Effective Training Scheme for Deep Neural Network in Edge Computing Enabled Internet of Medical Things (IoMT) Systems	IEEE access	2,020	cc-by	10,707	SPECIAL SECTION ON EDGE COMPUTING AND NETWORKING FOR UBIQUITOUS AI Received May 24, 2020, accepted June 1, 2020, date of publication June 5, 2020, date of current version June 18, 2020. Received May 24, 2020, accepted June 1, 2020, date of publication June 5, 2020, date of current version June 18, 2020. Digital Object Identifier 10.1109/ACCESS.2020.3000322 IRINA VALERYEVNA PUSTOKHINA 1, DENIS ALEXANDROVICH PUSTOKHIN2, DEEPAK GUPTA 3, ASHISH KHANNA3, K. SHANKAR 4, AND GIA NHU NGUYEN 5,6, (Member, IEEE) 1Department of Entrepreneurship and Logistics, Plekhanov Russian University of Economics, Moscow 117997, Russia 2Department of Logistics, State University of Management, Moscow 109542, Russia 3Department of Computer Science and Engineering, Maharaja Agrasen Institute of Technology, Delhi 110086, India 4Department of Computer Applications, Alagappa University, Karaikudi 630003, India 5Graduate School, Duy Tan University, Da Nang 550000, Vietnam 6Faculty of Information Technology, Duy Tan University, Da Nang 550000, Vietnam Corresponding author: Gia Nhu Nguyen (nguyengianhu@duytan.edu.vn) Corresponding author: Gia Nhu Nguyen (nguyengianhu@duytan.edu.vn) ABSTRACT At present times, the real-time requirement on the multiaccess healthcare monitoring system, information mining, and efﬁcient disease diagnosis of health conditions is a difﬁcult process. The recent advances in information technology and the internet of medical things (IoMT) have fostered extensive utilization of the smart system. A complex, 24/7 healthcare service is needed for effective and trustworthy monitoring of patients on a daily basis. To accomplish this need, edge computing and cloud platforms are highly required to satisfy the requirements of smart healthcare systems. This paper presents a new effective training scheme for the deep neural network (DNN), called ETS-DNN model in edge computing enabled IoMT system. The proposed ETS-DNN intends to facilitate timely data collection and processing to make timely decisions using the patterns that exist in the data. Initially, the IoMT devices sense the patient’s data and transfer the captured data to edge computing, which executes the ETS-DNN model to diagnose it. The proposed ETS-DNN model incorporates a Hybrid Modiﬁed Water Wave Optimization (HMWWO) technique to tune the parameters of the DNN structure, which comprises of several autoencoder layers cascaded to a softmax (SM) layer. The SM classiﬁcation layer is placed at the end of the DNN to perform the classiﬁcation task. The HMWWO algorithm integrates the MWWO technique with limited memory Broyden–Fletcher-Goldfarb-Shannon (L-BFGS). Once the ETS-DNN model generates the report in edge computing, then it will be sent to the cloud server, which is then forwarded to the healthcare professionals, hospital database, and concerned patients. The proposed ETS-DNN model intends to facilitate timely data collection and processing to identify the patterns exist in the data. An extensive set of experimental analysis takes place and the results are investigated under several aspects. The simulation outcome pointed out the superior characteristics of the ETS-DNN model over the compared methods. INDEX TERMS Deep neural network, Internet of Medical Things, edge computing, training scheme, healthcare, optimization. INDEX TERMS Deep neural network, Internet of Medical Things, edge computing, training scheme, healthcare, optimization. This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0 I. INTRODUCTION a minor fatal disease especially for individuals affected with diabetes. Mostly, people who are suffering from diabetes and CVD are highly vulnerable to the ailments such as cardiomy- opathy, stroke, peripheral arterial disease, and neuropathy. The preventive health tracking, as well as illness detection of these patients, are essential since the later detection of disease leads to pathetic situations for elderly people. The cautious metrics for such disease forecasting could be attained with The health care system plays a vital role in remote health sensing which is useful in preventing the emergency condi- tions of people and extends the lifetime to a greater extent. Here, cardiovascular disease (CVD) is considered to be The associate editor coordinating the review of this manuscript and approving it for publication was Xiaofei Wang . 107112 VOLUME 8, 2020 VOLUME 8, 2020 I. V. Pustokhina et al.: Effective Training Scheme for DNN in Edge Computing Enabled IoMT Systems FIGURE 1. The layered architecture of edge computing. the help of a health monitoring system. Wearable internet of things (WIoT) [1] is one of the signiﬁcant objectives in the healthcare system that is used for scrutinizing the patient’s health. In the medical sector, IoT is typically named as IoMT, which has revolutionized the medical zone with the newly developed remote healthcare system about social merits, perception, and effective diagnosis of disease. Due to the persistent computation of IoT, it is simple to manage the clinical objectives like doctor’s advice, remedies, medical tools as well as patient’s records. The combination of IoT and Machine Learning (ML) makes the health monitoring system highly effective, and planned medical solutions are used by programmed events prior to human utilization. In line with this, smart healthcare allows telehealth, telerehabilita- tion, telesurgery, and telemedicine which approve the remote rigorous care as well as patients’ observation at any place. It is not applicable to deploy wearable tools for physiological estimation; however, it is an essential factor for smart health- care which develops a whole network where the medical nodes are ﬁxed to the human body, creating wireless body sensor networks (WBSN). It performs the action of transfer- ring medical data to the medical cloud using the internet of medical things (IoMT) models. It is comprised of 3 important components namely, BSN, Gateways, as well as data cloud center. In recent times, IoMT supports massive domains to provide healthcare services to distant stakeholders. I. INTRODUCTION Medical data generated from medical nodes are provided to respective administrators to validate the patient’s information whenever it is required. FIGURE 1. The layered architecture of edge computing. aggregated by them are computed locally using edge devices. Once the data processing is completed, it has been forwarded to CC to process maximum computational operations and memory storage. In the health monitoring system, the minimum-powered wearable sensor device is treated as edge devices. Such devices have limited power and hence, the models which decrease the computation and energy application without decreasing the model’s working function to develop edge dependent health care sectors. The cloud-edge computing approach is one of the capable techniques to incorporate agile computing in health monitoring systems. It is expanded to forward the computations among the CC and edge devices under the application of hierarchical structure with the advan- tages of the edge as well as CC environment which helps in the healthcare data examination from IoT devices. The embedding of edge computing makes possible and stronger delay-sensitive health care applications while the CC offers maximum resources to compute and memory storage. As an inclusion, cloud and edge generate the performance enhance- ment in health care domains. IoT-relied applications expand the boundaries of health- care and operated even at homes for primary disease pre- diction and extend the patient’s lifetime [2]. Such domains are mainly applied to give energy-effective, minimum cost, maximum satisfaction as well as lower latency services for healthcare participants. Massive traditional smart health mon- itoring methods depend upon the cloud environment [3]. This model is used to forward the health information produced from IoT devices to the cloud via the Internet and provides the diagnostic reports attained using Deep Learning (DL) approach used in the cloud. Unfortunately, it is insufﬁcient for health care services where low latency is one of the essential attributes. Hence, the healthcare support system needs a novel processing technique with delay-sensitive monitoring which has to be smart and stable management. Under the application of the latest computation approaches, DL has been widely employed for intelligence in numer- ous ﬁelds like image classiﬁcation, object recognition, and natural language processing. The self-taught as well as the compression ability of DL assists to learn the features of input data hierarchically and automatically that make sure to highlight the hidden patterns and abnormal patterns among them. I. INTRODUCTION As a result, the DL technique has become effective for a maximum number of IoT-based applications. Extensive DL models were employed in the decision-making process regarding the medical zone. The productiveness of DL can be accomplished using deep layers that are inbuilt in the struc- ture that makes the DL technologies highly intensive. Thus, the minimum-powered edge devices are not applicable to develop the DL method since it is not capable to satisfy max- imum computational cost requirements of the DL method. The major issue involved in deploying effective latency aware health monitoring system is based on the embedding of DL Edge computing is deﬁned as a prolonged kind of cloud computing (CC) where the data has been computed closer to the edge of the network from the place of data production [4]. Edge computation is considered as local processing which reduces the frequent data trafﬁc, distance, and latency. Since the edge devices are capable to acknowledge the data spon- taneously; it is a vital objective in latency-sensitive health care domains. The fundamental structure of edge computing is given in Fig. 1. The ﬁrst part of a network gathers the data to be processed using an IoT network or a Local Area Network (LAN) or a Radio Access Network in which the data 107113 VOLUME 8, 2020 I. V. Pustokhina et al.: Effective Training Scheme for DNN in Edge Computing Enabled IoMT Systems inference into edge device which has restricted computational abilities [5]. attain effective results with respect to reliability and energy utilization. It is one of the vital studies in the medical sector of multiple access physical monitoring system, that mostly concentrates on decreasing the people’s healthiness based risk factors. Some of the works have recommended that telehealth mod- ules are applied widely, but it does not provide any best results, and data-based methods are applied for predicting the multimodal alterations of physiology. This technique accom- plishes maximum predictive value, with higher accuracy and the disease diagnosis cannot be performed properly due to the existence of complications [7]. This paper presents a new effective training scheme for the deep neural network (DNN), called ETS-DNN model in edge computing enabled IoMT system. The IoMT devices transfer the captured data to edge computing, which executes the ETS-DNN model to diagnose it. I. INTRODUCTION The proposed ETS-DNN model incorporates a Hybrid Modiﬁed Water Wave Opti- mization (HMWWO) technique to tune the variables of DNN, which comprises many autoencoder (AE) layers that are placed in series a softmax (SM) classiﬁer layer. Then, the diagnostic report will be sent to the cloud server, which is then forwarded to the healthcare professionals, hospital database, and concerned patients. The proposed ETS-DNN model intends to facilitate timely data collection and process- ing to identify the patterns exist in the data. The proposed method identiﬁes the patterns exist in the data to determine the presence of disease from the gathered data using IoMT devices. A detailed experimental analysis is carried out to ensure the goodness of the ETS-DNN model. A team of developers from Stanford University carried out a study for examining the external actions as well as heart patterns of humans with the application of DL models that attained better simulation outcomes. In recent decades, Data- intensive analyses (DIA) were applied to offer the location- aware sensitive monitoring results with maximum power utilization and greater error rate. Recently, Bao et al. [8] have deployed condition-based monitoring (CBM) which is employed to observe the diverse signs of the human body in the multi-access physical monitoring system to mainly detect the errors and abnormal functions of the internal organs [9]. In this study, owing to the combination of irregular frequency, it is not applicable to give better data regarding the patient’s health [10]. In short, the key contributions of the study are provided as follows. Propose a new effective training scheme for the deep neural network (DNN), called ETS-DNN model in edge computing enabled IoMT system. Proposed ETS-DNN model incorporates a Hybrid Mod- iﬁed Water Wave Optimization (HMWWO) algorithm for tuning the parameters of DNN structure. The dynamic nature of this method leads to the worst diagnosing performance. The development of the DL sys- tem and maximum detection to classify the datasets and external data 18 is used as the challenging factor to resolve the multi-access physical monitoring system issue [11]. In Yang et al. [12], the IoT-based system was established and combined with wearable sensor nodes for the analysis of patients pain with the help of facial surface electromyogram, while the efﬁciency is minimum than the presented model Bassoli et al. I. INTRODUCTION [13], the energy application issue is reported by modern plug and play system under the application of IoT. Here, the researchers fall short to assume the trade-off among error and delay. Resolve the local optima problem in Water Wave Opti- mization (WWO) by the MWWO algorithm, which makes use of a new exploration parameter. The proposed HMWWO algorithm integrates the abil- ity of limited memory Broyden–Fletcher–Goldfarb– Shannon (L-BFGS) algorithm in searching the high dimensional space and the competence of MWWO algorithm in the discovery of new probable candidate solutions. Finally, SM classiﬁcation layer is placed at the end of the DNN to perform the classiﬁcation task. y Fong and Chung [14] provided the mobile CC for the medical system in which biomedical signals generated from various places are frequently gathered. Miranda et al. [15] presented a new environment that enables the control of healthcare systems. It is executed with the utilization of various methods that is sampled for 8 months and result esti- mations approved the merits using IoT in the healthcare appli- cation. Jansen and Reijers [16] deployed a reconstruction of a mental healthcare model by applying colored Petrinets. Consequently, a maximum function is reached; a service and ﬂow time are limited and improves the system efﬁciency. The major complexity in healthcare systems is overcrowding of the immediate department, and it refers that sources and workﬂows should be normalized. Dotoli et al. [17] project a Petri Nets approach to enhance the structure and dynamics of ED at GH of Bari, Italy. The technique describes the patient’s ﬂow control and introduced the best conclusion with novel The upcoming portions of the study are arranged as follows. Section 2 derives the existing works related to IoMT in edge computing in a clear and classiﬁed way. Section 3 discusses the proposed ETS-DNN model and validates the model in section 4. Lastly, conclusions are drawn in section 5. II. LITERATURE SURVEY In recent times, edge computing [6] and fog computing were assumed as applicable methodologies to examine the data sources from diverse applications of the healthcare domain. Also, mobile edge computing is an emerging model that is applied currently for multi-access external monitoring approaches. Even though the models are able to provide optimal outcomes, it has the limitations of delay and detection rate in sending the healthcare dataset through the system. The application of neural network (NN) relied arithmetic processing on health dataset computation is not suitable to 107114 107114 VOLUME 8, 2020 I. V. Pustokhina et al.: Effective Training Scheme for DNN in Edge Computing Enabled IoMT Systems neural network (PFFBPNN), and particle swarm-optimized radial basis function network (PSRBFN). Though several works have been available in the literature, there is still a need to develop a new method for edge computing enabled IoMT system with improved performance. neural network (PFFBPNN), and particle swarm-optimized radial basis function network (PSRBFN). Though several works have been available in the literature, there is still a need to develop a new method for edge computing enabled IoMT system with improved performance. resource dimensions that ensures the patient ﬂow. Literature by Mahulea et al. [18] approved that synchronization, as well as concurrency, makes Petri nets an effective device to design and examine the healthcare systems. These modules have proposed a method for patient ﬂow, with the application of Petri nets to allocate the resources which depend upon the required actions. III. THE PROPOSED MODEL FOR EDGE COMPUTING ENABLED IoMT SYSTEMS An alternative model for healthcare with Petri nets was presented by Augusto and Xie [19]. A new technique named as MedPRO is developed and concatenated with results to report the medicinal issues. Fanti et al. [20] implied another path to resolve the ED overcrowding, where the primary discharge from ED and establish a home care choice. It has been presented with a combined system under the application of Petri nets to observe the patients from home, and make sure the data transmission between patients, physicians, caretak- ers, and emergency care units. The overall working principle of the proposed work is depicted in Fig. 2. As shown, the proposed model involves a set of three data collection from patients, preprocessing, and classiﬁcation at edge computing and data transmission to the cloud server. Then, it sends information to emergency care, hospitals, doctors, and patients. These processes are detailed in the subsequent subsections. FIGURE 2. The overall process of proposed ETS-DNN. Then, the developers used Petri net simulation to learn the patient ﬂow and manage the resource while service assign- ment is a vital aspect. The Petri net modeling is comprised of improved healthcare business process and workﬂow. A novel simulation tool has been presented by Davidrajuh et al. [21] to decrease the size of Petri net methods for the tedious system. Also, the application of CC for tackling and com- pute healthcare data and resources, while edge computation would be the major activator of intelligent healthcare for modern cities. An IoT based medical device used to collect the information related to the patients before and after heart disease is done in [22]. The collected data has been seam- lessly sent to the healthcare center and undergo processing by the use of higher-order Boltzmann deep belief neural net- work (HOBDBNN). This method learns the features of heart disease from earlier investigation and attains effectiveness through efﬁcient manipulation of complex data. FIGURE 2. The overall process of proposed ETS-DNN. A. DATA COLLECTION Initially, IoMT devices are utilized for gathering the health details of the patients and the interlinked devices communi- cate with other gadgets during the transmission of healthcare data. When the IoT devices are kept in a body, it collects the medical details like electrocardiogram (ECG), heart rate, blood pressure, glucose level, cholesterol, and pulse rate. These details are sent to the edge computing to analyze the patient’s health conditions. With respect to the IoMT devices and gathered data, wearable and ﬁtness watches can also be used to record the patient data and their physical activities. For examining the outcome, the patient’s data from the UCI repository is also used. The gathered data undergo prepro- cessing and then examined by the proposed ETS-DNN model at edge computing. In [23], a Joint Deep Learning and IoMT Driven Framework for Elderly Patients has been presented. The authors have introduced an effective self-adaptive power control-based enhanced efﬁcient-aware approach (EEA) for minimizing energy dissipation and enhancing battery life- time. Then, a joint DL-IoMT model is presented for the car- diac image processing of remote elderly patients. Next to that, DL driven layered architecture for IoMT is presented. In [24], an effective integrated approach for adequate heart failure risk prediction is presented. This method is based on hierarchical neighborhood component-based-learning (HNCL) and adap- tive multi-layer networks (AMLN). In [25], a new model for protecting medical information from external threats with the dissipation of less probable resources of low-power operated healthcare gadgets. Here, the ML-based biometric security model is presented where the feature extraction from Elec- trocardiogram (ECG) signals for the training stage. a: AE NETWORK (2) AE is composed of single input, hidden and output layers. The AE undergoes training in an unsupervised fashion to generate the corresponding input at the resultant phase with a lower erection error. Thus, the level of output is identical to the level of input. Also, AE is mainly undergone training to incorporate the input to feature spaces, that involves the dimension is minimum when compared with the input space. Therefore, the dimensions of a code space might be selected as a max- imum when compared to the input space for enhancing the classiﬁcation speed in certain events. At this point, the AE tries to offer an optimal presentation of the input vector under the replacement of proper code [26]. In this approach, ˆf refers to the activation function of decoder neurons. The input-output correlation of the decoder might be implied by ˆu = gD( ˆW, ˆb; c). The networking structure of AE is depicted in Fig. 4 and the outcome of AE is implied by ˆu = gAE(W, b, ˆW, ˆb; u). FIGURE 4. Layers in cascading encoder/decoder. FIGURE 4. Layers in cascading encoder/decoder. . Layers in cascading encoder/decoder. FIGURE 3. The network structure of AE. The objective function of AE is described as given below: Esparse = EZ + β N X q=1 KL(ρ∥ˆρ) (3) (3) The predeﬁned cost function is comprised of 2 portions. Initially, the EZ is referred to as the objective function of a NN. The β denoted as the weight of a sparsity penalty in Eq. (3): The predeﬁned cost function is comprised of 2 portions. Initially, the EZ is referred to as the objective function of a NN. The β denoted as the weight of a sparsity penalty in Eq. (3): EZ = 1 Z Z X k=1 e2 k + λ 2 ∥W∥+ ˆW (4) (4) FIGURE 3. The network structure of AE. where λ represents the regularization term, it is mainly applied to eliminate the problem of over-ﬁtting. The error vector is deﬁned as variations among the required outputs as well as the original output as shown below: Fig. 3 implies the network of AE, in which the count of neurons from the outcome layer is similar to the input values [26]. The M dimension input vector is described by u(1), u(2) . . . 1) DEEP NEURAL NETWORKS (DNN) The right-handed portion of AE is named as the decoding unit where the input is the result of the hidden layer (c) and outcome ˆu is the simulation outcome of AE. The decoding device is composed of a weight matrix ˆW and ˆb vector transforms the provided code vector to the actual input vector which products to lower error. The relationship between input and outcome of the encoding unit is given in the following: DL methods are capable to obtain high-dimensional features from the input dataset. Thereby, the features collected from DNN have been applied to enhance the function of classiﬁ- cation models. The commonly employed DL module is the DNN classiﬁer that is built under the combination of a stack of AE system using an SM classiﬁer. ˆu = ˆf ˆb + ˆWc (2) B. PREPROCESSING The initial stage in the IoMT data preprocessing involves the elimination of noise and missing values exist in the gathered data. The data with zero noise helps to achieve a better detec- tion rate in the diagnosis process. In this paper, the undesir- able data is discarded using the median studentized residual method due to the fact it correctly investigates the relativity among the data in the dataset. It will result in an enhanced Some other related methods available in the literature are genetic algorithm-based trained recurrent fuzzy neural net- works (GA-TRFNN), swarm optimized convolution neural network along with the support vector algorithm (SCNN- SVM), particle optimized feed-forward backpropagated 107115 107115 VOLUME 8, 2020 I. V. Pustokhina et al.: Effective Training Scheme for DNN in Edge Computing Enabled IoMT Systems outcome associations of the encoder can be represented as c = gE(W, b; u) and expressed as given in Eq. (1): detection rate in the diagnosis process. Firstly, the data will be overlooked in terms of rows and columns for replacing the missing ones with the median value. Then, the normalization of data takes place in the range of 0 to 1 for minimizing the complexity lies in the diagnosis process. The normalization process is carried out by the use of multiple distributions of the data. detection rate in the diagnosis process. Firstly, the data will be overlooked in terms of rows and columns for replacing the missing ones with the median value. Then, the normalization of data takes place in the range of 0 to 1 for minimizing the complexity lies in the diagnosis process. The normalization process is carried out by the use of multiple distributions of the data. c = f b + WZu (1) (1) where f implies the activation function of encoding neurons. The weight of an encoder is deﬁned by W matrix that links the inputs of hidden layers and b vector which comprised of neuron bias. U vector is deﬁned as the input of the encoding part and vector c is referred to as encoder’s output. a: AE NETWORK u(Z) and neuron count present in a hidden layer is N where M and N deﬁne the set of positive integers. In this model, T is the count of input vectors. The left portion of an AE is called an encoder, in which the input is deﬁned as input of an AE, and the outcome is the result of a hidden layer. The encoder transforms an input vector as a code that should be highly effective for input vectors. The input and ek = u(k) −ˆu (5) (5) where k = 1, 2, . . . Z. It is simple to monitor that the EZ is an expression denoting the internal weight of the AE in which where k = 1, 2, . . . Z. It is simple to monitor that the EZ is an expression denoting the internal weight of the AE in which EZ = EAE W, b, ˆW, ˆb . (6) (6) 107116 107116 VOLUME 8, 2020 VOLUME 8, 2020 I. V. Pustokhina et al.: Effective Training Scheme for DNN in Edge Computing Enabled IoMT Systems The latter portion of Eq. (3), is denoted below: c2,(p)(p = 1, 2, . . . , Z) of the encoding unit of the sub- sequent AE layer while the target output measures are the actual class labels v(p)(p = 1, 2, . . . , Z) reached from the actual training dataset. KL(ρ∥ˆρq) = ρlog ρ ˆρq + (1 −ρ) log 1 −ρ 1 −ˆρq (7) (7) 4. Consequently, the training is ﬁnished by performing the tuning process to enhance the classiﬁer function of a DNN using HMWWO algorithm, which is discussed in the next section. 4. Consequently, the training is ﬁnished by performing the tuning process to enhance the classiﬁer function of a DNN using HMWWO algorithm, which is discussed in the next section. where ρ indicates sparsity value and ˆρ is value as given in Eq. (8): ˆρj = 1 Z Z X p=1 fq u(i) (8) (8) b: SAE NETWORK The encoding device is composed of several AE, which are linked together for developing SAE, as demonstrated in Fig. 5. Under the reformation of input-output association of AE SAE network along with L cascaded AEs might be attained simply as given in the following [26]: gSAE = g1 Eog2 Eo · · · ogL E (9) (9) the SAE model is developed by an encoder portion of trained AE. The decoding portions of AEs were not applied in creat- ing SAE since it is desired for training AE, which is deﬁned as follows. 2) HMWWO ALGORITHM The AE units are interconnected to develop a stacked autoen- coder (SAE) network, which is discussed in the subsequent section. The AE units are interconnected to develop a stacked autoen- coder (SAE) network, which is discussed in the subsequent section. The proposed HMWWO algorithm integrates the ability of with L-BFGS algorithm in searching the high dimensional space and the competence of the MWWO algorithm in the discovery of new probable candidate solutions. The detailed explanations of the various processes are discussed below. FIGURE 5. Layers in cascading encoder/decoder. a: WWO ALGORITHM FIGURE 5. Layers in cascading encoder/decoder. The WWO was evolved from shallow water wave methods to solve optimization issues. With no loss of generality, there is a maximization issue with objective function f. The solu- tion space U is said to be similar to a seabed region, and ﬁtness of a point u ∈U can be estimated conversely by the seabed depth: as lower the distance to present water levels, the ﬁtness becomes high f(u). The 3-D space of seabed has been normalized to n-dimension space. Due to the massive presence of EAs, WWO retains the population of solutions, every solution is analogous to a ‘‘wave’’ which has the height h ∈Z+ as well as a wavelength λ ∈R+. Initially, h is ﬁxed with a constant hmx and λ is 0.5. In the problem-solving process, 3 kinds of tasks have been assumed namely, Propa- gation, Refraction, and Breaking. For every generation, every wave has to be propagated exactly. The propagation operator develops a novel wave u′ by moving every dimension d of actual wave x as given below [27]. c: TRAINING PROCEDURE u′(d) = u(d) + ran(−1, 1) · λL(d) (10) (10) The typical training step of DNN is processed under the application of dataset such as u(1), u(2), . . . , u(Z) . It refers to T input vectors while v(1), v(2), . . . , v(Z) and implies the desired values that denote the classes related to the parallel input vector. The key objective of the training task is the process of tuning the inner parameters of DNN to achieve effective classiﬁcation performance. A specialized training step is applied to attain the optimal function. At this point, every layer of a DNN undergoes training autonomously. For instance, the training procedure of 2 AEs are 1 SM layer and DNN is comprised of given steps: where ran (−1, 1) indicates a random function, and L(d) deﬁnes the length of dth dimension (1 ≤d ≤n). When a novel position is external with a possible range, then it may be at a random position at a speciﬁc range. The propagation is estimated using a ﬁtness of offspring wave u′. When f(u′) > f(u), u is interchanged by u′, and a wave height of u′ has been ﬁxed again to hmx. Else, u is maintained as well, however, the height h is limited by 1 that reﬂects the power dissipation. For every iteration, the wavelength of all waves u can be upgraded as given in the following: 1. Initially, the primary AE layer of a DNN has been trained as deﬁned previously. The actual input vec- tors u(p)(p = 1, 2, . . . , Z) from a training data set were employed as input and desired vectors. λ = λ · α−(f(x)−fmn+γ )/(fmx−fm15+γ ) (11) (11) where fmx and fmn represents the higher and lower ﬁtness measures from the recent population, α is a wavelength reducing coefﬁcients, and e shows n lower positive values to eliminate divisible by 0. Eq. (11) assures that maximum ﬁtness waves consist of wavelengths, and propagates with minimum values. 2. Next, the subsequent AE layer undergoes training under the application of output vectors c1,(p)(p = 1, 2, . . . , Z) of an encoder part of the trained AE layer as a training data. 3. Then, the SM layer of a system has been trained. The input training vector is deﬁned as the output vector 3. d: MWWO ALGORITHM where αmx and αmn indicate the maximum and minimum wavelength reducing coefﬁcients, iteration is deﬁned as the recent generation value and iTrmx is the higher generation number of a technique. The value of α enhances the func- tion of exploration with maximum values. If the iteration is enhanced, then α gets decreased, resulting in the optimal exploitation process. Hence, it develops the best management from exploration to exploitation. The previous studies state that the best management among exploration and exploitation is highly essential to accomplish the global as well as the local searching process where the searching process is limited in a recent space locally. It is assumed to be the major problem as the emphasizing factor. There are many attempts to reach proper control between exploration and exploitation which is highly complex in every optimizing task. WWO algorithm is composed of the lim- itation of smart methods. At the initial stage, if the local exploitation capability is assumed to be sufﬁcient, then the global exploration becomes vulnerable. Later, WWO meets the problem of earlier convergence; in which the searching task is terminated in local optima for the multimodal objective function and leaves the diversity. The previous studies state that the best management among exploration and exploitation is highly essential to accomplish the global as well as the local searching process where the searching process is limited in a recent space locally. It is assumed to be the major problem as the emphasizing factor. There are many attempts to reach proper control between exploration and exploitation which is highly complex in every optimizing task. WWO algorithm is composed of the lim- itation of smart methods. At the initial stage, if the local exploitation capability is assumed to be sufﬁcient, then the global exploration becomes vulnerable. Later, WWO meets the problem of earlier convergence; in which the searching task is terminated in local optima for the multimodal objective function and leaves the diversity. Hybridization of MWWO With L-BFGS Model: L-BFGS is said to be the effective optimization model according to the BFGS method that is acquired from the Quasi-Newton family for higher-level optimizing issues. The quasi-Newton approach is vastly deployed to encounter required models to develop hessian or inverse hessian of function (f) that is to be reduced. These L-BFGS and BFGS apply similar meth- ods for optimizing a function leaving the extended schemes of the Hessian matrix. c: BREAKING α develops reasonable management among exploration and exploitation, at the same time it improves the impact of propagation operator. If the wave shifts to a location in which the depth of the water is lesser than a predeﬁned value, then wave crest velocities go beyond the wave celerity. Finally, the crest is sharper and wave breaks pieces of lonely waves. In WWO, the breaking task is on wave u, which identiﬁes the optimal solution and performs local searching with u∗to accelerate wave break- ing. Also, the random selection of k dimensions and every dimension d produce a lonely wave u′ as given below, The main goal is to eliminate the trapping of local optima and the method has to apply exploration in the primary itera- tion. Thus, it is a vital problem in a population-based heuristic approach. In MWWO algorithm, the exponential adaptive α principle is applied for all iterations, then α is upgraded as given below: u′(d) = u(d) + N(0, 1) · βL(d) (14) (14) α(iteration) = αmx.(itrmx −iteration + 1 itrmx )θ θ = log( αmn αmx ) log( 1 itrmx ) (15) α(iteration) = αmx.(itrmx −iteration + 1 itrmx )θ θ = log( αmn αmx ) log( 1 itrmx ) (15) where β refers to breaking coefﬁcients. When there are no other lonely waves as possible as u∗, u∗is retained; then u∗ can be replaced by effective one from the lonely waves. (15) c: TRAINING PROCEDURE Then, the SM layer of a system has been trained. The input training vector is deﬁned as the output vector 107117 VOLUME 8, 2020 I. V. Pustokhina et al.: Effective Training Scheme for DNN in Edge Computing Enabled IoMT Systems Algorithm 1 MWWO Algorithm Randomly initiate a populace P of n solutions While termination criteria is not met do Upgrade α with novel α For every wave U ∈P do propagate U to a new U′; If f (U′) > f (U) then If f (U′) > f (Ubest) then Break U′ into new waves Upgrade (Ubest) with U′ Replace U with U′ Else Here, the refraction on waves has been processed with reduced heights to 0, and employs a simpler method of calcu- lating the location once the refraction process gets completed: u′ (d) = N u∗(d) + u (d) 2 , \|u∗(d) −u (d)\| 2 (12) (12) where u∗deﬁnes an optimal solution, and N(µ, σ) shows a where u∗deﬁnes an optimal solution, and N(µ, σ) shows a Gaussian arbitrary number with mean µ and SD σ. A novel location is an arbitrary value-centered partially among the actual location and well-known best location, and SD that is similar to the original value of the difference. These estima- tions are modiﬁed as competing for one for complex arith- metic optimization issues. Afterward, the wave height of uf has been reset to hmx, and a wavelength is ﬁxed with where u∗deﬁnes an optimal solution, and N(µ, σ) shows a Gaussian arbitrary number with mean µ and SD σ. A novel location is an arbitrary value-centered partially among the actual location and well-known best location, and SD that is similar to the original value of the difference. These estima- tions are modiﬁed as competing for one for complex arith- metic optimization issues. Afterward, the wave height of uf has been reset to hmx, and a wavelength is ﬁxed with U. h=U.h-1; If U. h = 0 then Refract U to a new U′ Upgrade the wavelength Return the optimal solution which has been identiﬁed. λ′ = λ f(u) f (u′) (13) (13) b: REFRACTION Algorithm 1 MWWO Algorithm Randomly initiate a populace P of n solutions While termination criteria is not met do Upgrade α with novel α For every wave U ∈P do propagate U to a new U′; If f (U′) > f (U) then If f (U′) > f (Ubest) then Break U′ into new waves Upgrade (Ubest) with U′ Replace U with U′ Else U. h=U.h-1; If U. h = 0 then Refract U to a new U′ Upgrade the wavelength Return the optimal solution which has been identiﬁed. d: MWWO ALGORITHM The L-BFGS requires lower storage when compared with BFGS models thus the L-BFGS is rapid than BFGS. To resolve the problem, an adaptive α has been presented to enhance the exploration ability of WWO that is termed as MWWO. Besides, the function of propagation in WWO was boosted which tends to minimize the possibility in ter- minating local optima for multimodal operations. This pro- cess leads to maximize the function of WWO at the time of switching out of local optima. The projected adaptive The above-mentioned models acquire higher storage and time to evaluate the hessian and inverse Hessian matrix under the application of existing methodologies. Initially, a positive 107118 VOLUME 8, 2020 VOLUME 8, 2020 I. V. Pustokhina et al.: Effective Training Scheme for DNN in Edge Computing Enabled IoMT Systems FIGURE 6. Sensitivity analysis of different models. deﬁnite and sparse symmetric matrix H0 is acquired from the f function, which has to be normalized. Then, Hk is obtained with the help of m BFGS update to H0 by applying data gathered from m prior rounds if k is superior to m. The process involved in the L-BFGS model can be represented as uk and the GD of a function is implied as gk. Then, Hk+1 = VZ kHkVk + ρksksZ k (16) (16) where ρk = 1/v τsk k , Yk = I−ρ ksksZ k , sk = uk+1−uk and yk = gk+1 −gk. The L-BFGS is capable to resolve the processing requirements due to the massive scale problems, such as DNN training. Also, L-BFGS is rapid and requires only minimum storage for large-scale issues. Hence, the HMWWO algo- rithm could be attained under the application of the L-BFGS model that enhances the efﬁciency of the MWWO approach. y pp The HMWWO algorithm is deﬁned by the inclusion of the L-BFGS algorithm to the MWWO for improvising the convergence rate of the quality of solutions in the MWWO algorithm. To achieve this, a solution generated by propa- gation is executed by the L-BFGS model until it gets stuck into local minima or each maximum iteration count. Once the exploration procedure of L-BFGS gets done, the L-BFGS offers optimal solutions and a vector with iterative evolution, which is employed for determining the common validation of the MWWO algorithm. D. SM CLASSIFIER It is a classiﬁer used for multi-label classiﬁcation issues. It performs the function of mapping the input vector c from N-dimensional space into K class labels, as deﬁned below. y p y Though the PSRBFN and HOBDBNN models have shown competitive results with the sensitivity values of 98.21% and 99.23% respectively, the proposed ETS-DNN model has out- performed the earlier models with the maximum sensitivity of 99.56%. For example, with the existence of 1500 patients, the presented ETS-DNN model has provided a higher sen- sitivity of 99.91% while the GA-TRFNN approach has reached a lower sensitivity of 96.89%. Simultaneously, the SCNN-SVM as well as PFFBPNN methodologies, have accomplished a somewhat maximum sensitivity of 97.20% and 97.87% correspondingly. Although the PSRBFN and HOBDBNN techniques have resulted in competing out- comes with the sensitivity of 98.89% and 99.83% corre- spondingly, the projected ETS-DNN approach has performed quite well than previous methods with the better sensitivity of 99.91%. vq = exp(θZ q c) PK k=1 exp θZ k c (q = 1, 2, . . . K) (17) (17) where θk = [θk1 θk2 . . . θkN]Z are the weights, that should be tuned by an effective optimization model. A. IMPLEMENTATION DATA The system applies the IoT based data and dataset from UCI repository data which is comprised of 123 instances and 23 attributes gathered from 10 patients with the appli- cation of 3 sensing gadgets. At the time of implementation, the dataset is partitioned into training and testing with the ration of 7:3 to verify the effectiveness of the presented model. Around 1500 patient records were gathered; such IV. PERFORMANCE VALIDATION In this section, the effectiveness of the ETS-DNN model has been investigated in the diagnosis of normal and abnormal heart patients to identify the presence of heart diseases. d: MWWO ALGORITHM Here, the internal parameters of DNN structure is optimized by the HMWWO algorithm for avoiding the local optimal problem in the AEs and SM for obtaining near-optimal DNN is not dependent of the initial- ized values of the network variables, the L-BFGS is employed for local searching of the parameter vectors to ﬁne-tune it. FIGURE 6. Sensitivity analysis of different models. information is computed with the help of the applied process to estimate the efﬁciency of the model. B. SENSITIVITY ANALYSIS Fig. 6 shows the analysis of the results of the ETS-DNN model in terms of sensitivity under a varying number of patients. The values present in the table conﬁrmed that the ETS-DNN model has shown effective performance over the compared methods. At the same time, the GA-TRFNN model has depicted worse outcomes over all the existing methods. For instance, under the presence of 500 patients, the pro- posed ETS-DNN model has offered a maximum sensitivity of 99.56% whereas the GA-TRFNN model has attained a minimum sensitivity of 95.23%. At the same time, the SCNN- SVM and PFFBPNN models have resulted in slightly higher sensitivity values of 95.23% and 96.35% respectively. C. SPECIFICITY ANALYSIS For sample, under the utilization of 1500 patients, the newly projected ETS-DNN model has attained a higher speciﬁcity of 99.42% while the GA-TRFNN model has achieved a lower speciﬁcity of 94.21%. Simultaneously, the SCNN-SVM and PFFBPNN methodologies have displayed moderate speciﬁcity values of 95.03% and 96.30% respectively. Additionally, PSRBFN and HOBDBNN schemes have depicted competitive results with the speciﬁcity values of 97.02% and 99.04% corre- spondingly. Therefore, the proposed ETS-DNN model has performed well than the existing techniques with a higher speciﬁcity of 99.42%. E. EXECUTION TIME ANALYSIS Fig. 9 analyze the time complexity analysis of the proposed and existing algorithms under a varying number of patients. The attained results ensured that the ETS-DNN model has revealed minimal time complexity over the other algorithms whereas higher time complexity has been exhibited by the GA-TRFNN algorithm. C. SPECIFICITY ANALYSIS Fig. 7 shows the analysis of the results of the ETS-DNN method with respect to speciﬁcity under various numbers 107119 107119 VOLUME 8, 2020 I. V. Pustokhina et al.: Effective Training Scheme for DNN in Edge Computing Enabled IoMT Systems FIGURE 7. Specificity analysis of different models. FIGURE 8. F-measure analysis of different models. FIGURE 8. F-measure analysis of different models. FIGURE 8. F-measure analysis of different models. FIGURE 7. Specificity analysis of different models. FIGURE 7. Specificity analysis of different models. FIGURE 8. F-measure analysis of different models. FIGURE 7. Specificity analysis of different models. projected ETS-DNN model has depicted greater F-measure of 98.94% and the GA-TRFNN model has accomplished the least F-measure of 93.92%. Meantime, the SCNN-SVM and PFFBPNN models have achieved considerable F-measure values of 94.66% and 95.78% correspondingly. Though the PSRBFN and HOBDBNN models have deﬁned com- peting results with the F-measure values of 97.24% and 98.49% respectively. Thus, the proposed ETS-DNN model has performed well when compared with alternate models by reaching a higher F-measure of 98.94%. For instance, under the existence of 1500 patients, the proposed ETS-DNN model has given a maximum F-measure of 99.89% while the GA-TRFNN model has achieved low F-measure of 95.55%. Meanwhile, the SCNN-SVM and PFFBPNN models have accomplished manageable F-measure values of 96.11% and 97.08% correspondingly. Though the PSRBFN and HOBDBNN models have showcased equivalent results with the F-measure of 97.95% and 99.43% respectively, the pro- posed ETS-DNN model has outperformed the existing mod- els with the maximum F-measure of 99.89%. of patients. The measures given in the table proved that the ETS-DNN model has implemented a successful func- tion than other approaches. Meanwhile, GA-TRFNN tech- nology has exhibited poor results when compared with alternate models. For illustration, under the application of 500 patients, the developed ETS-DNN model has pro- vided an optimal speciﬁcity of 98.32% and the GA-TRFNN model has accomplished lower speciﬁcity of 93.60%. Con- currently, the SCNN-SVM and PFFBPNN techniques have shown better speciﬁcity values of 94.10% and 95.21% respectively. Also, the PSRBFN and HOBDBNN approaches have showcased relative results with the speciﬁcity values of 96.28% and 97.76% correspondingly. Hence, the pro- posed ETS-DNN model has reached good results with the speciﬁcity of 98.32% than the previous models. D. F-SCORE ANALYSIS For illustration, under the application of 500 patients, the proposed ETS-DNN model has provided a lower time com- plexity of 4.43s and the GA-TRFNN model has reached a higher time complexity of 15.78s. Concurrently, the SCNN-SVM and PFFBPNN methods have shown better time complexity of 14.24s and 12.97s correspondingly. Though the PSRBFN and HOBDBNN models have deﬁned com- petitive results with the time complexity values of 12.45s Fig. 8 show the analysis of the results of the ETS-DNN model utilizing F-measure using diverse patient records. The values applied in the table conﬁrmed that the ETS-DNN model has exhibited the best performance than other mod- els. Concurrently, the GA-TRFNN model has shown inef- fective results when compared with previous approaches. For example, under the consumption of 500 patients, the 107120 VOLUME 8, 2020 VOLUME 8, 2020 I. V. Pustokhina et al.: Effective Training Scheme for DNN in Edge Computing Enabled IoMT Systems FIGURE 9. Time complexity analysis under a varying number of patients. FIGURE 10. Latency analysis under a varying number of patients. FIGURE 10. Latency analysis under a varying number of patients. FIGURE 9. Time complexity analysis under a varying number of patients. FIGURE 10. Latency analysis under a varying number of patients. FIGURE 9. Time complexity analysis under a varying number of patients. FIGURE 10. Latency analysis under a varying number of patients. FIGURE 9. Time complexity analysis under a varying number of patients. of 0.054s has been incurred by the proposed model with WEC and a maximum of 0.079s has been attained by the WOEC system. Similarly, under all cases, the proposed model with WEC has shown better results in terms of latency. After observing the above-mentioned tables and ﬁgures, it is obvi- ous that the ETS-DNN model is superior to other mod- els under several aspects. The proposed model has offered enhanced outcomes with maximum sensitivity, speciﬁcity, and F-score. At the same time, the proposed ETS-DNN model has achieved minimum time complexity and latency over the existing methods under a varying number of patients. and 6.89s respectively, the presented ETS-DNN model has performed well than traditional approaches with the greater time complexity value of 4.43s. For example, under the consumption of 1500 patients, the projected ETS-DNN model has provided a least time complexity value of 8.82s while the GA-TRFNN model has achieved a greater time complexity measure of 20.34s. V. CONCLUSION This paper has introduced a new ETS-DNN model in edge computing enabled IoMT system. At ﬁrst, the IoMT devices observe the patient’s data and transmit the captured data to edge computing, which subsequently performs the ETS-DNN model for diagnosis. The training scheme of DNN involves an HMWWO algorithm, which tunes the parameters involved in the DNN structure. The application of the new explo- ration parameter in the MWWO algorithm has resolved the local optima problem and its integration into the L-BFGS model has resulted in the discovery of efﬁcient solutions. Then, the SM layer is applied to perform the classiﬁcation task, which assigns the class labels appropriately. Finally, the diagnostic report is transmitted to the cloud server, which is then forwarded to the healthcare professionals, hospital database, and concerned patients. For experimenting with the performance analysis of the ETS-DNN model, a series of simulations were carried out and the results are determined under different aspects. In the future, the proposed ETS-DNN model can be implemented in hospitals to monitor and diag- nose the patients living in remote areas. VOLUME 8, 2020 D. F-SCORE ANALYSIS Meanwhile, the SCNN-SVM and PFFBPNN technologies have shown reasonable time complexity values of 18.08s and 15.63s respectively. Though the PSRBFN and HOB- DBNN models have implied competitive results with the time complexity rates of 15.24s and 10.89s correspondingly, the newly developed ETS-DNN model has outperformed the previous models with the optimal time complexity value of 8.82s. F. LATENCY ANALYSIS Table 1 and Fig. 10 examine the latency analysis of the edge computing (WEC) and without edge computing (WOEC) sys- tems to determine the effective performance on the inclusion of edge computing in healthcare. The table values indicated that the latency gets increased with an increase in the number of patients. TABLE 1. Latency Analysis (sec) of WEC and WOEC. REFERENCES [1] S. PB and C. 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His research inter- ests include enterprise logistics planning, artiﬁcial intelligence, big data, the Internet of Things, and reverse logistics network design. [17] M. Dotoli, M. P. Fanti, G. Iacobellis, L. Martino, A. M. Moretti, and W. Ukovich, ‘‘Modeling and management of a hospital department via Petri nets,’’ in Proc. IEEE Workshop Health Care Manage. (WHCM), Feb. 2010, pp. 1–6. [18] C. Mahulea, L. Mahulea, J.-M. Garcia-Soriano, and J.-M. Colom, ‘‘Petri nets with resources for modeling primary healthcare systems,’’ in Proc. 18th Int. Conf. Syst. Theory, Control Comput. (ICSTCC), Oct. 2014, pp. 639–644. 107122 VOLUME 8, 2020 I. V. Pustokhina et al.: Effective Training Scheme for DNN in Edge Computing Enabled IoMT Systems a Reviewer and a Session Chair of the IEEE international conference ICCCA 2016 and 2017. He has designed the syllabus for cloud computing, Java Programming, and distributed systems for GGSIP University. He was a Guest Editor of the IEEE Conference-IC3TSN-2017 and managing a special ses- sion on Parallel and Distributed Network-based Computing Systems. He was a Guest Editor in Springer Conference at ICDMAI-2018 and managing a special session on Computational Intelligence for Data Science. DEEPAK GUPTA received the Ph.D. degree from Inatel, Brazil, and the Ph.D. degree from Dr. A. P. J. Abdul Kalam Technical Univer- sity. He is currently an Eminent Academician, plays versatile roles, and responsibilities juggling between lectures, research, publications, consul- tancy, community service, Ph.D., and Postdoctoral Supervision. REFERENCES He is also working as an Assistant Professor with the Maharaja Agrasen Institute of Technology (GGSIPU), Delhi, India. He is also a Postdoctoral Researcher with the University of Valladolid, Spain. With 12 years of rich expertise in teaching and two years in industry, he focuses on rational and practical learning. He has contributed massive literature in the ﬁelds of human–computer interaction, intelligent data analysis, nature- inspired computing, machine learning, and soft computing. He has served as the Editor-in-Chief, a Guest Editor, and an Associate Editor of SCI and various other reputed journals (Elsevier, Springer, Wiley, and MDPI). He has actively been part of various reputed International conferences. He is not only backed with a strong proﬁle but his innovative ideas, research’s end- results and notion of implementation of technology in the medical ﬁeld is by and large contributing to the society signiﬁcantly. He has authored/Edited 33 books with National/International level publisher (Elsevier, Springer, Wiley, and Katson). He has published 101 scientiﬁc research publications in reputed International Journals and Conferences including 49 SCI Indexed Journals of IEEE, Elsevier, Springer, and Wiley. He is the Editor-in-Chief of OA Journal-Computers, an Associate Editor of Expert Systems (Wiley), Intelligent Decision Technologies (IOS Press), the Journal of Computational and Theoretical Nenoscience, an Honorary Editor of the ICSES Transactions on Image Processing and Pattern Recognition. He is also a Series Editor of Smart Sensors Technology for BioMedical Engineering (Elsevier), Intelli- gent Biomedical Data Analysis (De Gruyter, Germany), and Computational Intelligence for Data Analysis (Bentham Science). He is also associated with various professional bodies, such as ISTE, IAENG, IACSIT, SCIEI, ICSES, UACEE, Internet Society, SMEI, IAOP, and IAOIP. He has invited as a Faculty Resource Person/Session Chair/Reviewer/TPC member in different FDP, conferences, and journals. He is the Convener of ICICC conference series. K. SHANKAR is currently a Postdoctoral Fel- low with Alagappa University, Karaikudi, India. He has authored or coauthored more than 41 ISI Journal articles (with total Impact Factor 107.749) and more than 150 Scopus Indexed Articles. His current research interests include healthcare applications, secret image sharing scheme, dig- ital image security, cryptography, the Internet of Things, and optimization algorithms. He has guest-edited several special issues at many jour- nals published by Inderscience and MDPI. He has served as a Guest Editor and an Associate Editor of SCI, Scopus indexed journals, such as Elsevier, Springer, Wiley, and MDPI. ASHISH KHANNA ASHISH KHANNA received the B.Tech. and M.Tech. degrees from GGSIP University, Delhi, in 2004 and 2009, respectively, and the Ph.D. degree from the National Institute of Technology, Kurukshetra. He is currently a highly qualiﬁed individual with around 15 years of rich exper- tise in Teaching, Entrepreneurship, and Research and Development with specialization in computer science engineering subjects. He is currently an Assistant Professor with the Maharaja Agrasen Institute of Technology (GGSIPU), Delhi, India. He has been a part of various seminars, article presentations, research article reviews, and con- ferences, as a Convener, a Session Chair, and a Guest Editor of journals. He has coauthored several books in publication house and papers in national journals, international journals, and conferences. He has published many research papers in reputed journals and conferences. He also has articles in SCI-indexed and Springer journals. He has coauthored ten Text books and edited books, i.e., Distributed Systems, Java Programming and Website Development, Java Programming, Computer Graphics, Computer Graph- ics and Multimedia, Computer Networks, Computer Networks and Data Communication Networks, Success Mantra for IT interviews, and Funda- mental of Computing. He has also an edited book in Lambert publication. He recently successfully managed Smart India Hackathon in 2017 at MAIT, GGSIP University with teams under him winning prices at their distributed systems, cloud computing, vehicular ad hoc networks, and opportunistic networks. He displayed vast success in continuously acquiring new knowl- edge and applying innovative pedagogies and has always aimed to be an effective educator and have a global outlook which is the need of today. He is also associated with some Springer and the IEEE conferences and managing special sessions for them and looking forward for some more challenging tasks. He was a Reviewer of some SCI indexed journals, such as Cluster Computing (Springer) and the IEEE conferences. He is also GIA NHU NGUYEN (Member, IEEE) received the Ph.D. degree in computer science from the Ha Noi University of Science, Vietnam National University, Vietnam. He is currently the Dean of the Graduate School, Duy Tan University, Vietnam. He has a total academic teaching expe- rience of 20 years with more than 50 publications in reputed international conferences, journals, and online book chapter contributions (Indexed By: SCI, SCIE, SSCI, and Scopus). His areas of research include network communication, security and vulnerability, net- work performance analysis and simulation, cloud computing, and image processing in biomedical. REFERENCES He has authored/edited Conference Proceedings, Book Chapters, and two books published by Springer. He has been a part of various seminars, article presentations, research article reviews, convener, and a session chair of the several conferences. He displayed vast success in continuously acquiring new knowledge and applying innovative pedagogies and has always aimed to be an effective educator and have a global outlook. ASHISH KHANNA He is also an Associate Editor of the International Journal of Synthetic Emotions (IJSE). 107123 VOLUME 8, 2020 VOLUME 8, 2020
https://openalex.org/W2783103058	https://eprints.gla.ac.uk/153391/1/153391.pdf	English	null	Exploring masculinities, sexual health and wellbeing across areas of high deprivation in Scotland: The depth of the challenge to improve understandings and practices	Health and place/Health & place (Online)	2,018	cc-by	12,298	Exploring masculinities, sexual health and wellbeing across areas of high deprivation in Scotland: The depth of the challenge to improve understandings and practices mera,⁎, Lesley McMillanb, Lisa McDaidc, Dona Milned, Siân Russella,1, Kate Huntc Karen Lorimera,⁎, Lesley McMillanb, Lisa McDaidc, Dona Milned, Siân Russella,1, a Glasgow Caledonian University, School of Health & Life Sciences, Glasgow G4 0BA, Scotland, United Kingdom b Glasgow Caledonian University, Glasgow School for Business & Society, Glasgow G4 0BA, Scotland, United Kingdom c MRC/CSO Social & Public Health Sciences Unit, University of Glasgow, 200 Renﬁeld Street, Glasgow G2 3QB, Scotland, United Kingdom d NHS Lothian, Waverley Gate, 2-4 Waterloo Place, Edinburgh EH1 3EG, United Kingdom A R T I C L E I N F O Within and across areas of high deprivation, we explored constructions of masculinity in relation to sexual health and wellbeing, in what we believe to be the ﬁrst UK study to take this approach. Our sample of 116 heterosexual men and women age 18–40 years took part in individual semi-structured interviews (n = 35) and focus group discussions (n = 18), across areas in Scotland. Drawing on a socio-ecological framework, ﬁndings revealed experience in places matter, with gender practices rooted in a domestically violent milieu, where localised, socio-cultural inﬂuences oﬀered limited opportunities for more egalitarian performances of masculinity. We discuss the depths of the challenge in transforming masculinities in relation to sexual health and wellbeing in such communities. Keywords: Masculinities Sexual health Area depriviation Gender Violence wanted pregnancies and sexual dysfunction (Coker, 2007; de Visser et al., 2014; Ellsberg et al., 2008; Garcia-Moreno et al., 2005; McMillan, 2013; World Health Organisation, 2013), so it is right that the WHO oﬀer a broader holistic deﬁnition. Such a broad deﬁnition has been taken up in policy frameworks, including the Scottish Sexual Health and Blood Borne Virus Framework 2015–2020, developed to promote key outcomes including in relation to STIs and unintended pregnancies, inequalities, and sexual violence (Scottish Government, 2015). Health & Place 50 (2018) 27–41 Health & Place 50 (2018) 27–41 Contents lists available at ScienceDirect ⁎ Corresponding author. 1 Present address: Newcastle University, Institute of Health and Society, Newcastle upon Tyne NE2 4AX, United Kingdom. E-mail addresses: Karen.lorimer@gcu.ac.uk (K. Lorimer), Lesley.mcmillan@gcu.ac.uk (L. McMillan), Lisa.mcdaid@glasgow.ac.uk (L. McDaid), Dona.Milne@nhslothian.scot.nhs.uk (D. Milne), Sian.russell@newcastle.ac.uk (S. Russell), kate.hunt@glasgow.ac.uk (K. Hunt). https://doi.org/10.1016/j.healthplace.2017.12.002 Received 6 April 2017; Received in revised form 1 November 2017; Accepted 8 December 2017 ⁎ Corresponding author. 1 Present address: Newcastle University, Institute of Health and Society, Newcastle upon Tyne NE2 4AX, United Kingdom. E-mail addresses: Karen.lorimer@gcu.ac.uk (K. Lorimer), Lesley.mcmillan@gcu.ac.uk (L. McMillan), Lisa.mcdaid@glasgow.ac.uk (L. McDaid), Dona.Milne@nhslothian.scot.nhs.uk (D. Milne), Sian.russell@newcastle.ac.uk (S. Russell), kate.hunt@glasgow.ac.uk (K. Hunt). 1353-8292/ © 2017 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/B https://doi.org/10.1016/j.healthplace.2017.12.002 Received 6 April 2017; Received in revised form 1 November 2017; Accepted 8 December 2017 1353-8292/ © 2017 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/BY/4.0/). 1.1. Levels of inﬂuence on sexual health and wellbeing, and the importance of neighbourhoods and communities Connell deﬁnes ‘hegemonic masculinity’ as ‘the conﬁguration of gender practice which embodies the currently accepted answer to the problem of the legitimacy of patriarchy, which guarantees (or is taken to guarantee) the dominant position of men and the subordination of women’ (Connell, 1995, p. 77). For a brief theoretical overview of hegemonic masculinity, and its relationship with other masculinities (e.g., protest masculinity, hypermasculinity) see Jewkes et al. (2015a, 2015b), or for more detail see Connell (1995). Here, we emphasise three points, which are particularly pertinent to our study: ﬁrstly, masculinity is embodied, structurally positioned and ‘performed’ (Archer and Yamashita, 2003); secondly, masculinities are relational – hegemonic masculinity is ‘a particular form of masculinity in hierarchical relation to a certain form of femininity and to nonhege- monic masculinities’ (Connell and Messerschmidt, 2005), and thirdly; gender does not operate on its own but in relation to other social dynamics such as class, race and sexuality (Connell and Messerschmidt, 2005). As Courtenay has stated, the ‘social structuring of ethnicity, sexuality and class is intimately and systematically related to the social structuring of gender and power (Courtenay, 2009). Thus, eﬀorts to improve sexual health and wellbeing should be premised upon the understanding of gender as ‘a way of structuring social practice’ and ‘unavoidably involved with other social structures’ (Connell, 1995, p. 75). Connell deﬁnes ‘hegemonic masculinity’ as ‘the conﬁguration of gender practice which embodies the currently accepted answer to the problem of the legitimacy of patriarchy, which guarantees (or is taken to guarantee) the dominant position of men and the subordination of women’ (Connell, 1995, p. 77). For a brief theoretical overview of hegemonic masculinity, and its relationship with other masculinities (e.g., protest masculinity, hypermasculinity) see Jewkes et al. (2015a, 2015b), or for more detail see Connell (1995). Here, we emphasise three points, which are particularly pertinent to our study: ﬁrstly, masculinity is embodied, structurally positioned and ‘performed’ (Archer and Yamashita, 2003); secondly, masculinities are relational Immediate determinants of sexual health and wellbeing include, for example, individuals’ knowledge of sexual risks; however, although knowledge improvement is important for behaviour change, it is insuﬃcient on its own to eﬀect signiﬁcant change as inﬂuences upon sexual health stem from factors beyond individual knowledge. At the more distal level, poverty is a signiﬁcant contributor to various forms of gender-based violence (Jewkes, 2002). 1.1. Levels of inﬂuence on sexual health and wellbeing, and the importance of neighbourhoods and communities Epidemiological data reveal the impact of low socio-economic status (SES) upon sexual health (Arnold et al., 2011; Denning et al., 2011); this is compounded by those in low SES often being part of sexual networks with high underlying rates of STI's and HIV (Denning et al., 2011). The provision of laws (and law enforcement) to protect people from discrimination, violence and poverty can signiﬁcantly improve the success of individual behaviour change strategies (Coates et al., 2008). For example, the funding associated with the 1994 US Violence Against Women Act resulted in signiﬁcant eﬀects on sexually violent behaviour (DeGue et al., 2014). By challenging dominant norms, change can occur that results in improved gender equity and reductions in sexual risks, violence and coercion. However, structural factors that can inﬂuence sexual health and wellbeing (e.g., poverty) tend to go beyond speciﬁc domains of health (e.g., HIV prevention), and tackling such issues are commonly for governments to implement across policy ﬁelds; as such, interven- tions to improve sexual health and wellbeing more commonly operate at community- or individual-level. Berg and Longhurst's review, ‘Placing Masculinities and Geography’, provides an excellent overview of the masculinities and geography research from its beginnings, so we opt not to rehash that here (Berg and Longhurst, 2003). We do draw attention to the lack of studies that bring together a focus on masculinities, place and sexual health. So on the one hand, spatial studies have explored relationships between area- and individual-level risks and individual HIV status (Feldacker et al., 2010); how the built environment inﬂuences young people's sexual risk behaviours (Burns and Snow, 2012); and where and how to place STI screening services (Balfe et al., 2010; Goldenberg et al., 2008). On the other, studies have focused on masculinities but not sexual health, such as those exploring rural and urban inﬂuences on masculinities and gender practices (Bye, 2009; Lysaght, 2002; Ni Laoire C and Fielding, 2006). Lysaght's study, for example, revealed the performative character of dominant and subordinate masculinities in Belfast, focusing on the way that spatial context aﬀects the performance of gender identities (Lysaght, 2002). A scoping review (McDaid et al., 2012) underpinning our research, identiﬁed speciﬁc research gaps relating to intervention studies with adult heterosexual men from deprived areas. 1.1. Levels of inﬂuence on sexual health and wellbeing, and the importance of neighbourhoods and communities y Given the geographical variations in sexual behaviours and HIV risks and acquisition (Wadsworth et al., 1996), what are the ‘chains of causation that might link place of residence with health outcomes’ (Macintyre et al., 2002). The ‘broken windows’ theory applied to STIs found deteriorated physical conditions of local neighbourhoods were associated with gonorrhoea rates, independent of poverty (Cohen et al., 2000). The acquisition of STIs has been associated with exposure to neighbourhood poverty during adolescence (Ford and Browning, 2014). Data from the US National Longitudinal Study of Adolescent Health also found neighbourhood inﬂuences upon earlier sexual initiation (Cubbin et al., 2005). Exposure to community violence has been associated with increased sexual risk behaviours (Cooper et al., 2015; Senn et al., 2016; Voisin et al., 2014). Communities in which violence in the family is acceptable experience increased likelihood of such violence (Pinchevsky and Wright, 2012). These studies are examples from an evidence-base that has begun to point strongly towards the association between community violence, peer acceptance of norms as well as acceptance of certain sexual behaviours with sexual health and wellbeing outcomes within com- munities. Indeed, a systematic review of the relationships between neighbourhood characteristics and ‘intimate partner violence’ (a common term used in the USA to refer to what more commonly referred to in the UK as domestic abuse), found ‘ample evidence to indicate that some aspects of neighbourhood may be risk markers or risk factors for IPV’ (Beyer et al., 2013, p. 41). However, other systematic reviews, examining risk and protective factors for sexual violence, have concluded there is little evidence on how community level factors are associated with sexual violence (DeGue et al., 2014; Tharp et al., 2013), and have noted that there are no included studies from Europe, and all studies are cross-sectional, highlighting an important gap in evidence. We would argue that qualitative work is needed to begin to bridge this gap and illuminate experiences in places (Popay et al., 2003), particularly experience of sexual health in relation to masculinity constructions. Causal pathways link structural factors and sexual health and wellbeing outcomes; hence, for example, tackling gender inequalities can improve equitable interpersonal relationships (Taukobong et al., 2016) or reduce sexual risk behaviours (Gupta et al., 2008). 1.2. Masculinities and sexual health 1.2. Masculinities and sexual health health ﬁeld, and they provide a useful vehicle through which to convey the social embeddedness of behaviours, contextualised within environ- ments of strain and adversity. We sought to explore masculinity constructions within and across areas of high deprivation, in order to focus on local gender dynamics and the importance of experiences in places (Manzo, 2005), for the way these inﬂuence sexual health understandings and behaviours. 1.1. Levels of inﬂuence on sexual health and wellbeing, and the importance of neighbourhoods and communities To develop eﬀective interventions, particularly those designed to improve gender relations so as to impact on sexual health outcomes, we require further research to identify ‘causal or contextual factors that are malleable and have greatest scope for change’, as well as the level at which intervening is possible within existing systems (Wight et al., 2016, p. 2). 1. Introduction In a widely-cited, although not oﬃcially endorsed, deﬁnition from the World Health Organisation (WHO), sexual health is: “…a state of physical, emotional, mental and social well-being in relation to sexuality; it is not merely the absence of disease, dysfunction or inﬁrmity. Sexual health requires a positive and respectful approach to sexuality and sexual relationships, as well as the possibility of having pleasurable and safe sexual experiences, free of coercion, discrimina- tion and violence. For sexual health to be attained and maintained, the sexual rights of all persons must be respected, protected and fulﬁlled.” (WHO, 2006) In this article, one of a series of articles from our ‘DeMaSH’ study (Deprivation, Masculinities and Sexual Health), we ﬁrst draw upon a social determinants of health framework as it intersects with our analysis of how masculinities in places inﬂuence sexual health and wellbeing. Here we draw out the importance of neighbourhood and community level factors, citing examples of how we might engage in interruptions in ecological systems (Hawe et al., 2004); elsewhere we focus on ﬁndings relating to holistic sexual health understandings, the blaming of women for sexual violence, and alcohol and sexual consent understandings. Thus, in this paper we only mention such ﬁndings tangentially. Here we prioritise narratives of violence because they often receive less emphasis than ‘bugs and babies’ within the sexual There has been an increasing shift towards holistic deﬁnitions of sexual health, rather than a limited focus on sexually transmitted infections (STIs), blood borne viruses and unplanned pregnancies. The most recent National Survey of Sexual Attitudes and Lifestyles (Natsal- 3) survey, conducted across Britain with 15,162 people age 16–74 years found 1 in 10 women and 1 in 71 men reported an experience of ‘non-volitional’ sex (i.e., sex against their will) (Macdowall et al., 2013). Physical, psychological and sexual abuse is associated with sexual health outcomes such as sexually transmitted infections (STIs), un- K. Lorimer et al. Health & Place 50 (2018) 27–41 2. Methods Individual semi-structured interviews and focus group discussions were conducted with men and women living within the same geogra- phical localities, as described in more detail below. We anticipated that men, and women, might be more willing to talk about some sensitive issues, or personal experiences (e.g., experiences of domestic abuse, 28 Health & Place 50 (2018) 27–41 K. Lorimer et al. sexual consent negotiation, sexual histories etc.), in individual inter- views rather than in a group context. By contrast, as we anticipated that the ways in which understandings of both sexual health and socially appropriate performances of masculinity and femininity are co-created in interaction with others, we considered focus group discussions to be more appropriate to explore negotiations of such meanings between participants. We chose to conduct the focus groups as single sex discussions. Since our primary interest was in the interrelationships between constructions of masculinities and sexual health, attitudes and behaviours, we ﬁrst conducted focus groups with groups of men who were friends or who knew each other, so that we could capture men's ‘performances’ of masculinities in the presence of other men (De Visser et al., 2009; O'Brien et al., 2005; O’Brien et al., 2009). As sexual behaviours and attitudes are often related to peer inﬂuence, we anticipated that groups of friends may share some similar views, attitudes and behaviours, or be comfortable talking about some diﬀerences in this context (Hyde et al., 2005). The all-male focus groups were conducted in parallel with individual interviews with men (Phase 1 data collection). Towards the end of this phase we began to recruit women from the same localities to all-female focus groups as well as to individual interviews (Phase 2 data collection). In practice, the data were collected semi-sequentially, as there was a small temporal overlap between phases 1 and 2. Men's accounts were presented back to women (in both individual interviews and focus groups) in anonymised form, mostly in terms of generalisations (e.g., “Some men have told us that …what do you think of that?”). Our strategy was to present an alternative to the women's views, for example if they oﬀered a negative view of men we presented a positive comment a man or men had made to facilitate deeper exploration of women's experiences. time to build trust and rapport, but proved the most eﬀective means of recruitment. 2.4. Interviews and focus groups discussion topics Sexual risk-taking and sexual safety are constituted in social relations of heterosexuality (Holland et al., 1998). As such, we felt it essential to explore how women in these communities view men's accounts. The exclusion of women's perspectives would neglect the interplay between women's and men's perceptions, expectations and experiences of masculinities, and the ways in which men ‘perform’ their gender with sexual partners, within and beyond the purview of other men. However, we fully acknowledge that masculinities act in relation to each other, and how men relate to other men is also fundamentally important. We began by asking participants about their experiences of living/ growing up in the area (‘what was it like growing up in [area]?’), guiding respondents towards discussions that enabled us to explore the ways in which ontological identity (sense of self) and biographies are bound up with the places in which people live (Popay et al., 2003). We sought to understand inﬂuences on masculinity and gender practice formation in these speciﬁc localities, characterised, as described above, by high levels of social deprivation. SR conducted interviews and focus groups, with another researcher present during focus groups taking notes [KL]. This was followed by questions on their views towards men and women (using the 12 images noted in 2.3 above), probing for similarities and diﬀerences to people in their own area when growing up and also at present. The ﬁve images depicting gender-based violence were then used to prompt discussions on their understandings of sexual consent as well as views towards, and experiences of, physical, sexual and verbal abuse. Participants were then asked about their sexual health understandings, knowledge and behaviours (e.g., ‘how did you learn about sex?’, ‘what does sex mean to you?’, views towards condoms). 2.1. Participants We recruited men and women, aged 18–40 years, who resided in areas classiﬁed as the most deprived. Participants lived in major cities within Scotland (Glasgow, Edinburgh, Dundee) or rural/semi-rural settings in the Highlands. We selected these locations based on a combination of sexual health outcomes (e.g., rates of unintended pregnancies) and the degree of urbanicity/rurality, as assessed by the Scottish Government's 6-fold Urban/Rural Classiﬁcation, 2011–2012 (Scottish Government). We then focused on small areas classed as within the most deprived deciles of deprivation within Scotland, using the Scottish Index of Multiple Deprivation (SIMD). At the time of interview, all participants resided in a SIMD 1 area, i.e., within areas classed as the 10% most deprived. 2.3. Developing the topic guide In this study we embraced a holistic deﬁnition of sexual health, which encompasses disease prevention but also positive relationships free of coercion and violence (WHO, 2006). Our pilot work focused on developing the topic guides for focus groups and individual interviews. Following earlier research on men and masculinities (De Visser et al., 2009; Gill et al., 2005), we anticipated that the use of images could facilitate discussions around social constructions of masculinities and femininities; the pilot stage aﬀorded a valuable opportunity to test and reﬁne the selection of images (see Appendix A). We experimented with a variety of images and settled on a set of images of 6 men and 6 women, as well as carefully selected images to encourage discussions of gender-based violence. This latter set included images of: 1) an unconscious woman being carried by three young men, allegedly after her drink had been ‘spiked’ (using Rohypnol, a ‘date rape’ drug); 2) an image from a Police Scotland campaign depicting a rugby player with the text ‘I’m the kind of guy who doesn’t have sex with a girl when she's too drunk. Are you?’; 3) three still images from a television campaign advert about rape within the context of a relationship; 4) a woman cowering, with bodily bruises evident, and a man standing over her clenching his ﬁst, with the accompanying text ‘Women deserve equal rights….and lefts’ and 5) AWARE Centre campaign image (a ﬁst emerging from a man's mouth grabbing a woman's hair) with accompanying text ‘Verbal abuse can be just as horriﬁc’. These images were chosen to reﬂect a range of gender based violence, to explore whether, and how, these images were perceived as gender based violence by participants, and to prompt discussion of what can be a very diﬃcult topic. The images also reﬂected the fact that across the spectrum of violence, men predominate as perpetrators (Fleming et al., 2015; Macdowall et al., 2013; World Health Organisation, 2013), but this is not saying all men are violent. The images were also selected to reﬂect the capacity for men to make positive choices to not engage in sexually violent practices. 2. Methods These early interactions also provided valuable insights into the areas and communities in which participants lived. Recruitment occurred between February 2014 and April 2015. Interviews lasted between 45 min and 2 h (1 h on average) and focus groups between 1 and 2 h (1.5 h on average). Each participant received a £20 gift voucher as a gesture of thanks for their time. 3. Findings We recruited 116 people aged 18–40 years to the study: 68 men (mean age 30) and 48 women (mean age 29) (see Table 1). We conducted 35 individual interviews (19 men, 16 women) and another 81 took part in a total of 18 focus group discussions (11 with men and 7 with women); no individual took part in both. Most participants (see Table 2) were: unemployed, living in social housing, with at most school level qualiﬁcations (around one quarter had no qualiﬁcations); and married /currently in a long-term relationship (43%) or single (49%) (a few described themselves as divorced/separated or in an open relationship). % rounded. a Missing data for some participants so numbers do not always add to 116(total). …probably the biggest thing is the look of people. Kinda people in [area] kinda look pretty grey, pretty thin, pretty miserable looking to be honest. (Angela, age 34, Glasgow, Int3). The language many men and women used to describe their areas was, at times, stark (‘hellhole’,’ shithole’, ‘rough’), mirroring ﬁndings from other work, and sometimes even the exact same terms (Deuchar, 2010). People, and more often women, invoked strong emotional responses when recalling the youth violence in these areas (e.g., feeling ‘petriﬁed’), although many oﬀered accounts of change, such as reduc- tions in gang violence or drug misuse problems, in their areas. 2.6. Ethics Ethical approval was given by Glasgow Caledonian University, School of Health and Life Sciences research ethics committee (REF: SYEC12/APP202). 2.5. Analysis Table 2 Table 2 Demographic data for interview and focus group participants, by gender. Interviews and focus group discussions were transcribed and anno- tated to link, where possible, to our observations of relevant non-verbal communication. Transcripts were analysed using the Framework Approach (Ritchie and Lewis, 2003), focusing both on a priori areas of interest drawn from the topic guide whilst also allowing for unanticipated themes to be systematically identiﬁed inductively (Pope et al., 2000). Using QSR NVivo software we employed an iterative approach to our thematic analysis: a selection of the transcripts was read independently and repeatedly by two research team members to generate an initial coding framework; other team members independently reviewed this in light of their own readings of selected transcripts related to their areas of expertise. The coding framework was then further reﬁned through extensive discussion between the whole team, before all data were coded. Data were then indexed, charted systematically and organised using matrices in which each participant is represented by a row and each theme by a column. Constant comparison was carried out to ensure the analysis represented all perspectives, including any ‘deviant’ cases and to avoid individual or small group researcher bias. Throughout we have used pseudonyms and note the type of interview with Int (interview) or FG (focus group) in parentheses, along with the participants’ age at interview. 3.1. Neighbourhood/community: environments of strain and adversity As per our inclusion criteria, all participants were resident in an area of high deprivation, but most also grew-up in the same or a similar area. Childhood experiences, and many of the accounts of contemporary life, were thick descriptions of poverty, material deprivation and hostile environments. In addition to examples of experiencing a lack of basic nourishment and money, many also described how manifestations of poverty aﬀected the way people looked. For example, Angela said Yeah there was a lot o’ gang ﬁghts back then, yeah. There was a lot o’ like from scheme [housing estate] to scheme. It's not like that anymore but yeah, each scheme used to ﬁght, have a day that they went up and fought each other, sort of thing. There was a lot o’ gang violence back then. (Emma, age 32, Dundee, Int1) Overall, what emerged strongly in relation to accounts of manifesta- tions of poverty within places was people's habituation to them, often expressed through shrugs of shoulders and a neutral tone of voice. Angela (age 34, Glasgow, Int3) talked of growing up with poverty but “you just kinda deal with it”. Across the many descriptions of drug misuse, violence and criminality they experienced or were aware of in their locality, we failed to read such habituation as dispassion. Of course, not all participants who resided in these areas of high deprivation necessarily lived in poverty themselves. Nevertheless, we were struck by the shadow cast by poverty, drugs and violence, which appeared never to be far from the doorsteps of almost all participants, and for many it was an intrinsic part of negotiating everyday life. Hence, the stresses and strain of residing in some challenging environments varied across accounts but appeared present in some form. Table 1 Number of interviews and focus groups conducted, by recruitment area and gender. Recruitment area Total Glasgow Edinburgh Dundee Highlands Focus groups 18 5 5 4 4 Men 11 3 3 3 2 Women 7 2 2 1 2 Individual interviews 35 9 9 8 9 Men 19 5 4 5 5 Women 16 4 5 3 4 2.2. Recruitment We adopted a multi-faceted recruitment strategy, which included: passive approaches such as ﬂyers, postcards (which included a QR code and details of how people could ﬁnd out more about the study by texting or visiting the study website); and, more proactive approaches, such as talks in community settings, approaching potential participants directly in pubs and community settings, and engaging gatekeepers within community organisations. The proactive approaches required 29 K. Lorimer et al. Health & Place 50 (2018) 27–41 Table 2 Demographic data for interview and focus group participants, by gender. Total, n (%) Men, na Women, n Age, years 18–25 35 (30) 21 14 26–30 32 (28) 17 15 31–35 19 (16) 9 10 36–40 30 (26) 21 9 Relationship status (a1 missing) Long-term relationship/married 50 (43) 24 26 Single 56 (49) 36 20 Separated/Divorced 6 (5) 4 2 Open relationship 3 (2) 3 – Highest educational qualiﬁcation (a1 missing) None 25 (22) 14 11 Standard grades 51 (44) 31 20 Vocational/apprenticeship 3 (2) 3 – Scottish Highers/college qualiﬁcation (e.g., NC, HNC) 22 (19) 11 11 Undergraduate degree 3 (2) 3 – Prefer not to say 10 (9) 4 6 Employment status (a1 missing) Unemployed 71 (62) 45 26 Part-time 20 (17) 6 14 Full-time 19 (16) 14 5 Prefer not to say 5 (4) 2 3 Housing situation (a9 missing) No ﬁxed address 7 (6) 6 1 Living rent free (parents, friends) 19 (18) 13 6 Renting/LAH 75 (70) 39 36 Owner occupier 5 (4) 5 0 % rounded. a Missing data for some participants so numbers do not always add to 116(total). 2.5. Analysis 3.2.1. Youth violence The ‘doing’ of gender was protective, chivalrous and potentially violent: Thomas challenged the notion that individuals can easily change their behaviour: people just don't realise what it's like tae live and kind o' grow up in some o' these places and I think that they're kind o' ignorant when they think that they can just change a couple o' things and it'll make everything awright (Thomas, age 32, Glasgow, Int5) people just don't realise what it's like tae live and kind o' grow up in some o' these places and I think that they're kind o' ignorant when they think that they can just change a couple o' things and it'll make everything awright (Thomas, age 32, Glasgow, Int5) it would be forty, ﬁfty o' us boys hanging aboot a street at night so there'd maybe be four or ﬁve girls hanging aboot with us. But if anybody had came doon and tried tae ﬁght with any o' the girls and they had a whole big, massive squad o' us tae ﬁght (Thomas, age 32, Glasgow Int5) The breeding of alienation was evident in the class injury experi- enced by many respondents: the way in which class narratives were woven through narratives of structural disadvantage highlights the way in which masculinities were, for some, constructed in relation to class identity. Ally, for example, interwove his politics throughout his interview, from challenging housing regeneration to immigration: Such performances of violence, as a response to their environment, can be ways to gain power, and so demonstrate gains from adherence to hegemonic masculinity. However, such behaviours are more com- monly deemed a ‘protest’ masculinity whereby displays of hyper- masculine behaviours draw upon the hegemonic ideal but fail to reap the full rewards of it and certainly it is not a contestation of hegemony (Connell, 1995). That said, we also noted men sought emotional support in these collectives; thus, agency enhancement was sought through non-performative aspects. Neither were these ﬁxed practices, as the desistance literature has made clear in relation to ‘growing out’ of crime (Maruna, 1997; McNeill et al., 2012), even within circum- stances of continuing structural constraints. Each of these men conveyed a trigger for the break: facing jail time (Luke); gaining employment in a middle-class environment, lending itself to desistance from gang violence and embracing new hobbies (Ally); desire for a long-term relationship (Billy); becoming a father (Thomas). 3.2.1. Youth violence This underscores masculinities as dynamic, rather than ﬁxed (Dworkin et al., 2015). I feel like a lot o' this immigration is a way tae drive doon wage costs, you know what I mean? Rather than any great plan tae, you know, multiculturalise Britain, … No negative eﬀect on doctors or middle class people, you know, getting their cheap coﬀee or, you know, their nanny or whatever it is…(Ally, age 40, Glasgow Int1). I feel like a lot o' this immigration is a way tae drive doon wage costs, you know what I mean? Rather than any great plan tae, you know, multiculturalise Britain, … No negative eﬀect on doctors or middle class people, you know, getting their cheap coﬀee or, you know, their nanny or whatever it is…(Ally, age 40, Glasgow Int1). There was a tendency for men's recalled practices and beliefs to point towards a traditional hegemonic masculinity structuring their gender relations. However, at the intersection of gender, poverty and class (i.e., how these systems overlap), we read corporeal performances – sexual behaviours and violence – as reﬂecting responses to impo- verished situations by both adhering to and contesting this traditional hegemonic masculinity. Connell has described a ‘Protest masculinity [which] is a marginalised masculinity, which picks up themes of hegemonic masculinity in the society at large but reworks them in a context of poverty' (Connell, 1995, p. 114). The redemption scripts some men oﬀered conveyed a disruption and contestation of hegemo- nic masculinity, but not in line with protest masculinity. Through abstaining from youth violence or embracing a monogamous long-term relationship rather than multiple sexual partners, these ‘changed men’ challenged the hegemonic gender norms they had absorbed, even if not disrupting them completely. This focuses our attention on gender as ‘contextually bound and responsive to changing circumstances and relations’ (Doull et al., 2013, p. 340). Women's accounts also conveyed the negotiation of such violent milieus, sometimes evoking strong emotional responses (feeling ‘pet- riﬁed’), and likened gang violence to a ‘war zone’. However, what stood out across the women's data was their perceptions of high social cohesion in their very localised community, although this tended to be in sororities, with female neighbours and other women to whom they were not related (e.g., nursery/school staﬀ, other mothers). K. Lorimer et al. Health & Place 50 (2018) 27–41 Participants’ accounts often noted the inﬂuence of wider structural level inﬂuences bearing down on them. Luke, for example, pointed out that individuals’ alcohol consumption should not be detached from the inﬂuence of poverty mances of masculinity felt possible. Two particular types of violence narratives emerged across our data: youth violence and domestic abuse. The former was not a speciﬁc question in our topic guide whereas the latter was, via the images we used (see 2.4). L: Well, I think… in most non-aﬄuent areas it's like that. It's just people get desperate, don't they? And then they get drunk tae try an' hide their desperations and their reality sort of. 3.2.1. Youth violence In such relational accounts, we discerned the living of almost separate lives by women and men across these communities, impacting on how the sexes understood, and communicated with, each other. 3.2.1. Youth violence Men who discussed their experiences of engaging in youth violence –commonly, but not exclusively, our Glasgow participants – gave a variety of reasons for this, from boredom to the acquisition of status, mirroring others’ ﬁndings (Deuchar, 2010; Deuchar and Holligan, 2010). In these narratives, we read a mixture of choice (“Just dinnae [didn’t] feel coerced intae [into] it, just actively joined in”) and obligation (often even at diﬀerent points in the same interview), suggesting that their violent behaviours were unavoidable and normal- ised in their community. Int: So when you say they get desperate, how do you mean? Int: So when you say they get desperate, how do you mean? L: Well, you see… now you see all these people going tae food banks an' stuﬀlike that 'cause they cannae feed their kids or they've no' got a job so they sit aboot the hoose all day bored so they just go 'Ah, fuck it, I'll get drunk' (Luke, age 21, Edinburgh, Int3) Daniel spoke with passionate concern about Dundee's level of mortality related to drug misuse but added gang ﬁghts, just fae [from] where you’re fae – diﬀerent side o’ the street… everybody else was daen [doing] it, but it was the only thing there was to dae at the time (Jim, age 22, Glasgow Int2) building a’ that [new cultural facilities] on the waterfront [in Dundee]. What's that for? It's no’ for us. That's no’ for us. It might beneﬁt the city in general wi’ tourists and things, but when you come out o’ Dundee you’ll see it, it's a nice place, but behind that it's still Dundee. It's still the shithole that's here. (Daniel, age 38, Dundee, Int2) Peer group networks are a key site for the construction and (re) production of masculinity: young men engage in speciﬁc types of (hetero) sexual practice in order to gain social approval from peers (Richardson, 2010). For many men in this study, a collective mascu- linity centered on youth violence, appeared crucial for their embodied heteromasculinity. Table 1 Table 1 Number of interviews and focus groups conducted, by recruitment area and gender. 30 3.2. Violent milieux Across interviews and focus groups, it was clear that men's and women's biographies commonly conveyed gender practices rooted in a domestically violent milieu. We heard men talk about domestic abuse as a common feature of their communities, or personally witnessing A particularly prominent account of ‘place’ – commonly associated with growing up, but also peppering accounts of contemporary life – emphasised the violent milieus, in which a narrow range of perfor- 31 K. Lorimer et al. Health & Place 50 (2018) 27–41 domestic abuse. Here Ally talks about it within his community domestic abuse. Here Ally talks about it within his community The group context could have increased embarrassment such that humour may have been used as a way to cover discomfort. However, these were not one-oﬀjokes, smirks, sniggers or laughs. When challenged to explain their laughter, smiles or smirks, a level of back-tracking took place and discussions often, but not always, moved towards a more condoning tone. Men may have been performing in front of each other, or occasionally felt awkward in front of a female researcher, but they were not awkward when stating with certainty that many women ‘cry rape’. It's just something that I've seen for years, aye [yes]. It's a common thing, aye. You know? You might no' see the physical acts o' violence. You dae [do] sometimes. But you see the way women are. (Ally, age 40, Glasgow Int1)Those who spoke of more personal experiences, including witnessing domestic abuse as a child, mostly described certain incidents. to explain their laughter, smiles or smirks, a level of back-tracking took place and discussions often, but not always, moved towards a more condoning tone. Men may have been performing in front of each other, or occasionally felt awkward in front of a female researcher, but they were not awkward when stating with certainty that many women ‘cry rape’. I grew up wae [with] my mum, domestic violence, seen it when I was a child and I don’t like it. I remember being stuck in a room, my bedroom, and I could hear my mum getting battered oﬀall the walls (Ryan, age 36, Dundee Int4)Another man recalled an extreme episode of violence perpetrated on a woman he had been in a relationship with by her ex-partner, which resulted in him later being diagnosed with PTSD [we provide no further details here to preclude deductive disclosure of either party's identity]. 3.2. Violent milieux One man spoke of an aunt being subject to what has been referred to as ‘intimate terrorism’ – the sustained use of physical and other tactics to exert control (Stark et al., 2013). During these discussions, we witnessed some men's clear upset when recalling such incidents, particularly when recounting witnessing domestic abuse towards their mothers. For example, in one focus group a man had tears in his eyes, and in another a man's tone of voice contained anger, which was not present when discussing other issues throughout the focus group. Men who had witnessed domestic violence as a child, and/or in later life towards sisters or other female relations, were somewhat more likely to be empathetic towards women and stauncher in their condemnation of such violence. Despite the commonality of witnessing domestic abuse, and perceiving it as prevalent within their communities, most men conveyed a strong view against such violence, often in descriptions of what was acceptable for a man to do (invoking the ‘real man’ trope) (Salter, 2015) – the appropriate etiquette for ‘real’ men. Well, you don’t want tae wake up the next day and then the lassie's Well, you don’t want tae wake up the next day and then the lassie's like that, ‘What happened last night? Did we have sex? Blah, blah, blah.’ And then she goes away and says ‘I got raped last night.’ Know what I mean? ‘Cause she's too drunk to remember. (Dundee FG2, Men) Well, you don’t want tae wake up the next day and then the lassie's like that, ‘What happened last night? Did we have sex? Blah, blah, blah.’ And then she goes away and says ‘I got raped last night.’ Know what I mean? ‘Cause she's too drunk to remember. (Dundee FG2, Men) Some women also oﬀered similar views, such as Denise (age 32, Edinburgh, Int3): “if she's too drunk she doesnae know what she's doing and in the morning she could forget and obviously wake up next tae him, cry rape”. The blaming of women for incidents of sexual violence was salient in the data. Irrespective of whether the assignment of blame was nuanced with some ambiguity, or categorically stated from the outset, the caveats and considerations that followed almost always led back to attribution of blame to women. This was particularly marked in men's discussion, while women were more likely to attribute some blame to men. 3.2. Violent milieux It may be that the women's greater levels of personal experience of gender-based violence aﬀected responses. Indeed, we heard horriﬁc stories of abuse involving stabbings, brutal beatings and sustained coercive control. I was in a relationship where I suﬀered domestic violence, I put up with it for 4 years and I was ashamed. I felt so ashamed, I couldn’t tell anybody. So I’d need to stay in and I’d need to put foundation over my bruises. And it wasn’t just physical abuse, it was a mental abuse. He made me feel like I was worth, worth nothing. Sometimes I wanted to kill myself it was that bad. (Jodie, age 29, Dundee Int2) I just think men like that [who domestically abuse] are bullies, cowards. Total cowards. That they have tae hide behind doors and tell lies and, aye, it's disgusting, tae be honest wi' you. And that's a' [all] part o' the way that we grew up as well – if you were gonnae ﬁght wi' somebody then you had a ﬁght wi' them. You stood oot on the street and the two o' youse fought wi' each other like men “Thomas, age 32, Glasgow Int5”. Jade (age 36, Dundee Int3), viewed image 3 and said If I had a’ known, years ago, that you can actually get a boyfriend done with rape, then I would have done it – it's as simple as that. But obviously, it's hard to establish. You’re in a relationship; you’ve got a child to the person. Even when presented with some of the more positive views expressed by the men, many women expressed doubts about their veracity, sincerity and honesty, and denied that they were reﬂective of their own experiences of men in their areas. Thus, in contrast to the youth violence that many felt was normal- ised and in which some revealed they partook, far more men rejected the practice of violence towards a woman. Despite this, it remains prevalent. I would actually say that if the girl doesnae ﬁt the bill, men, boys, are, they are cruel. They are. I mean, you'll get… they'll call them names or they'll be nasty tae them. Yeah, I think that. It's just the way the world is in society. (Shannon, age 33, Edinburgh, Int1). P1: No, honestly. I’m no’ being funny about it, I’m no’, honestly. It just seems like that kinda thing. (Dundee FG2, Men) 3.2. Violent milieux Thus, for many of the men, the primary role model for their developing gender identity and practice was not a biological father, with women or even the state being a key inﬂuence. Littered through the childhood stories were substance use issues (alcoholic mother or violent drunken father), violence – particu- larly domestic abuse – and fragmented relationships. As previously stated, the emotional connection sought in certain masculine collectives was perhaps for some a way to gain a sense of family. However, we discerned no clear line between family structures and egalitarian/non-egalitarian views. Similarly, a weak association has been found between ‘father absence’ and physical and verbal aggres- sion (Boothroyd and Cross, 2017). Five male interviewees seemed utterly lacking in empathy for women, as was particularly prominent during the discussions using images of abuse. These men oﬀered consistently non-egalitarian views resting on a strong gender division of labour, and believed a positive relationship was, simply, one in which women were not hit. We sought to determine whether these men's biographies diﬀered from men we deemed ‘mixed-egalitarian’ (who oﬀered views mostly consistent with gender equality). Most of the non-egalitarians shared a biography of chaotic family structures, such as Ryan and Rab raised in and out of the care system. Ryan, an interviewee with a profound lack of empathy for women (despite witnessing domestic abuse as a child), seemed bereft of aspiration for a positive romantic relationship, almost wary of expecting much, but elsewhere he was animated in his aspiration for employment: ‘clean- cut, suit, tie, probably a good job, nice car’. Yet we also interviewed Connor (age 20, Dundee, Int1), who was raised by his grandmother from age 5. He oﬀered two passionate and engaged responses when discussing women in his life and his thoughts and aspirations for a positive relationship. Given our respondents were age 18–40 years, we have focused on a portion of the lifecourse which has been under-researched (McDaid et al., 2012). Whilst we gathered similar narratives to those found in other work, around men's claims to power through sex and violence (Courtenay, 2000), we also heard how some men, when shifting towards desiring intimacy, were often devastatingly unprepared. The masculinity norms adopted and enacted throughout youth appeared to have a lasting negative impact on these men as they were aging. 3.2. Violent milieux and indirectly associated with risks for STIs and HIV, as well as with various forms of violence (Brown et al., 2005; Fiaveh et al., 2015; Fleming et al., 2015). Whilst a range of masculinities may theoretically be available to men in any social context, the men in this study constructed their gender in circumstances in which they had little access to economic resources. Some seemed to recall practicing a ‘protest masculinity’, whilst others reproduced aspects of hegemonic masculinity. It was clear that localised, socio-cultural inﬂuences did not appear to enable more egalitarian expressions of masculinity; indeed we experienced an undertone of simmering resentment towards women within much of the men's data, and at times a more ‘gentriﬁed misogyny’ was evident (Glosswitch, 2016). We were struck by the level of blame attributed to women for incidents of sexual violence, which regardless of the extent of their discussions, with caveats and con- siderations, seemed always to lead to some level of blame upon women; although, some of the most aggressive views towards women emerged in relation to women who were deemed to have transgressed gender norms for ‘appropriate’ femininity in relation to sexuality and sex. Phipps suggests, ‘it is possible that the working-class woman may be more at risk of sexual violence as a punishment for unfeminine behaviour…’ (Phipps, 2009, p677). Men's and women's exposure to these localised, socio-cultural inﬂuences were delineating boundaries of acceptable social roles within romantic relationships. We are not suggesting here that women lack agency, but we note again that 13 of our 16 women interviewees recalled directly witnessing and/or being a survivor of domestic abuse. These experiences of power convey a social order, and can act to silence: silencing of men's desires for intimacy and silencing of women's aspirations for more equitable and fulﬁlling relationships. and parental relationships (Creighton and Oliﬀe, 2010). Most of the men in this study did not grow up within a conventional nuclear family. Ryan (age 36, Dundee Int4) and Rab (age 23, Highland Int2), for example, recalled growing up in and out of the care system. Many others did not grow up with their biological fathers present in their lives, but instead were parented by a mother or other female relative (e.g., grandmother), such as Connor (age 20, Dundee, Int1). 3.2. Violent milieux Across interviews and focus groups, the image we used to facilitate discussions of domestic abuse (Image 4) commonly resulted in men expressing wholehearted agreement that women had a right to be treated equally, without the spectre of violence: At ﬁrst I just seen ‘women deserve equal rights’, like yeah, yeah women deserve equal rights, but then I seen ‘and lefts.’ A woman doesnae deserve to be beaten "Scott, age 40, Dundee Int5". Of the sixteen women we interviewed, thirteen revealed that they had witnessed and/or been subject to personal experiences of domestic abuse. It is perhaps not unsurprising given such biographies of violence, and negotiating their femininity in relation to such violence, that women held such views towards men. Almost all of these comments were made with a serious tone of voice, with the exception of one focus group in which the women collectively laughed at a man's positive statement read to them. Nevertheless, many wanted such sentiments from men to be true. However, it is also important to note that we also heard women refer to other women as whores, tarts and bitches, and some also blamed victims of sexual violence, thus using language that supports inequalities and the gender order. However, at times, humour was wielded, in ways that minimized the seriousness of the issue, particularly during focus group discussions, as in this discussion of image 4 (domestic abuse), in which the image of a bruised and cowering woman led to claims of her being a ‘golddigger’: P2: You can see the pain in her face, you know what I mean? P2: You can see the pain in her face, you know what I mean? P1: Or it could be that, eh? But then I think she’ll end up selling the book later on, sorta thing, like, telling her story. P1: Or it could be that, eh? But then I think she’ll end up selling the book later on, sorta thing, like, telling her story. (Laughing) 3.3. Intrapersonal: family structures P1: No, honestly. I’m no’ being funny about it, I’m no’, honestly. It just seems like that kinda thing. (Dundee FG2, Men) Creighton and Oliﬀe recognise the importance of men's peer, partner 32 Health & Place 50 (2018) 27–41 K. Lorimer et al. 3.2. Violent milieux Such constrained masculinities work against positive gender relations, and for the achievement of even more positive and intimate relationships. We reiterate Connell's clear articulation that masculinities are not ﬁxed (Connell, 1995), so contexts in which the practice of heteromasculinity requires particularly gender/sexual behaviour (Messerschmidt, 2012b) are subject to change. Indeed, across the ‘redemption scripts’ we heard we heard change occurring amidst the same set of more distal, upstream, determinants. However, such change tended to occur around signiﬁcant life events, such as a threat of jail – a jolt rather than an evolution in attitudes and behaviours. For men to attain the positive, intimate relationships that some said they desire, the masculinity norms they embrace need to shift towards respect for gender equality. De Visser et al. (2009) found that aspects of ‘masculine capital’ can be traded to allow men to compensate for ‘non-masculine’ behaviours (De Visser et al., 2009). However, most of the men in this study lacked economic resources, and signiﬁers of masculinity they valued (e.g., car, being a ‘breadwinner’), so in order to adopt practices of masculinity that include making themselves emotionally vulnerable in an intimate relationship, what scope can they be oﬀered in the trade of masculine capital whilst the dominant localised socio-cultural constructions of masculinity devalue intimacy and gender equality? As the ‘Stepping Stones’ intervention work shows, ‘men's improving livelihoods aﬀorded men the opportunity to materially demonstrate the social changes – in the form of shifts in masculinity’ (Gibbs et al., 2015). Connor: ‘You don’t want somebody that's gonnae be cleaning hooses and that, no, you want somebody that's independent as well…You don’t want them tae just come hame, cook, clean, and give the bairn [child] their dinners…’ Interviewer: So why not? Connor: ‘Cause it's maybe a family if people dae things together. 5. Conclusions Drawing upon a social determinants of health framework, we found a complex picture of multi-level inﬂuences upon masculinity construc- tion across the life course: gender norms within contexts of poverty are reinforced at a local level to create a gendered environment, which is then taken on in individual behaviours and attitudes. The accounts revealed through this research highlight the depth of the challenge in improving understandings and practices of sexual health, tackling gender norms and helping foster more positive and equal relationships in these contexts. Localised, socio-cultural inﬂuences did not appear to enable more egalitarian expressions of masculinity, and were setting out boundaries of acceptable social roles within romantic relationships. For men to attain the positive, intimate relationships that some said they desire, the masculinity norms they embrace need to shift towards respect for gender equality. We strongly recommend interventions occur across all levels of inﬂuence, drawing upon evidence from multi- disciplinary and multi-agency approaches, due to the limits of what individual-level interventions can achieve. However, the contexts our participants recalled and described are the real-world ones that policies need to work within; therefore, the importance of experiences in places needs to be better understood if sexual health and wellbeing interven- tions are to achieve more than incremental gains. p y Our work has limitations. Having a female interviewer/moderator for the focus groups and interviews meant we were not able to investigate whether responses would have diﬀered with a male inter- viewer. Our focus on relationality as between women and men – although we have not completely excluded relations between men – reﬂected our primary interest in sexual health among heterosexual adults in this study. However, our study is enhanced by the large sample size and plurality of voices, including women's, those from urban as well as semi-rural areas and age ranges. We also employed both focus groups and individual interviews, so whilst we observed ‘performance’ of gender in group discussions, for example sexist comments backed with laughter, we also noted similar, if a little less boisterous, attitudes and beliefs in individual interviews. We took several steps to ensure the credibility of this work: we adopted methods, questions and analysis that are widely accepted; we piloted our topic guide and the use of speciﬁc images to prompt discussion; we did not recruit from one setting nor via one type of approach; and speciﬁc ploys were utilized during questioning. 4. Discussion Our study focused on masculinity constructions within and across areas of high deprivation, to focus on the importance of experience in places. Masculinities originating from strain and adversity have been explored both theoretically and empirically (Connell, 1995; Jewkes et al., 2015b). However, as a scoping review found a signiﬁcant gap on work that brings together masculinities, deprivation and sexual health, with adult heterosexual men (McDaid et al., 2012), we believe this is the ﬁrst UK study operating at this intersection. As has been made clear by others (Berg and Longhurst, 2003; Connell, 1995; Messerschmidt, 2012a), and we reiterate, it is important not to decouple masculinities from their social context. However, causal pathways in relation to complex problems are often diverse and interwoven (Wight et al., 2016). As such, a challenge within this study was to identify such pathways given the conﬂuence of factors such as social gender norms, peer cultures and early years’ experiences of violence (Jewkes et al., 2015b). When such wider socio-structural forces work against positive and more egalitarian gender relations, and inhibit men acting on aspira- tions for intimate relationships, individual-level interventions alone will not bring about transformative change. Tackling gender inequal- ities can improve equitable interpersonal relationships (Taukobong If we return to the WHO deﬁnition of sexual health, we are reminded of its holistic nature, where disease prevention sits alongside positive, respectful and pleasurable relationships, free of coercion, discrimination and violence (WHO, 2006). Masculinities are directly 33 Health & Place 50 (2018) 27–41 K. Lorimer et al. with a smirk or a disbelieving ‘aye, right’. Indeed, our own experience of conducting the ﬁeldwork included interactions that indicated an acceptance and normalisation of problematic attitudes and behaviours. et al., 2016) or reduce sexual risk behaviours (Gupta et al., 2008). Interventions are needed to tackle normative roles and assumptions regarding gender and masculinities that conﬂict with respect for equality, and cast collective agency as arising from shared and mutual experiences with structural features. Men's sexual risk behaviours have reduced through interventions which have addressed the negative impact of traditional gender norms (Hatcher et al., 2014; Jewkes et al., 2008; Solorzano et al., 2008). Such gender transformative interventions have sought to transform social norms, and systems that sustain gender inequality and violence (Fleming et al., 2014; Jewkes et al., 2015a). Funding and acknowledgements The DeMASH Project was funded by the Scottish Government Health Directorate (ref: CZH/4/925). Lisa McDaid/Kate Hunt are funded by the UK Medical Research Council/CSO (MC_UU_12017/ 11, MC_UU_12017/12, SPHSU11, SPHSU12). We thank all of the men and women who took part in focus groups and interviews, and the community organisations that helped to facilitate recruitment. 4. Discussion This and other research illustrates potential for change, but this is likely to lead only to incremental gains in the absence of measures to also tackle underlying structural constraints, such as poverty. 5. Conclusions We used iterative questioning and probes to elicit detailed data and remained alert to apparent contradictions which were followed-up. When men or women oﬀered a negative view, we oﬀered a vignette of a more positive perspective, and vice versa (conveying a negative/positive view as credible and creating space for people to revise their view) to allow us to probe participants’ understandings more fully. Whether men expressed more egalitarian views and rejected all violence towards women, for example, in order to present a socially acceptable mascu- linity to a female researcher is questionable given only one man of 68 in the study oﬀered consistently egalitarian views. 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https://openalex.org/W4366082762	https://www.biorxiv.org/content/biorxiv/early/2022/11/20/2022.09.20.508670.full.pdf	English	null	Ventral striatum dopamine release encodes unique properties of visual stimuli in mice	eLife	2,023	cc-by	18,984	. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 20, 2022. ; https://doi.org/10.1101/2022.09.20.508670 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 20, 2022. ; https://doi.org/10.1101/2022.09.20.508670 doi: bioRxiv preprint ARTICLE 1 2 TITLE: Ventral striatal dopamine encodes unique properties of visual stimuli in mice 3 4 AUTHORS: L. Sofia Gonzalez1,3,5,6, Austen A. Fisher1,3,6, Shane P. D’Souza2,4, Evelin M. 5 Cotella1,3, Richard A. Lang2,4, and J. Elliott Robinson1,3 6 7 1Rasopathy Program, Division of Experimental Hematology and Cancer Biology, Department of 8 Pediatrics, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, United States 9 2The Visual Systems Group, Abrahamson Pediatric Eye Institute, Cincinnati Children's Hospital 10 Medical Center, Cincinnati, OH, United States 11 3Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH, United 12 States 13 4Department of Ophthalmology, University of Cincinnati College of Medicine, Cincinnati, OH, 14 United States 15 5Neuroscience Graduate Program, University of Cincinnati College of Medicine, Cincinnati, OH, 16 United States 17 6These authors contributed equally. 18 19 To whom correspondence should be addressed: 20 Dr. J. Elliott Robinson 21 Division of Experimental Hematology and Cancer Biology 22 Department of Pediatrics 23 Cincinnati Children’s Hospital Medical Center 24 3333 Burnet Ave. 25 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 20, 2022. ; https://doi.org/10.1101/2022.09.20.508670 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 20, 2022. ; https://doi.org/10.1101/2022.09.20.508670 doi: bioRxiv preprint TITLE: Ventral striatal dopamine encodes unique properties of visual stimuli in mice 3 4 AUTHORS: L. Sofia Gonzalez1,3,5,6, Austen A. Fisher1,3,6, Shane P. D’Souza2,4, Evelin M. 5 Cotella1,3, Richard A. Lang2,4, and J. Elliott Robinson1,3 6 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 20, 2022. ; https://doi.org/10.1101/2022.09.20.508670 doi: bioRxiv preprint 3 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 20, 2022. ; https://doi.org/10.1101/2022.09.20.508670 doi: bioRxiv preprint . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 20, 2022. ; https://doi.org/10.1101/2022.09.20.508670 doi: ioRxiv preprint ABSTRACT: 1 The mesolimbic dopamine system is an evolutionarily conserved set of brain circuits that 2 plays a role in attention, appetitive behavior, and reward processing. In this circuitry, ascending 3 dopaminergic projections from the ventral midbrain innervate targets throughout the limbic 4 forebrain, such as the ventral striatum/nucleus accumbens (NAc). Dopaminergic signaling in the 5 NAc has been widely studied for its role in behavioral reinforcement, reward prediction error 6 encoding, and motivational salience. Less well characterized is the role of dopaminergic 7 neurotransmission in the response to surprising or alerting sensory events. To address this, we 8 used the genetically encoded dopamine sensor dLight1 and fiber photometry to explore the ability 9 of striatal dopamine release in to encode the properties of salient sensory stimuli in mice, such 10 as threatening looming discs. Here, we report that NAc lateral shell (LNAc) dopamine release 11 encodes the rate and magnitude of environmental luminance changes rather than visual stimulus 12 threat level. This encoding is highly sensitive, as LNAc dopamine could be evoked by light 13 intensities that were imperceptible to human experimenters. We also found that light-evoked 14 dopamine responses are wavelength-dependent at low irradiances, independent of the circadian 15 cycle, robust to previous exposure history, and involve multiple phototransduction pathways. 16 Thus, we have further elaborated the mesolimbic dopamine system’s ability to encode visual 17 information in mice, which is likely relevant to a wide body of scientists employing light sources 18 or optical methods in behavioral research involving rodents. 19 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 20, 2022. ; https://doi.org/10.1101/2022.09.20.508670 doi: bioRxiv preprint . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 20, 2022. ; https://doi.org/10.1101/2022.09.20.508670 doi: ioRxiv preprint INTRODUCTION: 1 ; https://doi.org/10.1101/2022.09.20.508670 doi: ioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 20, 2022. ; https://doi.org/10.1101/2022.09.20.508670 doi: bioRxiv preprint can induce NAc dopamine release (Robinson et al., 2019; Kutlu et al., 2021), the striatal dopamine 1 response to visual threats is not well characterized in mice. Additionally, it is unknown what visual 2 stimulus characteristics – if any – are encoded by NAc dopamine. Thus, one cannot fully interpret 3 the significance of aberrant responses in neurodevelopmental disease models without a more 4 thorough understanding of visual stimulus encoding by mesolimbic dopamine release in typically 5 developing subjects. 6 In this Research Article, we sought to probe ventral striatal dopaminergic responses to 7 arousing visual stimuli, including looming visual threats. Given the ability of dopaminergic neurons 8 to signal stimulus saliency (Bromberg-Martin et al., 2010b), we hypothesized that looming discs 9 would induce ‘alerting’ NAc dopamine release whose magnitude would scale proportionately with 10 perceived threat intensity. To test this hypothesis, we utilized the genetically-encoded sensor 11 dLight1 (Patriarchi et al., 2019) to monitor dopamine release in the NAc lateral shell (LNAc) of 12 freely moving adult C57Bl/6J mice with fiber photometry, as performed previously (Robinson et 13 al., 2019). The LNAc was chosen because dopamine release and/or VTA dopaminergic axon 14 terminal activity encode both stimulus valence and prediction errors in this region (de Jong et al., 15 2018; Robinson et al., 2019; Yuan et al., 2019). Here, we report that LNAc dopamine release 16 reliably reads out unique visual stimulus properties in mice, a phenomenon that is likely relevant 17 to a wide body of scientists employing light sources or optical methods in behavioral research. 18 19 RESULTS: 20 INTRODUCTION: 1 The mesolimbic dopamine system is an evolutionarily conserved set of circuits that plays 2 a role in approach and avoidance, appetitive behavior, and reward processing (Wise, 2004; Everitt 3 and Robbins, 2005; Alcantara et al., 2022). In this circuitry, ascending dopaminergic projections 4 from the ventral midbrain, including the ventral tegmental area (VTA), innervate targets 5 throughout the limbic forebrain, such as the ventral striatum/nucleus accumbens (NAc). 6 Dopaminergic signaling in the NAc has been widely studied for its involvement in motivational 7 salience, behavioral reinforcement, and reward prediction error encoding (Schultz et al., 2015; 8 Berridge and Robinson, 2016; Watabe-Uchida et al., 2017; Berke, 2018). Less well characterized 9 is the role of dopaminergic neurotransmission in the response to unpredicted or alerting sensory 10 events, which may encourage investigation or prime motivated behavioral responses to these 11 stimuli (Horvitz, 2000; Bromberg-Martin et al., 2010a; Schultz, 2010). While many previous 12 studies have reported phasic firing of dopaminergic neurons in response to light flashes in 13 laboratory animals (Horvitz et al., 1997; Comoli et al., 2003; Dommett et al., 2005), it is unclear 14 how NAc dopamine release encodes the properties and/or emotional valence of arousing visual 15 stimuli, such as visual threats. 16 Across a range of species (Ball and Tronick, 1971; Sun and Frost, 1998; Maier et al., 17 2004; Nakagawa and Hongjian, 2010; Yilmaz and Meister, 2013; Temizer et al., 2015), rapidly 18 approaching objects or looming visual threats elicit automatic defensive or avoidance responses. 19 In mice, presentation of an expanding, overhead, black disc that simulates aerial predator 20 approach (a looming stimulus) promotes rapid escape to an available shelter, followed by long 21 periods of freezing (Yilmaz and Meister, 2013). In our previous work published in eLife (Robinson 22 et al., 2019), mice modeling cognitive dysfunction associated with neurofibromatosis type 1 (NF1) 23 exhibited more vigorous escape in responses to looming stimulus presentation. Additionally, NAc 24 dopamine release evoked by a white light stimulus was enhanced in NF1 model mice, which was 25 correlated with behavioral conditioning abnormalities. Despite the demonstration that white light 26 . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 20, 2022. Dopaminergic responses to looming visual threats. 21 To explore the encoding of visual threats by LNAc dopamine, we first measured dLight1 22 signals evoked by looming discs (Figure 1A-D) using a custom Bonsai-controlled (Lopes et al., 23 2015) setup for programmable visual stimulus presentation on an overhead liquid crystal display 24 (LCD) within a light and sound-attenuating chamber (see Materials and Methods for details). 25 . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 20, 2022. ; https://doi.org/10.1101/2022.09.20.508670 doi: ioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 20, 2022. ; https://doi.org/10.1101/2022.09.20.508670 doi: bioRxiv preprint During photometry recordings, mice were exposed to trains of five overhead, black, looming discs 1 on a light gray background that we empirically determined produce short-latency escape in 2 C57Bl/6J mice (Figure 1E, Video 1), consistent with previous studies (Evans et al., 2018; Yilmaz 3 and Meister, 2013). As controls, we presented mice with trains of black discs that do not reliably 4 evoke defensive responses (Figure 1E), such as a static disc (a fixed 30.5 cm black disc on a 5 light gray background), a receding disc (a black disc that contracted from 30.5 cm to 0 cm on a 6 light gray background), and contrast inverted discs (light gray static, looming, or receding discs 7 on a black background). We observed that looming discs induced low-amplitude dopamine 8 transients at the onset of the first stimulus in each train that – contrary to our hypothesis – were 9 not significantly different from the dLight1 responses to non-threatening static and receding discs 10 (Figure 1B-C). Surprisingly, repeating these experiments with contrast-inverted discs that do not 11 induce escape (Figure 1E) evoked ~3 to 6-fold greater dopamine release than black discs (Figure 12 1B-C). This raised the possibility that LNAc dopamine release tracks stimulus brightness rather 13 than threat intensity. 14 Dopaminergic responses to rapid changes in environmental lighting conditions. 16 Because inverted looming discs, in which the number of bright overhead pixels ramps as 17 the disc expands, produced lower amplitude (Figure 1C), longer latency (Figure 1D) dLight1 18 responses than static or receding inverted discs with an instantaneous pixel change, we 19 hypothesized that LNAc dopamine may encode the rate of change of dark-to-light transitions. To 20 test this possibility, we exposed mice to full screen, instantaneous transitions from black to light 21 gray during dLight1 recordings, which eliminated disc edge motion as a contributing visual 22 stimulus property. We found that instantaneous dark-to-light transitions produced a high 23 amplitude (10.38 ± 0.43 z-score), short duration (full width a half maximal amplitude: 143 ± 9.7 24 ms) dopamine transient that peaked 434 ± 3.3 ms after transition onset (Figure 2A). Lengthening 25 the dark-to-light transition time (i.e. the fade-in time) to full screen illumination (Figure 2B) non- 26 . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 20, 2022. ; https://doi.org/10.1101/2022.09.20.508670 doi: ioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 20, 2022. ; https://doi.org/10.1101/2022.09.20.508670 doi: bioRxiv preprint linearly decreased the magnitude of the dLight1 peak and increased the peak latency (Figure 2C). 1 For transition times less than ~500 ms, the dopamine peak latency closely matched the fade-in 2 time, above which peak response occurred hundreds of milliseconds to seconds before full field 3 illumination was reached (Figure 2C). When transition times were greater than 1 s, evoked 4 dLight1 transients were often too small to accurately resolve from the fluorescent baseline for 5 individual mice. However, averaging the fluorescence trace from all mice prior to peak detection 6 allowed signals to be resolved for longer transition times. Thus, results are presented as both the 7 fluorescence peak(s) derived from the photometry trace averaged across all mice (Figure 2C) and 8 individual mice (Figure 2-Figure Supplement 1), which showed high concordance for transition 9 times of 1 second or less (Figure 2-Figure Supplement 1). Dopaminergic responses to rapid changes in environmental lighting conditions. 16 No dLight1 response was reliably 10 evoked by a ten-second dark-to-light transition despite the stimulus ramping to the same number 11 of bright pixels as trials with shorter transition times (Figure 2B). 12 Next, we examined whether LNAc dopamine release also reads out the magnitude of 13 environmental lighting changes by measuring the dLight1 response to ten-second, instantaneous 14 exposures to white light across a range of intensities (0.2 nW/cm2 – 5.0 W/cm2, measured at 15 mouse level) generated by a light emitting diode (LED) (Figure 2D-F). High irradiance LED 16 illumination (5 W/cm2) evoked a dLight1 transient at stimulus onset (Figure 2D) that was similar 17 to transients evoked by the LCD monitor (Figure 2A-B; irradiance at mouse level: 11 W/cm2). 18 This response did not depend on the time of testing within the vivarium day-night cycle (Figure 19 2G) and was larger than the response to auditory tones (80 dB; 1 – 16 kHz; Figure 2-Figure 20 Supplement 1). When LED irradiance was reduced, we observed an intensity-dependent 21 decrease in the magnitude of the dLight1 peak and increase in the response latency (Figure 2E- 22 F), consistent with Bloch’s law of temporal summation in mammalian photoreceptors 23 (Scharnowski et al., 2007; Donner, 2021). Significant dopaminergic responses were observed at 24 all irradiances tested, including 0.2 nW/cm2, which was not perceptible to the human 25 . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 20, 2022. ; https://doi.org/10.1101/2022.09.20.508670 doi: ioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 20, 2022. ; https://doi.org/10.1101/2022.09.20.508670 doi: bioRxiv preprint experimenter. As a point of reference, the lock screen of a Samsung S21 smart phone on the 1 lowest brightness setting had an irradiance of 20 nW/cm2 when placed in the same position as 2 the white LED. Likewise, time-locked dopamine release could be evoked by simply uncovering 3 the enclosure peephole that allows users to observe mouse behavior (irradiance: 17 nW/cm2; 4 Figure 2-Figure Supplement 1). Dopaminergic responses to rapid changes in environmental lighting conditions. 16 These results were not likely caused by mouse movement, as 5 illumination of a white LED that was 1000-fold more intense (5 mW/cm2) than the highest 6 irradiance tested had little effect on behavior when freely exploring mice entered a target zone 7 within a dark arena (Video 2). Thus, LNAc dopamine release is sensitively evoked by ambient 8 light and reliably encodes the speed of these lighting transitions over short timescales. 9 10 Dopaminergic responses to repeated light stimuli. 11 Previous literature suggests that dopaminergic neuron firing in response to sensory events 12 habituates as the novel stimulus becomes familiar (Schultz, 1998). In order to test if the dLight1 13 response to white light is affected by a history of previous exposures, we exposed mice to twenty 14 consecutive one-second white light pulses over five trials (100 pulses total) across a range of 15 interstimulus intervals (ISIs; 10 ms to 10 s; Figure 3A). We found that light-evoked dopamine 16 transient magnitude decayed logarithmically as a function of the ISI duration (Figure 3B). When 17 the ISI was short (e.g. 10 – 100 ms), dLight1 responses habituated rapidly. This is exemplified by 18 the dopaminergic response to 40 Hz light flicker, which is used therapeutically to enhance neural 19 activity in the context of Alzheimer’s disease (Singer et al., 2018). Presentation of a sixty-second 20 40 Hz white LED flicker (5.0 W/cm2 irradiance, 50% duty cycle) induced a dopamine transient 21 only at stimulus onset (Figure 3C) that was indistinguishable from the response to constant 22 illumination (Figure 2D). Conversely, in shorter experiments when the ISI was long (100 s), no 23 habituation of the dopamine response to light was observed from stimulus-to-stimulus (Figure 24 3D). Repeating this experiment immediately after three hundred consecutive one-second 25 exposures with a one-second ISI produced an ~20% reduction in the dLight1 response to 100 s 26 . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 20, 2022. ; https://doi.org/10.1101/2022.09.20.508670 doi: ioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 20, 2022. ; https://doi.org/10.1101/2022.09.20.508670 doi: bioRxiv preprint ISI light; however, this effect did not reach statistical significance (p = 0.08) (Figure 3D). Therefore, 1 habituation of the dopamine response to repeated light stimuli is much more strongly influenced 2 by stimulus frequency than the total number of previous exposures. Dopaminergic responses to repeated light stimuli. 11 3 During repeated stimulus experiments, the greatest reduction in the peak LNAc dopamine 4 response to light stimuli occurred between the first and second light pulse in each stimulus train. 5 In order to further characterize this phenomenon, we varied the duration of the first stimulus to 6 determine if the total amount of initial light exposure modulates the dopaminergic response to a 7 subsequent stimulus (Figure 3E). We found that the dopaminergic response to a 1 second white 8 LED test stimulus was not significantly different when preceded by either a 300 second or 1 9 second preconditioning light stimulus one second earlier (Figure 3F). No difference in dLight1 10 response to the preconditioning stimulus was observed between conditions (Figure 3F). We did 11 observe, however, that the 300-second preconditioning stimulus produced a dopaminergic 12 response at light offset, whereas the 1-second preconditioning stimulus did not (Figure 3G). 13 Taken together, our findings indicate that ISI is a more significant determinant of stimulus-to- 14 stimulus dopamine release habituation than light stimulus duration. 15 16 Wavelength and photoreceptor contributions to the dopaminergic response to light. 17 In these and previous experiments (Robinson et al., 2019), we employed a white LED light 18 to induce striatal dopamine release; however, this light source is composed of multiple 19 wavelengths throughout the visible spectrum. Therefore, we next investigated if light-evoked 20 dopamine release exhibits wavelength specificity. This is additionally germane given the 21 widespread use of molecular and optical technologies in rodents that require delivery of specific 22 wavelengths of visible light in order to probe neural activity, structure, or biology (Fenno et al., 23 2011; Resendez and Stuber, 2015; Sabatini and Tian, 2020). In order to determine if the dLight1 24 response varied by wavelength, we measured dopamine release induced by ten-second 25 exposures to environmental ultraviolet (UV; 360 nm), blue (475 nm), green (555 nm), red (635 26 . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 20, 2022. ; https://doi.org/10.1101/2022.09.20.508670 doi: ioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 20, 2022. ; https://doi.org/10.1101/2022.09.20.508670 doi: bioRxiv preprint nm), and far-red (730 nm) light across a 100,000-fold range of irradiances (1 nW/cm2 to 100 1 W/cm2). These experiments revealed broad sensitivity of the mesolimbic dopamine system to 2 light across the visual spectrum (Figure 4A-B). The dopamine response was least sensitive to UV 3 and red light when the irradiance was low (1 nW/cm2; Figure 4A-B), and far-red light (730 nm) 4 only induced dopamine release when the irradiance was high (100 W/cm2; Figure 4-Figure 5 Supplement 1). The ability of red light to induce dopamine release at intensities as low as 0.1 6 W/cm2 is consistent with research that rodents are better at perceiving red wavelengths than is 7 commonly acknowledged (Danskin et al., 2015; Nikbakht and Diamond, 2021; Vinberg et al., 8 2019). Whereas the dLight1 response to UV and red light was irradiance-dependent, the response 9 to blue and green light remained robust across the entire irradiance range (Figure 4A). Wavelength and photoreceptor contributions to the dopaminergic response to light. 17 These 10 experiments indicate that the mesolimbic dopamine system is responsive to all visible 11 wavelengths yet is most sensitive to blue and green light. 12 The mouse visual system utilizes numerous opsin proteins for image-forming and non- 13 imaging forming phototransduction with unique wavelength sensitivities. These include the rod 14 opsin rhodopsin for scoptopic vision (λmax ~500 nm) and short (λmax ~360 nm) and medium/long 15 wavelength (λmax ~508 nm) cone opsins for photopic vision. Additionally, melanopsin (λmax ~480 16 nm) is expressed in intrinsically photosensitive retinal ganglion cells (ipRGCs) that mediate 17 circadian entrainment, the pupillary light reflex, and light-regulated changes in mood (Panda et 18 al., 2003; Hattar et al., 2003; Fernandez et al., 2018). While it has been hypothesized that ipRGCs 19 engage VTA dopamine neurons via hypothalamic intermediates (Zhang et al., 2021), the role of 20 melanopsin in the dopaminergic response to light is unknown. In order to parse the role of visual 21 opsins versus melanopsin in the mesolimbic response to dark-to-light transitions, we performed 22 LNAc dLight1 recordings in Opn4 (melanopsin) knockout mice and Gnat1/Gnat2 double knockout 23 mice (Gnat1/2-dKO). Gnat1 and 2 knockout mice lack expression of rod and cone α-transducin, 24 respectively, and exhibit loss of signal transduction through these photoreceptors (Deng et al., 25 . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 20, 2022. ; https://doi.org/10.1101/2022.09.20.508670 doi: ioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 20, 2022. ; https://doi.org/10.1101/2022.09.20.508670 doi: bioRxiv preprint 2009; Yao et al., 2018). Compared with wildtype littermates, Gnat1/2-dKO mice displayed a robust 1 reduction in the dopaminergic response to a 5 W/cm2 white LED (Figure 4C) and an increase in 2 the dLight1 response latency (Figure 4-Figure Supplement 1). Light-evoked dopamine release 3 was not abolished, however, in these mice (Figure 4C). Spectral analysis indicated that Gnat1/2- 4 dKO mice retain sensitivity to UV light (Figure 4D, Figure 4-Figure Supplement 1), which may be 5 indicative of residual cone-based vision (Allen et al., 2010). Wavelength and photoreceptor contributions to the dopaminergic response to light. 17 Conversely, loss of melanopsin 6 expression in Opn4 knockout mice (Panda et al., 2002) did not affect the dLight1 response to the 7 white light stimulus (Figure 4E, Figure 4-Figure Supplement 1). These findings indicate that light- 8 evoked dopamine release is rod and cone-dependent and does not involve melanopsin. 9 DISCUSSION 11 In these investigations, we used the genetically-encoded dopamine sensor dLight1 to 12 demonstrate that LNAc dopamine release can encode rapid changes in luminance but not 13 looming threat intensity. We found that rapid dark-to-light transitions evoked time-locked 14 dopamine responses at stimulus onset at irradiances as low as 0.2 nW/cm2, which is in line with 15 findings that mice see over a 100 million-fold range of light intensity beginning at ~4 cd/m2 16 (Umino et al., 2008). The magnitude of these dopaminergic responses was highly dependent on 17 light stimulus frequency and transition rate rather than duration or novelty. In fact, high amplitude 18 LNAc dLight1 responses to a white LED persisted after hundreds of exposures and months of 19 regular testing. Although mesolimbic dopamine systems regulate wakefulness (Eban-Rothschild 20 et al., 2016) and exhibit circadian oscillation (Korshunov et al., 2017), the time of testing did not 21 appear to be a significant contributor to our findings. Sudden dark-to-light transitions are highly 22 salient to nocturnal rodents that must avoid detection by visual predators (Thompson et al., 2010), 23 so it is possible that the dopaminergic response to light represents a specialized saliency signal. 24 Previously, phasic dopamine responses to unexpected, unconditioned sensory events have been 25 . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 20, 2022. ; https://doi.org/10.1101/2022.09.20.508670 doi: ioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 20, 2022. ; https://doi.org/10.1101/2022.09.20.508670 doi: bioRxiv preprint conceptualized in the context of novel action discovery (Redgrave and Gurney, 2006), the need 1 to alert to stimuli that require motivated responses (Schultz and Romo, 1990; Horvitz, 2000), 2 mechanisms of associative learning (Lisman and Grace, 2005), etc. Thus, further studies will be 3 needed to firmly establish the ethological and neurobiological importance of dopaminergic 4 responses to environmental light. 5 We also found that LNAc dopamine is broadly evoked by wavelengths across the visual 6 spectrum. DISCUSSION 11 Given the high proportion of rods in the mouse retina (~97% of photoreceptors) (Jeon 7 et al., 1998) and the reduced sensitivity of dopaminergic responses to 360 and 635 nm light at 8 lower irradiances, it is probable that rod-based phototransduction is primarily responsible for 9 visually-evoked dopamine release under dim (scotopic) lighting conditions. Conversely, rod and 10 cone opsins likely contributed to dLight1 signals in the photopic range. These hypotheses are 11 supported by our observation that genetic disruption of rod and cone-based signaling in Gnat1/2- 12 dKO mice substantially attenuated the dopaminergic response to light. Gnat1/2-dKO mice 13 retained sensitivity to high irradiance UV light, which was most likely caused by incomplete loss 14 of cone-based vision in this model (Allen et al., 2010). We cannot, however, rule out the 15 involvement of UV-sensitive non-visual opsins in our observed findings, such as neuropsin 16 (Opn5), which is maximally activated by 380 nm light (Tarttelin et al., 2003). Neuropsin-expressing 17 retinal ganglion cells project to multiple limbic regions (Sasaki et al., 2021), and this opsin 18 promotes thermogenesis via intrinsically light-sensitive glutamatergic neurons in the preoptic area 19 (Zhang et al., 2020). While melanopsin-expressing ipRGCs are hypothesized to engage VTA 20 outputs via a disynaptic circuit involving the preoptic area (Zhang et al., 2021), we found that 21 Opn4 knockout had no effect on the ability of light to evoke LNAc dopamine. Given that ipRGCs 22 receive rod and cone input via the retinal synaptic network (Güler et al., 2008; Lall et al., 2010; 23 Altimus et al., 2010), it is possible that these neurons contribute to light-evoked dopamine release 24 independent of melanopsin. Thus, functional lesioning studies will be required to elucidate the 25 role of non-image forming visual pathways in the dopaminergic encoding of visual stimuli. 26 . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 20, 2022. ; https://doi.org/10.1101/2022.09.20.508670 doi: ioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 20, 2022. DISCUSSION 11 ; https://doi.org/10.1101/2022.09.20.508670 doi: bioRxiv preprint Visual information is conveyed from the retina to the brain via the axons of retinal ganglion 1 cells that synapse in downstream nuclei to mediate image processing, circadian entrainment, 2 pupillary reflexes, gaze orientation, etc. (Peirson et al., 2018). While thalamocortical visual 3 pathways are required for conscious visual perception, neither the primary visual cortex (V1) nor 4 the visual thalamus (e.g. lateral geniculate nucleus) significantly innervate ventral midbrain 5 dopamine neurons (Watabe-Uchida et al., 2012). Previous work by Redgrave and colleagues 6 suggest that dopaminergic responses to light are driven by the superior colliculus (SC) (Comoli 7 et al., 2003; Dommett et al., 2005; Takakuwa et al., 2017), which receives direct input from retinal 8 ganglion cells (Dhande and Huberman, 2014) in its superficial layers and promotes motivated 9 behavior via deep motor-output layers (Branco and Redgrave, 2020). SC glutamatergic projection 10 neurons directly synapse onto VTA (Solié et al., 2022) and substantia nigra pars compacta 11 dopamine neurons (Huang et al., 2021), both of which project to the LNAc (Beier et al., 2015; 12 Poulin et al., 2018). Likewise, optogenetic stimulation of SC neuron somata is sufficient to evoke 13 LNAc dopamine release in vivo (Robinson et al., 2019). While these observations support a role 14 for the SC in dopaminergic responses to light, the relative contribution of different visual 15 processing centers to our findings is an important area of future study. 16 1 Mesolimbic dopaminergic circuits are thought to play a role in the pathophysiology of 17 several neuropsychiatric conditions, including disorders of impulse control, schizophrenia, and 18 neurodevelopmental disorders (Li et al., 2006; Purper-Ouakil et al., 2011; Maia and Frank, 2017; 19 Robinson and Gradinaru, 2018), including NF1 (Brown et al., 2010; Diggs-Andrews et al., 2013; 20 Anastasaki et al., 2015). Patients with NF1 exhibit high rates of attention deficit/hyperactivity 21 disorder (Mautner et al., 2015; Miguel et al., 2015), in which difficulties with attentional orientation 22 are associated with a diminished ability to suppress distractive stimuli (Aboitiz et al., 2014) such 23 that irrelevant environmental cues are assigned exaggerated stimulus salience (Tegelbeckers et 24 al., 2015). Previously in eLife, we showed that dopaminergic responses to light are enhanced in 25 NF1 model mice and correlate with disruptions in the expression of conditioned behavior 26 . DISCUSSION 11 CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 20, 2022. ; https://doi.org/10.1101/2022.09.20.508670 doi: ioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 20, 2022. ; https://doi.org/10.1101/2022.09.20.508670 doi: bioRxiv preprint (Robinson et al., 2019). Our current findings suggest that these responses reflect changes in the 1 encoding of environmental lighting conditions and, given their correlation with phenotypic 2 expression, may reflect altered stimulus saliency. Aberrant sensory processing and motivational 3 dysregulation are common features of neurodevelopmental disorders, including syndromic and 4 non-syndromic forms of autism spectrum disorder (Behrmann et al., 2006; Tomchek and Dunn, 5 2007; Robinson and Gradinaru, 2018). Therefore, better characterization of the functional 6 interplay between visual processing and dopaminergic circuitry may improve our 7 pathophysiological understanding of these disorders. 8 . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 20, 2022. ; https://doi.org/10.1101/2022.09.20.508670 doi: ioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 20, 2022. ; https://doi.org/10.1101/2022.09.20.508670 doi: bioRxiv preprint MATERIALS AND METHODS 1 MATERIALS AND METHODS 1 Key Resources Table Reagent type (species) or resource Designation Source or reference Identifiers Additional information Recombinant DNA reagent pAAV-hSyn- dLight1.2 Addgene Cat#: 111068 RRID:Addgen e_111068 Produced by Addgene in the AAV5 serotype Software, Algorithm Python 3.8 Python Software Foundation RRID:SCR_0 08394 Software, Algorithm Fiber Photometry Trace Processing Tucker-Davis Technologies https://www.tdt. com/docs/sdk/of fline-data- analysis/offline- data- python/FibPhoE pocAveraging/ Software, Algorithm Bonsai 2.6.3 Bonsai Foundation CIC RRID:SCR_0 17218 Software, Algorithm Looming Visual Stimulus Generation Austen Fisher, Robinson Lab https://github.co m/jelliottrobinso n/Robinson_La b Software, Algorithm ABET II Software for Operant Control Lafayette Instrument Company Model 89501 Software, Algorithm Ethovision XT 17 Noldus Information Technology RRID:SCR_0 00441 Software, Algorithm GraphPad Prism 9 GraphPad Software, Inc. RRID:SCR_0 02798) Software, Algorithm Data Science Workbench 14.0.0.15 TIBCO Software, Inc. RRID:SCR_0 14213 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 20, 2022. ; https://doi.org/10.1101/2022.09.20.508670 doi: bioRxiv preprint Other Mono Fiber- Optic Cannula Doric Lenses, Inc. Cat#: MFC_400/430 - 0.66_6mm_M F1.25_FLT OD: 400 m, Length: 6 mm Other Mono Fiber- Optic Patch Cable Doric Lenses, Inc. Cat#: MFP_400/430 /1100- 0.57_1m_FC M- MF1.25_LAF, Doric Lenses Inc. OD: 400 m, Length: 1 m Experimental subjects were adult, male and female C57Bl/6J mice (the Jackson 3 Laboratory Stock No: 000664), homozygous Opn4 knockout mice (Panda et al., 2002), or 4 homozygous Gnat1/2 knockout mice (Gnat1-/-, Gnat2cpfl-3 mice; the Jackson Laboratory Stock No: 5 033163) that were greater than 12 weeks of age. Animals were pair or group housed (3-4 per 6 group) throughout the duration of the experiment in a vivarium on a 14-hour/10-hour light/dark 7 cycle (lights on at 0600 hrs, lights off at 2000 hrs) with ad libitum access to food and water. All 8 experiments were performed during the light phase of the vivarium light/dark cycle, except when 9 white LED exposure was performed 2-3 hours into the dark phase, as shown in Figure 2G. MATERIALS AND METHODS 1 Animal 10 husbandry and experimental procedures involving animal subjects were conducted in compliance 11 with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and 12 approved by the Institutional Animal Care and Use Committee (IACUC) and by the Department 13 of Veterinary Services at Cincinnati Children’s Hospital Medical Center (CCHMC) under IACUC 14 protocol 2020-0058. Mice were only excluded from studies if they could not complete an entire 15 experiment due to health concerns or loss of brain implant, if there was no dynamic photometry 16 signal six weeks after surgery, or if the location of the photometry fiber tip was histologically 17 determined to be outside the lateral nucleus accumbens (NAc). 18 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 20, 2022. ; https://doi.org/10.1101/2022.09.20.508670 doi: bioRxiv preprint 1 Surgical Procedures 2 Stereotaxic viral vector injections and optical fiber implantation surgeries for dLight1 were 3 performed as previously described (Robinson et al., 2019). This procedure was similar to the 4 published protocol of Tian and colleagues (Patriarchi et al., 2019). In brief, mice were 5 anesthetized with isoflurane (1 – 3% in 95% O2/5% CO2 provided via nose cone at 1 L/min), the 6 scalp was shaved and sterilized with chlorhexidine surgical scrub, the skull surface was exposed, 7 and a craniotomy hole was drilled over the lateral NAc (antero-posterior: 1.2 mm, medio-lateral: 8 1.6 mm relative to Bregma). 800-1000 nL of a AAV5-hSyn-dLight1.2 vector (~1 × 1013 viral 9 genomes/mL, obtained from Addgene; catalog #AAV5-111068) was delivered into the LNAc 10 (antero-posterior: 1.2 mm, medio-lateral: 1.6 mm, dorso-ventral: -4.2 mm relative to Bregma) 11 using a blunt or beveled 34 or 35-gauge microinjection needle within a 10 uL microsyringe 12 (NanoFil, World Precision Instruments) controlled by a microsyringe pump with SMARTouch 13 Controller (UMP3T-1, World Precision Instruments) over ten minutes. Following viral injection, a 14 6 mm long, 400 μm outer diameter mono fiber-optic cannula (MFC_400/430- 15 0.66_6mm_MF1.25_FLT, Doric Lenses Inc.) with a metal ferrule was lowered to the same 16 stereotaxic coordinates and affixed to the skull surface with C&B Metabond (Parkel Inc.) and 17 dental cement. MATERIALS AND METHODS 1 Mice were given 5mg/kg carprofen (s.c.) intraoperatively and for two days 18 postoperatively for pain. Mice were allowed a minimum of five weeks for surgical recovery and 19 virus expression prior to participation in behavioral studies. 20 Fluorescent signals were monitored using an RZ10x fiber photometry system from Tucker- 23 Davis Technologies, which allowed for dLight1 excitation and emission light to be delivered and 24 collected via the same implanted optical fiber. Our system employed a 465-nm LED for sensor 25 excitation and a 405-nm LED for isosbestic excitation. Light was filtered and collimated using a 26 . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 20, 2022. ; https://doi.org/10.1101/2022.09.20.508670 doi: ioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 20, 2022. ; https://doi.org/10.1101/2022.09.20.508670 doi: bioRxiv preprint six channel fluorescent MiniCube [FMC6_IE(400-410)_E1(460-490)_F1(500-540)_E2(555- 1 570)_F2(580-680)_S] from Doric Lenses, Inc., which was coupled to the implanted optical fiber 2 via a one-meter, low autofluorescence fiber optic patch cable (MFP_400/430/1100-0.57_1_FCM- 3 MF1.25LAF, Doric Lenses Inc.). The emission signal from 405 nm isosbestic excitation was used 4 as a reference signal to account for motion artifacts and photo-bleaching. A first order polynomial 5 fit was applied to align the 465 nm signal to the 405 nm signal. Then, the polynomial fitted model 6 was subtracted from the 465 nm channel to calculate ΔF values. The code for performing this 7 function was provided by Tucker-Davis Technologies, Dr. David Root (University of Colorado, 8 Boulder), and Dr. Marisela Morales (NIDA); it is available at: 9 function was provided by Tucker-Davis Technologies, Dr. David Root (University of Colorado, 8 Boulder), and Dr. Marisela Morales (NIDA); it is available at: 9 https://www.tdt.com/docs/sdk/offline-data-analysis/offline-data-python/FibPhoEpocAveraging/. 10 During behavioral experiments, the ΔF time-series trace was z-scored within epochs to 11 account for data variability across animals and sessions, as described by Morales and colleagues 12 (Barker et al., 2017). MATERIALS AND METHODS 1 CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 20, 2022. ; https://doi.org/10.1101/2022.09.20.508670 doi: ioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 20, 2022. ; https://doi.org/10.1101/2022.09.20.508670 doi: bioRxiv preprint chamber underneath the LCD. Looming discs expanded from 0 cm to 30.5 cm over 0.84 s and 1 froze at full expansion for 0.26 s, encompassing 61.9 degrees of visual angle, as previously 2 described (Evans et al., 2018; Yilmaz and Meister, 2013). Receding discs shrunk from 30.5 cm 3 to 0 cm over 0.84 s. Static discs maintained their 30.5 cm diameter throughout the duration of the 4 stimulus. During each stimulus train, five discs were shown consecutively with a 0.5 ms 5 interstimulus interval (ISI). Mice were exposed to five stimulus trains with a 600 s inter-trial interval 6 (ITI). Single full field fades from black to light gray (0 – 10 s fade duration) were delivered via the 7 LCD screen across 5 trials with a 120 s ITI. 8 White LED exposures were delivered via the house light of a modular conditioning 9 chamber (Model 80015NS, Lafayette Instruments Company) placed within the light and sound 10 attenuating box and controlled by ABET II software (Lafayette Instrument Company). A TTL 11 breakout adapter (Model 81510) was used to synchronize stimulus delivery with the photometry 12 recording. Single ten-second light stimuli were delivered across ten trials with a randomized ITI 13 between 90 and 180 s. Glass neutral density filters were used to attenuate the irradiance when 14 necessary (0.1 – 3.0 OD, HOYA Filter USA and/or Edmund Optics TECHSPEC filters). Trains of 15 20 one-second light stimuli with variable ISIs (10 ms – 10 s) were delivered across five trials (100 16 total exposures) with a 300 s ITI. One-second light stimuli with a 100 s ISI were delivered before 17 and after 300 one-second light stimuli with a one-second ISI; 100 s separated the 300 one-second 18 stimuli and each 100 s ISI stimulus train. MATERIALS AND METHODS 1 When fiber photometry was performed during sensory stimulus exposure 13 experiments, dLight1 signals were synchronized to stimulus onset via delivery of TTL pulses to 14 the photometry system. Peak data (magnitude, latency, and full width at half-maximal intensity) 15 was analyzed using Python. 16 17 Visual Stimulus Exposure 18 Visual stimuli were delivered to mice during fiber photometry recordings within a custom 19 setup that featured a 24 inch liquid crystal display (LCD) mounted 25.4 cm above mouse level in 20 a light and sound attenuating chamber (Model 83018DDP, Lafayette Instrument Company). 21 Stimuli (looming, static, and receding discs; full screen fades; etc.) were generated on the LCD 22 display using Bonsai (Lopes et al., 2015), which also controlled delivery of a TTL pulse to the 23 photometry system via a BNC cable to timestamp stimulus onset. The TTL pulse was generated 24 with an Arduino Uno Rev3 microcontroller. During each experiment, mice were placed within the 25 bottom of a clean shoebox cage with a thin layer cob bedding in the light and sound attenuating 26 https://www.tdt.com/docs/sdk/offline-data-analysis/offline-data-python/FibPhoEpocAveraging/. 10 During behavioral experiments, the ΔF time-series trace was z-scored within epochs to 11 account for data variability across animals and sessions, as described by Morales and colleagues 12 (Barker et al., 2017). When fiber photometry was performed during sensory stimulus exposure 13 experiments, dLight1 signals were synchronized to stimulus onset via delivery of TTL pulses to 14 the photometry system. Peak data (magnitude, latency, and full width at half-maximal intensity) 15 was analyzed using Python. 16 Visual stimuli were delivered to mice during fiber photometry recordings within a custom 19 setup that featured a 24 inch liquid crystal display (LCD) mounted 25.4 cm above mouse level in 20 a light and sound attenuating chamber (Model 83018DDP, Lafayette Instrument Company). 21 Stimuli (looming, static, and receding discs; full screen fades; etc.) were generated on the LCD 22 display using Bonsai (Lopes et al., 2015), which also controlled delivery of a TTL pulse to the 23 photometry system via a BNC cable to timestamp stimulus onset. The TTL pulse was generated 24 with an Arduino Uno Rev3 microcontroller. During each experiment, mice were placed within the 25 bottom of a clean shoebox cage with a thin layer cob bedding in the light and sound attenuating 26 . MATERIALS AND METHODS 1 Ten-second single wavelength stimuli were delivered in random order with a 5 randomized ITI (140 – 200 s) to achieve five total exposures per color per mouse. 6 7 Auditory Stimulus Exposure 8 Auditory stimulus exposures were performed in the modular testing chamber within the 9 light and sound attenuating enclosure similarly to single white LED exposures. A ten-second 80 10 dB tone (1 – 16 kHz; generated via Lafayette Instruments 7 Tone Generator Model 81415M) was 11 presented via a speaker (0.25 – 16 kHz; Model 80135M14, Lafayette Instrument Company) 12 across 5 trials with a randomized ITI (140 – 200 s). 13 14 Looming Stimulus Assay 15 The looming stimulus assay was performed as previously described (Yilmaz and Meister, 16 2013) using an apparatus built to the specifications of Evans et al. (2018). The apparatus featured 17 a 20.3 cm (w) x 61 cm (l) x 40.6 cm (h) clear, open, rectangular acrylic arena with a dark, infrared 18 (IR) light-transmitting shelter at one end and a ‘threat zone’ at the opposite end that housed a 9 19 cm clear plastic petri dish to encourage exploration outside of the shelter. A 15.6 inch monitor 20 was mounted above the arena so that discs (19.5 cm maximum diameter encompassing 27 21 degrees of visual angle) could be presented to the mice when they entered the threat zone. The 22 arena floor was backlit with an infrared light (880 nm back-lit collimated backlight, Advanced 23 Illumination) to improve mouse tracking under dim light conditions. The entire apparatus was 24 placed inside a custom light-attenuating enclosure for testing. During testing, mice were recorded 25 with a Basler acA2040-120 um camera with a Edmunds Optics TECHSPEC 6mm C Series fixed 26 MATERIALS AND METHODS 1 One minute of 40 Hz flicker exposure (50% duty cycle) 19 was repeated across 5 trials with a 120 s ITI. For paired light stimuli experiments, a one-second 20 white LED stimulus was delivered one second after a 300 s or 1 s light stimulus across five trials 21 with a 300 s ITI. 22 Individual wavelength light stimuli were generated with a Lumencor Aura III LED light 23 engine, which was triggered via TTL inputs from the Lafayette Instruments TTL breakout adapter 24 and controlled by ABET II. The liquid light guide that delivered the visual stimulus was positioned 25 in the approximate location of the white LED within the testing chamber. LED light power 26 . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 20, 2022. ; https://doi.org/10.1101/2022.09.20.508670 doi: ioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 20, 2022. ; https://doi.org/10.1101/2022.09.20.508670 doi: bioRxiv preprint (measured at mouse level with a Thor Labs PM100D optical power meter with S130VC 1 photodiode sensor) was modulated using the onboard Lumencor graphical user interface and, 2 when necessary, attenuated via the use of glass neutral density filters (0.1 – 3.0 OD, HOYA Filter 3 USA and/or Edmund Optics TECHSPEC filters) placed in front of the liquid light guide outlet within 4 a custom housing. Ten-second single wavelength stimuli were delivered in random order with a 5 randomized ITI (140 – 200 s) to achieve five total exposures per color per mouse. 6 (measured at mouse level with a Thor Labs PM100D optical power meter with S130VC 1 photodiode sensor) was modulated using the onboard Lumencor graphical user interface and, 2 when necessary, attenuated via the use of glass neutral density filters (0.1 – 3.0 OD, HOYA Filter 3 USA and/or Edmund Optics TECHSPEC filters) placed in front of the liquid light guide outlet within 4 a custom housing. Auditory Stimulus Exposure 8 Mous 3 position and velocity data was analyzed post hoc using Ethovision XT software (Noldu 4 Information Technology) and Python. Note: In Video 1, the clear, circular pedestals that separate 5 the infrared backlight from the apparatus base can be seen with the IR camera; they were below 6 the arena floor and inaccessible to the mouse. The setup was modified for spotlight experiment 7 so that the pedestals would not be visible in the captured videos. 8 Spotlight experiments were performed in the same apparatus using the same procedur 9 described above except that a high intensity white LED (5 mW/cm2 measured at mouse leve 10 positioned to illuminate the threat zone replaced the LCD monitor. 11 12 Statistical Analysis 13 Statistical analysis was performed using Python, GraphPad Prism 9 (GraphPad Software 14 Inc.), and/or Data Science Workbench 14 (for 3-way repeated measures ANOVA; TIBCO 15 Software, Inc.). All statistical tests performed on data presented in the manuscript are stated i 16 the figure captions and provided in detail with the corresponding source data in the Supplementa 17 Data and Statistical Analysis file. For each experiment, statistical tests were chosen based on th 18 structure of the experiment and data set. No outliers were removed during statistical analysis 19 Parametric tests were used throughout the manuscript. Sample size estimates were based o 20 studies in Robinson et al. (2019) and power analysis performed using the sampsizepwr() functio 21 in Matlab (MathWorks). When analysis of variance (ANOVA; 1-way, 2-way, 3-way, and/o 22 repeated measures) was performed, multiple comparisons were corrected using the Bonferron 23 correction. When repeated measures ANOVA could not be performed due to missing value 24 (Figure 2-Figure Supplement 1, panel C), data was analyzed by fitting a mixed model in GraphPa 25 Prism 9; this approach uses a compound symmetry covariance matrix and is fit using restricte 26 focal length lens, and real time position tracking was performed with Bonsai. This allowed for 1 presentation of the overhead looming, receding, or static disc stimulus to be automatically 2 triggered when the animal was in the threat zone following a ten-minute habituation period. Mouse 3 position and velocity data was analyzed post hoc using Ethovision XT software (Noldus 4 Information Technology) and Python. Auditory Stimulus Exposure 8 Auditory stimulus exposures were performed in the modular testing chamber within the 9 light and sound attenuating enclosure similarly to single white LED exposures. A ten-second 80 10 dB tone (1 – 16 kHz; generated via Lafayette Instruments 7 Tone Generator Model 81415M) was 11 presented via a speaker (0.25 – 16 kHz; Model 80135M14, Lafayette Instrument Company) 12 across 5 trials with a randomized ITI (140 – 200 s). 13 The looming stimulus assay was performed as previously described (Yilmaz and Meister, 16 2013) using an apparatus built to the specifications of Evans et al. (2018). The apparatus featured 17 a 20.3 cm (w) x 61 cm (l) x 40.6 cm (h) clear, open, rectangular acrylic arena with a dark, infrared 18 (IR) light-transmitting shelter at one end and a ‘threat zone’ at the opposite end that housed a 9 19 cm clear plastic petri dish to encourage exploration outside of the shelter. A 15.6 inch monitor 20 was mounted above the arena so that discs (19.5 cm maximum diameter encompassing 27 21 degrees of visual angle) could be presented to the mice when they entered the threat zone. The 22 arena floor was backlit with an infrared light (880 nm back-lit collimated backlight, Advanced 23 Illumination) to improve mouse tracking under dim light conditions. The entire apparatus was 24 placed inside a custom light-attenuating enclosure for testing. During testing, mice were recorded 25 with a Basler acA2040-120 um camera with a Edmunds Optics TECHSPEC 6mm C Series fixed 26 . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 20, 2022. ; https://doi.org/10.1101/2022.09.20.508670 doi: ioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 20, 2022. ; https://doi.org/10.1101/2022.09.20.508670 doi: bioRxiv preprint focal length lens, and real time position tracking was performed with Bonsai. This allowed fo 1 presentation of the overhead looming, receding, or static disc stimulus to be automatical 2 triggered when the animal was in the threat zone following a ten-minute habituation period. Auditory Stimulus Exposure 8 Note: In Video 1, the clear, circular pedestals that separated 5 the infrared backlight from the apparatus base can be seen with the IR camera; they were below 6 the arena floor and inaccessible to the mouse. The setup was modified for spotlight experiments 7 so that the pedestals would not be visible in the captured videos. 8 Spotlight experiments were performed in the same apparatus using the same procedure 9 described above except that a high intensity white LED (5 mW/cm2 measured at mouse level) 10 positioned to illuminate the threat zone replaced the LCD monitor. 11 Statistical Analysis 13 Statistical analysis was performed using Python, GraphPad Prism 9 (GraphPad Software, 14 Inc.), and/or Data Science Workbench 14 (for 3-way repeated measures ANOVA; TIBCO 15 Software, Inc.). All statistical tests performed on data presented in the manuscript are stated in 16 the figure captions and provided in detail with the corresponding source data in the Supplemental 17 Data and Statistical Analysis file. For each experiment, statistical tests were chosen based on the 18 structure of the experiment and data set. No outliers were removed during statistical analysis. 19 Parametric tests were used throughout the manuscript. Sample size estimates were based on 20 studies in Robinson et al. (2019) and power analysis performed using the sampsizepwr() function 21 in Matlab (MathWorks). When analysis of variance (ANOVA; 1-way, 2-way, 3-way, and/or 22 repeated measures) was performed, multiple comparisons were corrected using the Bonferroni 23 correction. When repeated measures ANOVA could not be performed due to missing values 24 (Figure 2-Figure Supplement 1, panel C), data was analyzed by fitting a mixed model in GraphPad 25 Prism 9; this approach uses a compound symmetry covariance matrix and is fit using restricted 26 . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 20, 2022. ; https://doi.org/10.1101/2022.09.20.508670 doi: ioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 20, 2022. ; https://doi.org/10.1101/2022.09.20.508670 doi: bioRxiv preprint maximum likelihood (REML). When results were compared to a pre-stimulus baseline, this value 1 was defined as the amplitude of the dLight1 peak that occurred 500 ms prior to stimulus delivery. 2 When results were compared to a ‘null’ stimulus, the value was defined as the dLight1 peak that 3 occurred at the onset of a TTL that timestamped a trial in which no stimulus was delivered. 4 Competing interests: The authors have no competing interests to declare. 4 Data and Materials Availability 6 Viral vector plasmids used in this study are available on Addgene. Codes used for fiber 7 photometry signal extraction and analysis are available at https://www.tdt.com/docs/sdk/offline- 8 data-analysis/offline-data-python/FibPhoEpocAveraging/. Codes used for visual stimulus 9 generation are available at https://github.com/jelliottrobinson/Robinson_Lab. Source data is 10 available in the provided Supplemental Data and Statistical Analysis file. 11 12 ACKNOWLEDGEMENTS 13 We would like to acknowledge Mary Claire Casper and Shiva Senthilkumar for assistance 14 with histological sample preparation and preliminary data analysis, respectively. We would also 15 like to thank Dr. Gregory Schwartz at Northwestern University Feinberg School of Medicine and 16 Dr. Diego Fernandez at the National Institute of Mental Health for helpful discussions regarding 17 technical considerations and/or interpretation of the experimental findings. This work was funded 18 by a Cincinnati Children’s Research Foundation Trustee Award, a Simons Foundation Autism 19 Research Initiative (SFARI) Bridge to Independence Award (663007), a SFARI Supplement to 20 Enhance Equity and Diversity (SEED) Award, and a Gilbert Family Foundation Neurofibromatosis 21 Gene Therapy Initiative Team Science Award to JER. 22 23 Competing interests: The authors have no competing interests to declare. 24 25 Viral vector plasmids used in this study are available on Addgene. Codes used for fiber 7 photometry signal extraction and analysis are available at https://www.tdt.com/docs/sdk/offline- 8 data-analysis/offline-data-python/FibPhoEpocAveraging/. Codes used for visual stimulus 9 generation are available at https://github.com/jelliottrobinson/Robinson_Lab. Source data is 10 available in the provided Supplemental Data and Statistical Analysis file. 11 ACKNOWLEDGEMENTS 13 We would like to acknowledge Mary Claire Casper and Shiva Senthilkumar for assistance 14 with histological sample preparation and preliminary data analysis, respectively. We would also 15 like to thank Dr. Gregory Schwartz at Northwestern University Feinberg School of Medicine and 16 Dr. Diego Fernandez at the National Institute of Mental Health for helpful discussions regarding 17 technical considerations and/or interpretation of the experimental findings. This work was funded 18 by a Cincinnati Children’s Research Foundation Trustee Award, a Simons Foundation Autism 19 Research Initiative (SFARI) Bridge to Independence Award (663007), a SFARI Supplement to 20 Enhance Equity and Diversity (SEED) Award, and a Gilbert Family Foundation Neurofibromatosis 21 Gene Therapy Initiative Team Science Award to JER. 22 26 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 20, 2022. ; https://doi.org/10.1101/2022.09.20.508670 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 20, 2022. ; https://doi.org/10.1101/2022.09.20.508670 doi: bioRxiv preprint REFERENCES: 1 Aboitiz F, Ossandón T, Zamorano F, Palma B, Carrasco X. 2014. Irrelevant stimulus processing in ADHD: 2 catecholamine dynamics and attentional networks. Front Psychol 5:183. 3 doi:10.3389/fpsyg.2014.00183 4 Alcantara IC, Tapia APM, Aponte Y, Krashes MJ. 2022. 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Nature 585:420–425. doi:10.1038/s41586-020-268 26 Zhang Z, Beier C, Weil T, Hattar S. 2021. The retinal ipRGC-preoptic circuit mediates the acu 27 light on sleep. Nat Commun 12:5115. doi:10.1038/s41467-021-25378-w 28 29 Thompson S, Recober A, Vogel TW, Kuburas A, Owens JA, Sheffield VC, Russo AF, Stone EM. 1 aversion in mice depends on nonimage-forming irradiance detection. Behav Neurosc 2 827. doi:10.1037/a0021568 3 Tomchek SD, Dunn W. 2007. Sensory processing in children with and without autism: a comp 4 study using the short sensory profile. Am J Occup Ther 61:190–200. doi:10.5014/ajot 5 Umino Y, Solessio E, Barlow RB. 2008. Speed, spatial, and temporal tuning of rod and cone vi 6 mouse. J Neurosci 28:189–198. doi:10.1523/JNEUROSCI.3551-07.2008 7 Vinberg F, Palczewska G, Zhang J, Komar K, Wojtkowski M, Kefalov VJ, Palczewski K. 2019. Se 8 Mammalian Cone Photoreceptors to Infrared Light. Neuroscience 416:100–108. 9 doi:10.1016/j.neuroscience.2019.07.047 10 Watabe-Uchida M, Eshel N, Uchida N. 2017. Neural Circuitry of Reward Prediction Error. Ann 11 Neurosci 40:373–394. doi:10.1146/annurev-neuro-072116-031109 12 Watabe-Uchida M, Zhu L, Ogawa SK, Vamanrao A, Uchida N. 2012. Whole-brain mapping of 13 to midbrain dopamine neurons. Neuron 74:858–873. doi:10.1016/j.neuron.2012.03. 14 Wise RA. 2004. Dopamine, learning and motivation. Nat Rev Neurosci 5:483–494. doi:10.103 15 Yao K, Qiu S, Wang YV, Park SJH, Mohns EJ, Mehta B, Liu X, Chang B, Zenisek D, Crair MC, De 16 B. 2018. Restoration of vision after de novo genesis of rod photoreceptors in mamm 17 retinas. Nature 560:484–488. doi:10.1038/s41586-018-0425-3 18 Yilmaz M, Meister M. 2013. Rapid innate defensive responses of mice to looming visual stim 19 23. doi:10.1016/j.cub.2013.08.015 20 Yuan L, Dou Y-N, Sun Y-G. 2019. Topography of Reward and Aversion Encoding in the Mesoli 21 Dopaminergic System. J Neurosci 39:6472–6481. 23. doi:10.1016/j.cub.2013.08.015 Yuan L, Dou Y-N, Sun Y-G. 2019. Topography of Reward and Aversion Encoding in the Mesolimbic 21 Dopaminergic System. J Neurosci 39:6472–6481. doi:10.1523/JNEUROSCI.0271-19.2019 22 Yuan L, Dou Y-N, Sun Y-G. 2019. Topography of Reward and Aversion Encoding in the Mesolimbic 21 Dopaminergic System. J Neurosci 39:6472–6481. doi:10.1523/JNEUROSCI.0271-19.2019 22 p g y Zhang KX, D’Souza S, Upton BA, Kernodle S, Vemaraju S, Nayak G, Gaitonde KD, Holt AL, Linne CD, Smith 23 AN, Petts NT, Batie M, Mukherjee R, Tiwari D, Buhr ED, Van Gelder RN, Gross C, Sweeney A, 24 Sanchez-Gurmaches J, Seeley RJ, Lang RA. 2020. Violet-light suppression of thermogenesis by 25 opsin 5 hypothalamic neurons. Nature 585:420–425. doi:10.1038/s41586-020-2683-0 26 Zhang KX, D’Souza S, Upton BA, Kernodle S, Vemaraju S, Nayak G, Gaitonde KD, Holt AL, Linne CD, Smith 23 AN, Petts NT, Batie M, Mukherjee R, Tiwari D, Buhr ED, Van Gelder RN, Gross C, Sweeney A, 24 Sanchez-Gurmaches J, Seeley RJ, Lang RA. 2020. Violet-light suppression of thermogenesis by 25 opsin 5 hypothalamic neurons. Nature 585:420–425. doi:10.1038/s41586-020-2683-0 26 Zhang Z, Beier C, Weil T, Hattar S. 2021. The retinal ipRGC-preoptic circuit mediates the acute effect of 27 light on sleep. Nat Commun 12:5115. doi:10.1038/s41467-021-25378-w 28 Zhang Z, Beier C, Weil T, Hattar S. 2021. The retinal ipRGC-preoptic circuit mediates the acute effect of 27 light on sleep. Nat Commun 12:5115. doi:10.1038/s41467-021-25378-w 28 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 20, 2022. ; https://doi.org/10.1101/2022.09.20.508670 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 20, 2022. ; https://doi.org/10.1101/2022.09.20.508670 doi: bioRxiv preprint VIDEO TITLES: 1 2 Video 1. Behavioral response to presentation of black looming discs on a light 3 background when mice entered the threat zone of a rectangular arena. 4 5 Video 2. Behavioral response to illumination of a spotlight when mice entered the target 6 zone of a rectangular arena. 7 8 9 Video 2. Behavioral response to illumination of a spotlight when mice entered the target 6 zone of a rectangular arena. 7 9 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 20, 2022. ; https://doi.org/10.1101/2022.09.20.508670 doi: bioRxiv preprint . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 20, 2022. ; https://doi.org/10.1101/2022.09.20.508670 doi: ioRxiv preprint FIGURES AND CAPTIONS 1 2 Figure 1. LNAc dopaminergic encoding of visual threats. 3 2 Figure 1. LNAc dopaminergic encoding of visual threats. 3 2 Figure 1. LNAc dopaminergic encoding of visual threats. 3 Figure 1. LNAc dopaminergic encoding of visual threats. 3 A. Fluorescent dopamine signals were recorded with dLight1 and fiber photometry in the nucleus 4 accumbens lateral shell (LNAc) during presentation of looming and control discs. B. Average 5 dLight1 response to trains of five black or contrast inverted discs ± standard error of the mean 6 (SEM). C. The dLight1 response to black or inverted discs was dependent on disc 7 color/background, disc type (static vs. looming vs. receding), and stimulus number (n = 11; 3-way 8 repeated measures ANOVA; F8,240 = 2.02, pdisc background x disc type x stimulus number = 0.045; F1,240 = 143.92, 9 pbackground < 0.001; F2,240 = 150.32, pdisc type < 0.001; F4,240 = 192.64, pstimulus number < 0.001). Bonferroni 10 post hoc tests revealed that contrast inverted discs evoked more dopamine than black discs. 11 Contrast inverted looming discs evoked less dopamine than inverted static and receding discs 12 A. Fluorescent dopamine signals were recorded with dLight1 and fiber photometry in the nucleus 4 accumbens lateral shell (LNAc) during presentation of looming and control discs. B. Average 5 dLight1 response to trains of five black or contrast inverted discs ± standard error of the mean 6 (SEM). C. The dLight1 response to black or inverted discs was dependent on disc 7 color/background, disc type (static vs. looming vs. receding), and stimulus number (n = 11; 3-way 8 repeated measures ANOVA; F8,240 = 2.02, pdisc background x disc type x stimulus number = 0.045; F1,240 = 143.92, 9 pbackground < 0.001; F2,240 = 150.32, pdisc type < 0.001; F4,240 = 192.64, pstimulus number < 0.001). Bonferroni 10 post hoc tests revealed that contrast inverted discs evoked more dopamine than black discs. 11 Contrast inverted looming discs evoked less dopamine than inverted static and receding discs 12 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 20, 2022. ; https://doi.org/10.1101/2022.09.20.508670 doi: bioRxiv preprint . FIGURES AND CAPTIONS 1 CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 20, 2022. ; https://doi.org/10.1101/2022.09.20.508670 doi: ioRxiv preprint after the first presentation. D. dLight1 transient peak latency was dependent on disc 1 color/background and disc type (n = 11; 2-way repeated measures ANOVA; F2,20 = 64.78, pdisc 2 background x disc type < 0.001; F1,20 = 25.69, pbackground < 0.001; F2,20 = 7.58, pdisc type = 0.01). Bonferroni 3 post hoc tests showed that contrast inverted static and receding discs evoked transients with 4 shorter latency compared to black discs. Additionally, transients evoked by contrast inverted 5 receding discs had shorter latency than contrast inverted looming discs. E. Escape velocity 6 following overhead disc presentation was dependent on disc color/background and disc type (n = 7 12; 2-way repeated measures ANOVA; F2,22 = 49.28, pdisc background x disc type < 0.001; F1,22 = 18.38, 8 pbackground = 0.001; F2,22 = 28.89, pdisc type < 0.001). Bonferroni post hoc tests showed that black 9 looming discs induced greater escape velocity than all other overhead discs. For panels C and D, 10 indicates p < 0.05 vs. black disc of the same type (e.g. black static disc vs. contrast inverted 11 static disc); + indicates p < 0.05 vs looming disc of the same color (e.g. black looming vs. black 12 receding disc). For panel E, * indicates p < 0.05 vs. other overhead discs. 13 14 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 20, 2022. ; https://doi.org/10.1101/2022.09.20.508670 doi: bioRxiv preprint 1 Figure 2. Dopaminergic responses to rapid dark to light transitions. 2 1 1 Figure 2. Dopaminergic responses to rapid dark to light transitions. 2 Figure 2. Dopaminergic responses to rapid dark to light transitions. 2 A. Instantaneous LCD screen transitions from dark to light evoked rapid dopamine release at 3 stimulus onset when compared to the pre-stimulus baseline (inset: baseline and stimulus-induced 4 dLight1 peak values for individual mice; n = 11; paired t-test; t10 = 7.01, p < 0.001). B. FIGURES AND CAPTIONS 1 CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 20, 2022. ; https://doi.org/10.1101/2022.09.20.508670 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 20, 2022. ; https://doi.org/10.1101/2022.09.20.508670 doi: bioRxiv preprint 1 Figure 3. Dopaminergic responses to repeated light stimuli. 2 1 Figure 3. Dopaminergic responses to repeated light stimuli. 2 Figure 3. Dopaminergic responses to repeated light stimuli. 2 A. (Left) Dopamine release evoked by 20 one-second white LED stimuli was reduced with 3 repeated exposures and was dependent on the interstimulus interval (ISI; 10 ms – 10 s; n = 9; 2- 4 way repeated measures ANOVA; F57,456 = 9.54, pstimulus number x interstimulus interval < 0.001; F19,456 = 72.98, 5 pstimulus number < 0.001; F3,456 = 63.91, pinterstimulus interval < 0.001). (Right) Averaged dLight1 fluorescent 6 traces showing the dopaminergic response to 20 one-second white LED light pulses with a 100 7 ms ISI (purple) or 10 s ISI (orange). B. Total habituation of the peak dLight1 response to repeated 8 stimuli (shown as the peak response to the 20th stimulus as a percentage of the 1st stimulus) is 9 dependent on the duration of the interstimulus interval (n = 9; 1-way repeated measures ANOVA; 10 F3,24 = 104.0, p < 0.001). Data shown with a semi-log fit (y-intercept: 48.92%, slope: 22.58 %s-1, 11 R2 = 0.86). C. (Left) Averaged dLight1 trace showing LNAc dopamine evoked by a 60 second 12 presentation of 40 Hz white LED flicker (inset: response during the first second after stimulus 13 onset) ± SEM. (Right) 40 Hz flicker only evoked significant dopamine release at stimulus onset (n 14 = 10; 1-way repeated measures ANOVA; F2,18 = 100.4, p < 0.001). Bonferroni post hoc tests 15 A. FIGURES AND CAPTIONS 1 dLight1 5 responses to the onset of LCD screen dark to light transitions at different transition lengths (0.05 6 – 2.0 s) ± SEM. C. The magnitude (pink) and latency (teal) of dopaminergic responses to dark- 7 to-light transitions varied non-linearly depending on transition speed (peak amplitude: one-phase 8 exponential decay, y0 = 8.84 z-score, plateau = 1.36 z-score, tau = 0.43 s, R2 = 0.90; peak latency: 9 one-phase exponential association, y0 = 0.43 ms, plateau = 0.59 ms, tau = 0.54 s, R2 = 0.98). D. 10 dLight1 responses to the onset ten-second white LED stimuli across a range of irradiances (0 11 W/cm2 – 0.01 W/cm2) ± SEM. E. The magnitude of the dopaminergic response to 10s white 12 LED stimuli was dependent on the stimulus irradiance (n = 7; 1-way repeated measures ANOVA; 13 F7,42 = 38.79, p < 0.001). Data shown with a one-phase exponential association fit (y0 = 1.20 z- 14 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 20, 2022. ; https://doi.org/10.1101/2022.09.20.508670 doi: bioRxiv preprint . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 20, 2022. ; https://doi.org/10.1101/2022.09.20.508670 doi: ioRxiv preprint score, plateau = 0.84 z-score, tau = 0.00074 W/cm2, R2 = 0.63). F. The latency of the 1 dopaminergic response to 10 s white LED stimuli was dependent on the stimulus irradiance (n = 2 7; 1-way repeated measures ANOVA; F6,36 = 47.35, p < 0.001). Data shown with a one-phase 3 exponential decay fit (y0 = 0.42 ms, plateau = 0.35 ms, tau = 0.0079 W/cm2, R2 = 0.76). G. The 4 dopaminergic response to 5.0 W/cm2 white light was not different (right) if measured at the 5 beginning of the vivarium dark (left) or light (center) phase of the day-night cycle (n = 11; paired 6 t-test; t10 = 1.27, p = 0.23). In all panels, * indicates p < 0.05. 7 8 9 . FIGURES AND CAPTIONS 1 Bonferroni post hoc tests revealed that the dLight1 13 response to the test stimulus onset was significantly smaller than the response to the onset of the 14 preconditioning stimulus, regardless of its duration. There was no difference between the dLight1 15 response to the onset of the preconditioning (p = 0.11) or test stimulus (p = 0.40) between 16 experiments. G. The dLight1 response to light offset was larger for a 300 s light stimulus compared 17 to a 1 s light stimulus (n = 11; paired t-test; t10 = 4.91, p < 0.001). In all panels, * indicates p < 18 0.05. 19 confirmed that the dLight1 peak at LED onset was greater than baseline and offset responses, 1 which did not differ from each other (p = 0.09). D. (Left) No habituation in the peak dLight1 2 response to repeated 1 s white LED stimuli was observed when the ISI was sufficiently long (100 3 s) – both before and after presentation of 300 one-second LED stimuli with a one-second ISI (n = 4 7; 2-way repeated measures ANOVA; F4,24 = 0.73, pstimulus number x exposure history = 0.52; F4,24 = 1.01, 5 pstimulus number = 0.40; F1,24 = 4.61, pexposure history = 0.08). (Right) Averaged dLight1 fluorescent traces 6 showing the dopaminergic response to 5 one-second white LED light pulses with a 100 s ISI 7 before (black) or after (pink) 300 one-second LED stimuli. E. Averaged dLight1 fluorescent traces 8 showing the dopaminergic response to a 1 s white LED stimulus one second after a 1 s (left) or 9 300 s (right) preconditioning stimulus ± SEM. F. The dLight1 response to a 1 s white LED test 10 stimulus was not dependent on the length of the preconditioning stimulus (n = 11; 2-way repeated 11 measures ANOVA; F1,10 = 0.27, pinitial stimulus length x stimulus Number = 0.61; F1,10 = 3.83, pinitial stimulus length = 12 0.08; F1,10 = 55.10, pstimulus number < 0.001). Bonferroni post hoc tests revealed that the dLight1 13 response to the test stimulus onset was significantly smaller than the response to the onset of the 14 preconditioning stimulus, regardless of its duration. There was no difference between the dLight1 15 response to the onset of the preconditioning (p = 0.11) or test stimulus (p = 0.40) between 16 experiments. G. FIGURES AND CAPTIONS 1 (Left) Dopamine release evoked by 20 one-second white LED stimuli was reduced with 3 repeated exposures and was dependent on the interstimulus interval (ISI; 10 ms – 10 s; n = 9; 2- 4 way repeated measures ANOVA; F57,456 = 9.54, pstimulus number x interstimulus interval < 0.001; F19,456 = 72.98, 5 pstimulus number < 0.001; F3 456 = 63.91, pinterstimulus interval < 0.001). (Right) Averaged dLight1 fluorescent 6 . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 20, 2022. ; https://doi.org/10.1101/2022.09.20.508670 doi: ioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 20, 2022. ; https://doi.org/10.1101/2022.09.20.508670 doi: bioRxiv preprint confirmed that the dLight1 peak at LED onset was greater than baseline and offset responses, 1 which did not differ from each other (p = 0.09). D. (Left) No habituation in the peak dLight1 2 response to repeated 1 s white LED stimuli was observed when the ISI was sufficiently long (100 3 s) – both before and after presentation of 300 one-second LED stimuli with a one-second ISI (n = 4 7; 2-way repeated measures ANOVA; F4,24 = 0.73, pstimulus number x exposure history = 0.52; F4,24 = 1.01, 5 pstimulus number = 0.40; F1,24 = 4.61, pexposure history = 0.08). (Right) Averaged dLight1 fluorescent traces 6 showing the dopaminergic response to 5 one-second white LED light pulses with a 100 s ISI 7 before (black) or after (pink) 300 one-second LED stimuli. E. Averaged dLight1 fluorescent traces 8 showing the dopaminergic response to a 1 s white LED stimulus one second after a 1 s (left) or 9 300 s (right) preconditioning stimulus ± SEM. F. The dLight1 response to a 1 s white LED test 10 stimulus was not dependent on the length of the preconditioning stimulus (n = 11; 2-way repeated 11 measures ANOVA; F1,10 = 0.27, pinitial stimulus length x stimulus Number = 0.61; F1,10 = 3.83, pinitial stimulus length = 12 0.08; F1,10 = 55.10, pstimulus number < 0.001). FIGURES AND CAPTIONS 1 The dLight1 response to light offset was larger for a 300 s light stimulus compared 17 to a 1 s light stimulus (n = 11; paired t-test; t10 = 4.91, p < 0.001). In all panels, * indicates p < 18 0 05 19 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 20, 2022. ; https://doi.org/10.1101/2022.09.20.508670 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 20, 2022. ; https://doi.org/10.1101/2022.09.20.508670 doi: bioRxiv preprint Figure 4. Dopaminergic responses to individual wavelengths across the visual spectrum. 1 Figure 4. Dopaminergic responses to individual wavelengths across the visual spectrum. 2 A. The dopaminergic response to UV (360 nm), blue (475 nm), green (555 nm), and red (635 nm) 3 LED light was wavelength and irradiance-dependent (n = 8; 2-way repeated measures ANOVA; 4 F12,84 = 9.63, pwavelength x irradiance < 0.001; F3,84 = 37.59, pwavelength < 0.001; F4,84 = 10.08, pirradiance = 5 0.004). Bonferroni post hoc tests revealed that dopamine evoked by UV and red light was smaller 6 than blue and green wavelengths at the lowest irradiance tested (0.001 W/cm2). The dLight1 7 response to the red LED was also significantly lower than blue and green LEDs at irradiances of 8 0.01 W/cm2 and 0.1 W/cm2. For comprehensive reporting of all significant post hoc tests across 9 irradiances and wavelengths, see the provided Supplemental Data and Statistical Analysis file. 10 B. Averaged dLight1 trace showing LNAc dopamine evoked by either 0.001 W/cm2 (1 nW/cm2) 11 or 10 W/cm2 UV, blue, green, or red LEDs ± SEM. C. The dopaminergic response to 5.0 W/cm2 12 white light was significantly reduced in Gnat1/2 double knockout (dKO) mice relative to wildtype 13 controls (nWT = 4, nKO = 6; unpaired t-test; t8 = 7.08, p < 0.001). D. The reduction in the dLight1 14 response to 10 W/cm2 light in Gnat1/2 dKO was wavelength dependent (2-way repeated 15 A. FIGURES AND CAPTIONS 1 The dopaminergic response to UV (360 nm), blue (475 nm), green (555 nm), and red (635 nm) 3 LED light was wavelength and irradiance-dependent (n = 8; 2-way repeated measures ANOVA; 4 F12,84 = 9.63, pwavelength x irradiance < 0.001; F3,84 = 37.59, pwavelength < 0.001; F4,84 = 10.08, pirradiance = 5 0 004) Bonferroni post hoc tests revealed that dopamine evoked by UV and red light was smaller 6 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 20, 2022. ; https://doi.org/10.1101/2022.09.20.508670 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 20, 2022. ; https://doi.org/10.1101/2022.09.20.508670 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is ma The copyright holder for this prepr this version posted November 20, 2022. ; https://doi.org/10.1101/2022.09.20.508670 doi: bioRxiv preprint measures ANOVA; F3,24 = 7.02, pgenotype x wavelength = 0.002; F3,24 = 17.54, pwavelength = 0.003; F1,24 = 1 85.80, pgenotype < 0.001). Bonferroni post hoc tests revealed that the dLight1 response to blue (475 2 nm), green (555 nm), and red (635 nm) light was lower in Gnat1/2 mice relative to wildtype 3 littermates. E. The dopaminergic response to 5.0 W/cm2 white light was not different in Opn4 4 (melanopsin) knockout mice relative to wildtype controls (nWT = 11, nKO = 6; unpaired t-test; t15 = 5 0.75, p = 0.46). 6 measures ANOVA; F3,24 = 7.02, pgenotype x wavelength = 0.002; F3,24 = 17.54, pwavelength = 0.003; F1,24 = 1 85.80, pgenotype < 0.001). Bonferroni post hoc tests revealed that the dLight1 response to blue (475 2 nm), green (555 nm), and red (635 nm) light was lower in Gnat1/2 mice relative to wildtype 3 littermates. E. FIGURES AND CAPTIONS 1 The dopaminergic response to 5.0 W/cm2 white light was not different in Opn4 4 (melanopsin) knockout mice relative to wildtype controls (nWT = 11, nKO = 6; unpaired t-test; t15 = 5 0.75, p = 0.46). 6 7 7 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 20, 2022. ; https://doi.org/10.1101/2022.09.20.508670 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 20, 2022. ; https://doi.org/10.1101/2022.09.20.508670 doi: bioRxiv preprint 1 Figure 2-Figure Supplement 1. 2 A. The magnitude of the dopaminergic response to dark-to-light transitions was dependent on the 3 stimulus transition time (n = 11; 1-way repeated measures ANOVA; F7,70 = 17.76, p < 0.001). B. 4 The latency of the peak dopaminergic response to dark-to-light transitions was dependent on the 5 stimulus transition time (n = 11; 1-way repeated measures ANOVA; F7,70 = 11.03, p = 0.007). In 6 A and B, pink circles (mean ± SEM) and gray dots (individual values) represent data derived from 7 fluorescent dLight1 traces averaged across trials for each individual mice. Shown for comparison, 8 black circles represent data derived from fluorescence traces averaged across all mice prior to 9 peak detection, which yielded a single value for each transition time. C. The dopaminergic 10 response to 80 dB tones was dependent on their frequency (n = 10; 1-way repeated measures 11 ANOVA; F5,45 = 10.11, p < 0.001) and was less than the response to a 5 W/cm2 LED light stimulus 12 (Mixed-effects ANOVA; F = 26.62, pstimulus < 0.001). Bonferroni post hoc tests revealed that the 13 dLight1 response to the light stimulus was significantly larger than all tone responses. D. (Left) 14 Opening the enclosure peephole evoked significant dopamine release at stimulus onset when 15 compared to the pre-stimulus baseline (n = 6; paired t-test; t5 = 5.72, p = 0.002). (Right) Averaged 16 1 Fi 2 Fi S l 1 1 Figure 2-Figure Supplement 1. 2 g g pp A. FIGURES AND CAPTIONS 1 The magnitude of the dopaminergic response to dark-to-light transitions was dependent on the 3 stimulus transition time (n = 11; 1-way repeated measures ANOVA; F7,70 = 17.76, p < 0.001). B. 4 The latency of the peak dopaminergic response to dark-to-light transitions was dependent on the 5 stimulus transition time (n = 11; 1-way repeated measures ANOVA; F7,70 = 11.03, p = 0.007). In 6 A and B, pink circles (mean ± SEM) and gray dots (individual values) represent data derived from 7 fluorescent dLight1 traces averaged across trials for each individual mice. Shown for comparison, 8 black circles represent data derived from fluorescence traces averaged across all mice prior to 9 peak detection, which yielded a single value for each transition time. C. The dopaminergic 10 response to 80 dB tones was dependent on their frequency (n = 10; 1-way repeated measures 11 ANOVA; F5,45 = 10.11, p < 0.001) and was less than the response to a 5 W/cm2 LED light stimulus 12 (Mixed-effects ANOVA; F = 26.62, pstimulus < 0.001). Bonferroni post hoc tests revealed that the 13 dLight1 response to the light stimulus was significantly larger than all tone responses. D. (Left) 14 Opening the enclosure peephole evoked significant dopamine release at stimulus onset when 15 compared to the pre-stimulus baseline (n = 6; paired t-test; t5 = 5.72, p = 0.002). (Right) Averaged 16 dLight1 trace showing LNAc dopamine evoked by opening the peephole on the door of the 17 behavioral testing chamber enclosure to observe mouse behavior ± SEM. The irradiance 18 associated with this manipulation was 17 nW/cm2 measured at mouse level. In all panels, * 19 indicates p < 0.05. 20 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 20, 2022. ; https://doi.org/10.1101/2022.09.20.508670 doi: bioRxiv preprint 1 Figure 4-Figure Supplement 1. 2 A. (Left) 100 W/cm2 far-red (730 nm) LED light evoked significantly greater dopamine release 3 compared to 10 W/cm2 light (n = 8; paired t-test; t7 = 2.54, p = 0.04). (Right) Averaged dLight1 4 trace showing LNAc dopamine evoked by a 100 W/cm2 far-red LED ± SEM. B. FIGURES AND CAPTIONS 1 (Left) The latency 5 of the peak dLight1 response to 5.0 W/cm2 white light was significantly greater in Gnat1/2 double 6 knockout (dKO) mice relative to wildtype controls (WT; nWT = 4, nKO = 6; unpaired t-test; t8 = 4.77, 7 p = 0.001). (Right) There was no difference in the latency of the peak dLight1 response to 5.0 8 W/cm2 white light in Opn4 knockout mice (KO) relative to wildtype controls (WT; black; nWT = 11, 9 nKO = 6; unpaired t-test; t15 = 0.16, p = 0.87). C. Averaged dLight1 trace showing LNAc dopamine 10 evoked by 10 W/cm2 UV (360 nm), blue (475 nm), green (555 nm), or red (635) LEDs ± SEM in 11 wildtype (orange) or Gnat1/2-dKO (black) mice. * indicates p < 0.05. 12
https://openalex.org/W2142044828	https://europepmc.org/articles/pmc4783449?pdf=render	English	null	Allocation of the S-genome chromosomes of Aegilops variabilis Eig. carrying powdery mildew resistance in triticale (× Triticosecale Wittmack)	Protoplasma	2,015	cc-by	11,108	Protoplasma (2016) 253:329–343 DOI 10.1007/s00709-015-0813-6 Protoplasma (2016) 253:329–343 DOI 10.1007/s00709-015-0813-6 ORIGINAL ARTICLE Allocation of the S-genome chromosomes of Aegilops variabilis Eig. carrying powdery mildew resistance in triticale (× Triticosecale Wittmack) M. Kwiatek1 & J. Belter1 & M. Majka1 & H. Wiśniewska1 Received: 23 February 2015 /Accepted: 25 March 2015 /Published online: 14 April 2015 # The Author(s) 2015. This article is published with open access at Springerlink.com Abstract It has been hypothesized that the powdery mildew adult plant resistance (APR) controlled by the Pm13 gene in Aegilops longissima Schweinf. & Muschl. (SlSl) has been evolutionary transferred to Aegilops variabilis Eig. (UUSS). The molecular marker analysis and the visual evaluation of powdery mildew symptoms in Ae. variabilis and the Ae. variabilis × Secale cereale amphiploid forms (2n=6x=42, UUSSRR) showed the presence of product that corresponded to Pm13 marker and the lower infection level compared to susceptible model, respectively. This study also describes the transfer of Ae. variabilis Eig. (2n=4x=28, UvUvSvSv) chro- mosomes, carrying powdery mildew resistance, into triticale (× Triticosecale Wittm., 2n=6x=42, AABBRR) using Ae. variabilis × S. cereale amphiploid forms. The individual chro- mosomes of Ae. variabilis, triticale ‘Lamberto’ and hybrids were characterized by genomic and fluorescence in situ hy- bridization (GISH/FISH). The chromosome configurations of obtained hybrid forms were studied at first metaphase of meiosis of pollen mother cells (PMCs) using GISH. The sta- tistical analysis showed that the way of S-genome chromo- some pairing and transmission to subsequent hybrid genera- tions was diploid-like and had no influence on chromosome pairing of triticale chromosomes. The cytogenetic study of hybrid forms were supported by the marker-assisted selection using Pm13 marker and visual evaluation of natural infection by Blumeria graminis, that allowed to select the addition or substitution lines of hybrids carrying chromosome 3Sv which were tolerant to the powdery mildew infection. Keywords Aegilops . Chromosome transfer . In situ hybridization . Molecular marker . Powdery mildew . Resistance genes . Triticale Introduction Powdery mildew caused by Blumeria graminis (DC.) E.O. Speer f. sp. Tritici Em. Marchal (Bgt) = Erysiphe graminis DC. Ex Merat f. sp. Tritici Em. Marchal is one of the wide- spread fungal diseases in cereals. This pathogen has recently infected triticale (× Triticosecale Wittm.), man-made, artificial cereal, which was created to combine the characteristics of cold, disease tolerance and adaptation to unfavourable soils and climates with the productivity and nutritional qualities (Woś et al. 2002). At the beginning of the triticale production, the diseases did not appear to be a serious limitation, probably because of lack of the appropriate, triticale-directed pathotypes of fungal pathogens. Moreover, the grown areas of this crop were incidental to cause serious shifts in the path- ogen virulence (Ammar et al. 2004). While the harvest area of triticale began to increase, the new hybrid pathotypes carrying virulence genes appeared (Arseniuk 1996). The new, resistant cultivars could eliminate the fungicides accumulation in grain Handling Editor: Heiti Paves * M. Kwiatek mkwi@igr.poznan.pl J. Belter jbel@igr.poznan.pl M. Majka mmaj@igr.poznan.pl H. Wiśniewska hwis@igr.poznan.pl 1 Institute of Plant Genetics, Polish Academy of Sciences, Strzeszyńska 34, 60-479 Poznań, Poland Handling Editor: Heiti Paves 1998) of Ae. variabilis (UvUvSvSv). Ae. variabilis has been used as a donor of desir- able genes to wheat through interspecific hybridization such as powdery mildew resistance (Spetsov et al. 1997), leaf rust resistance (Marais et al. 2008) and resistance to nematodes (Coriton et al. 2009). and reduce the crop losses caused by powdery mildew. Two types of resistance to powdery mildew have been identified so far (Flor 1971). First is called monogenic (vertical) or rac- specific resistance, which is effective for some isolates of the pathogen, but ineffective for others. Race-specific resistance is expressed in seedlings and involve single major R genes, in a gene-for-gene interaction (Chen and Chełkowski 1999). Race-specific resistance genes are widely used to combat the wheat diseases, yet the resistance is often short-lived, espe- cially when the genes are employed singly in new varieties (Marais et al. 2008). Second type of resistance to powdery mildew is known as an adult plant resistance (APR), also called ‘slow mildewing’ and ‘partial resistance,’ which decel- erates the infection, growth and reproduction of the pathogen in adult plants. APR to powdery mildew is more durable than race-specific resistance; therefore it is more desirable in breed- ing programmes. One of the APR genes is Pml3 powdery mildew resistance gene that ensures high tolerance to all known races of this disease in wheat. The Pm13 gene has been transferred from the chromosome 3S1 of Aegilops longissima Schweinf. & Muschl. (2n=2x=14 chromosomes; SlSl) into common wheat, Triticum aestivum L. cv. ‘Chinese Spring’ (Ceoloni et al. 1988). Considering the synteny in the genome construction of related species, which evolved from a com- mon ancestral gene by speciation, Cenci et al. (2003) hypoth- esized that the Pm13 marker linked with powdery resistant gene has a conservative character. On this basis, it can be assumed that species with S-genome chromatin such as tetra- ploids (Aegilops variabilis Eig.) and hexaploids (Aegilops vavilovi Zhuk.) could carry the genomic region responsible for powdery mildew resistance. What is more, Ae. longissima is considered as a donor of S-genome (Yu and Jahier 1992; Zhang et al. 1992; Badaeva et al. 1998) of Ae. variabilis (UvUvSvSv). Ae. variabilis has been used as a donor of desir- able genes to wheat through interspecific hybridization such as powdery mildew resistance (Spetsov et al. 1997), leaf rust resistance (Marais et al. 2008) and resistance to nematodes (Coriton et al. 2009). Handling Editor: Heiti Paves variabilis × Secale cereale (UvUvSvSvRR) in the crosses with triticale (AABBRR) will have a significant impact on F1 hy- brid stability because of R-genome chromosomes, which will be able to pair during prophase I of meiosis and will ensure the functional daughter cells formation and sufficient level of vital pollen grains as a consequence. In this purpose, four subsequent generations (F1 to BC2F2) of (Ae. variabilis × S. cereale) × triticale hybrids were obtain- ed. The chromosome composition during metaphase of mito- sis in root apical meristems and chromosome pairing during metaphase I (MI) of meiosis of the pollen mother cells (PMCs) were characterized using fluorescence and genomic in situ hybridization (FISH/GISH). Finally, the Pm13 marker (Cenci et al. 1998) was verified in the Ae. variabilis, parental components and in the hybrid plants and compared with visual evaluation of powdery mildew infection. Handling Editor: Heiti Paves Key message We have demonstrated the Pm13 resistance gene originally found on chromosome 3Sl of Ae. longissima has been transferred to Ae. variabilis. We have obtained 26 triticale plants carrying 3Sv chromosome(s) with the powdery mildew resistance. * M. Kwiatek mkwi@igr.poznan.pl J. Belter jbel@igr.poznan.pl M. Majka mmaj@igr.poznan.pl H. Wiśniewska hwis@igr.poznan.pl 1 Institute of Plant Genetics, Polish Academy of Sciences, Strzeszyńska 34, 60-479 Poznań, Poland * M. Kwiatek mkwi@igr.poznan.pl J. Belter jbel@igr.poznan.pl M. Majka mmaj@igr.poznan.pl H. Wiśniewska hwis@igr.poznan.pl 1 Institute of Plant Genetics, Polish Academy of Sciences, Strzeszyńska 34, 60-479 Poznań, Poland 330 M. Kwiatek et al. and reduce the crop losses caused by powdery mildew. Two types of resistance to powdery mildew have been identified so far (Flor 1971). First is called monogenic (vertical) or rac- specific resistance, which is effective for some isolates of the pathogen, but ineffective for others. Race-specific resistance is expressed in seedlings and involve single major R genes, in a gene-for-gene interaction (Chen and Chełkowski 1999). Race-specific resistance genes are widely used to combat the wheat diseases, yet the resistance is often short-lived, espe- cially when the genes are employed singly in new varieties (Marais et al. 2008). Second type of resistance to powdery mildew is known as an adult plant resistance (APR), also called ‘slow mildewing’ and ‘partial resistance,’ which decel- erates the infection, growth and reproduction of the pathogen in adult plants. APR to powdery mildew is more durable than race-specific resistance; therefore it is more desirable in breed- ing programmes. One of the APR genes is Pml3 powdery mildew resistance gene that ensures high tolerance to all known races of this disease in wheat. The Pm13 gene has been transferred from the chromosome 3S1 of Aegilops longissima Schweinf. & Muschl. (2n=2x=14 chromosomes; SlSl) into common wheat, Triticum aestivum L. cv. ‘Chinese Spring’ (Ceoloni et al. 1988). Considering the synteny in the genome construction of related species, which evolved from a com- mon ancestral gene by speciation, Cenci et al. (2003) hypoth- esized that the Pm13 marker linked with powdery resistant gene has a conservative character. On this basis, it can be assumed that species with S-genome chromatin such as tetra- ploids (Aegilops variabilis Eig.) and hexaploids (Aegilops vavilovi Zhuk.) could carry the genomic region responsible for powdery mildew resistance. What is more, Ae. longissima is considered as a donor of S-genome (Yu and Jahier 1992; Zhang et al. 1992; Badaeva et al. Plant material Glasshouse experiments were carried out in four subsequent vegetation seasons at Institute of Plant Genetics, Polish Academy of Sciences in Poznań, Poland. Seeds of Aegilops umbellulata Zhuk. (PI 222762; 2n=2x=14; UuUu) and Ae. longissima (PI 604112; 2n=2x=14; SlSl) were kindly sup- plied for the study from the National Small Grains Germplasm Research Facility, National Small Grains Collection (Aberdeen, Idaho, USA). Seeds of Ae. variabilis were received from the collection of Professor M. Feldman (The Weizmann Institute of Science, Israel). The Ae. variabilis × S. cereale amphiploids (UvUvSvSvRR, 2n=6x=42) were obtained by Wojciechowska and Pudelska (1999). The F1 (Ae. variabilis × S. cereale) × triticale hybrids were obtained by crossing of triticale cv. ‘Lamberto’ with Ae. variabilis × S. cereale amphiploids as a pollinator. Backcrosses with the triticale as a male parent were used to achieve following gen- erations (BC1F1 and BC2F1). Finally, the self-pollinations of BC2F1 hybrids were made to gain BC2F2 plants. The percent- age ratio of the total amount of seeds from each plant with the total amount of pollinated flowers of each plant was calculated (Table 1). The aims of this study were to: (1) evaluate the presence and the expression of Pm13 gene in Ae. variabilis; (2) to identify the individual chromosomes of Ae. variabilis respon- sible for powdery mildew resistance and (3) transfer them into triticale. The distant crossing between diploid Aegilops species and hexaploid triticale can be disturbed because of (1) different ploidy level of the parental components and (2) the expression of Ph1 gene located on chromosome 5B in wheat (or triticale), responsible for homologues chromosome pairing during mei- osis (Riley and Chapman 1958; Lukaszewski and Kopecký 2010). To avoid the unwanted crossing limitations connected with different chromosome number in parental forms and to circumvent the chromosome pairing system controlled by Ph1 gene, we assumed that using amphiploid forms of Ae. Chromosome preparation Seeds were germinated on moist filter paper in Petri dishes for 3–4 days. For mitosis metaphase accumulation, the root-tips were collected and stored in ice for 26 h. Afterwards, the plants were placed in the vernalisation chamber for 6 weeks and then located in the glasshouse until harvest. The fixation of the root-tips was made using ethanol and acetic acid (3:1, v/v). The chromosome preparations were made according to Triticale distant hybrids with powdery mildew resistance 331 Table 1 Results of distant crossing between hexaploid (2n=6x=42) forms of triticale ‘Lamberto’ with Ae. variabilis × S. cereale amphiploid and its progeny Table 1 Results of distant crossing between hexaploid (2n=6x=42) forms of triticale ‘Lamberto’ with Ae. variabilis × S. cereale amphiploid and its progeny Hybrid generation Cross combination Number of pollinated flowers Number of seeds obtained Crossability Number of adult plants with Pm13 marker Female parent Male parent F1 triticale (6x) Ae. variabilis × S. cereale (6x) 106 19 0.18 6 BC1F1 F1 Triticale (6x) 68 17 0.25 5 BC2F1 1 Triticale (6x) 46 3 0.07 0 3 Triticale (6x) 116 6 0.05 0 4 Triticale (6x) 82 11 0.13 11 6 Triticale (6x) 30 2 0.03 1 7 Triticale (6x) 56 3 0.05 3 BC2F2 4/1 Self 64 0 0 0 4/2 Self 44 0 0 0 4/3 Self 52 0 0 0 4/4 Self 48 0 0 0 4/5 Self 74 27 0.36 2 4/6 Self 64 3 0.05 3 4/7 Self 60 0 0 0 4/8 Self 40 0 0 0 4/9 Self 52 0 0 0 4/10 Self 73 10 0.13 10 4/11 Self 66 10 0.15 10 6/1 Self 68 0 0 0 7/1 Self 48 0 0 0 7/2 Self 17 0 0 0 7/3 Self 52 0 0 0 sing between hexaploid (2n=6x=42) forms of triticale ‘Lamberto’ with Ae. variabilis × S. cereale amphiploid and its wheat clone pTa794 (Gerlach and Dyer 1980) by polymerase chain reaction (PCR) with tetramethyl-rhodamine-5-dUTP (Roche) using universal M13 ‘forward’ (5′-CAG GGT TTT CCC AGT CAC GA-3′) and ‘reverse’ (5′-CGG ATA ACA ATT TCA CAC AGG A-3′) sequencing primers. The thermal cycling programme consist of the following: 94 °C for 1 min, 39 cycles of 94 °C for 40 s, 55 °C for 40 s, and 72 °C for 90 s, and 72 °C for 5 min. Chromosome preparation The 25S rDNA probe was made by nick translation of a 2.3-kb ClaI sub-clone of the 25-5.8-18S rDNA coding region of Arabidopsis thaliana (Unfried and Gruendler 1990) with digoxigenin-11-dUTP (Roche). It was used for detection of 25-5.8-18S rDNA loci. The pSc119.2 repetitive DNA sequence, kindly supplied from Dr Kubalaková (Laboratory of Molecular Cytogenetics and Cytometry, Inst itute of Experi mental Botany, Olomouc, Czech Republic), was amplified and labelled by PCR with digoxigenin-11-dUTP (Roche) by using universal M13 primers (Vrána et al. 2000). The probe pAs1 (Afa family) was amplified by PCR from the genomic DNA of Ae. tauschii and labelled with digoxigenin-11-dUTP (Roche) according to Nagaki et al. (1995). Digoxigenin detection was made using anti-digoxigenin-fluorescein antibody (Roche). Hasterok et al. (2006). The F1 to BC2F2 hybrids were grown in the nursery and their meiotic behaviour was analysed in PMCs at MI of meiosis. Anthers of the hybrids containing PMCs at MI were fixed in 1:3 (v/v) acetic acid/ethanol and stored at −20 °C for a maximum of 2 months. MI of meiosis prepara- tions were made according to Zwierzykowski et al. (2008). The anthers were squashed in 45 % acetic acid, and the slides were stored at 4 °C until in situ hybridization. PCR amplification of powdery mildew resistance gene marker Genomic DNA was extracted from fresh leaves of single plants using GeneMATRIX Plant & Funghi DNA Purification Kit (EURx Ltd.). Total genomic DNAs of F1 to BC2F2 hybrids were used as templates for PCR. The reaction was performed in 25 μl reaction mixture containing: 1.5 μl 50 ng/μl of DNA, 2.5 μl 10×PCR buffer (50 mM KCl, 1.5 mM MgCl2, 10 mM Tris-HCl, pH 8.8, 0.1 % Triton X-100), 1 μl 2.5 mM dNTPs (Thermo Fisher Scientific, Waltham, MA, USA), 12.5 pmol of each primer (UTV14 for- ward: CGC CAG CCA ATT ATC TCC ATG A and UTV14 reverse: AGC CAT GCG CGG TGT CAT GTG AA; Cenci et al. 1998) (Sigma), and 16 μl MQ H2O, 0.5 μl (2 U/μl) Taq Polymerase (Thermo Fisher Scientific). Amplifications were carried out in LabCycler thermocycler (SensoQuest Biomedizinische Elektronik, Goettingen, Germany). Amplification products were electrophoresed at 5 V/cm for about 3 h in 1.5 % agarose gel (Sigma), stained with ethidium bromide (Sigma), visualized under UV light and photographed (Syngen UV visualiser). Second FISH with pSc119.2 and pAs1 (labelled with digoxygenin-11-dUTP and tetramethyl-rhodamine-5-dUTP, respectively) was made with the same conditions after reprobing. After second reprobing, GISH was carried out ac- cording to Kwiatek et al. (2012) with modifications. Multicolour GISH was carried out using U-genome probe (from Ae. umbellulata), Sl-genome probe (from Ae. longissima) and unlabelled triticale genomic DNA which was used as specific blocker. The GISH mixture (40 μL per slide), containing 50 % formamide, 2×SSC, 10 % dextran sulphate, 90 ng each of the genome probes, and 4.5 μg blocking DNA, was denatured at 75 °C for 10 min and stored on ice for 10 min. In case of initial GISH on triticale ‘Lamberto’ chromosomes, the hybridization mix contained the following: A-genome probe generated from genomic DNA of Triticum monococcum L., R-genome probe (rye, S. cereale L.) and blocking DNA from B-genome (Aegilops speltoides Tausch; 2n=2x=14; SS). The chromosomal DNA denaturation, hybridization and immunodetection conditions were the same as above-mentioned. Mitotic and meiotic (MI) cells were examined with an Olympus XM10 CCD camera attached to an Olympus BX 61 automatic epifluorescence microscope. Image processing was carried out using Olympus Cell-F (version 3.1; Olympus Soft Imaging Solutions GmbH: Münster, Germany) imaging software and PaintShop Pro X5 software (version 15.0.0.183; Corel Corporation, Ottawa, Canada). In situ hybridization 2005; Wiśniewska et al. 2013). Single-factor analysis of var- iance and Tukey’s Honest Significant Difference (HSD) test was used to examine the differences of means of chromosome configurations between plants from respective generations and the differences of means of chromosome configurations between plants from BC2F1 with comparison to their progeny in BC2F2 generation. FISH was carried out to study the mitotic chromosomes of root meristems. On the other hand, GISH was used to examine both the mitotic chromosomes of root meristemes and meiotic chromosomes of PMCs. Four probes were subjected to in situ hybridization on the same chromosome preparations. First FISH was made according to Książczyk et al. (2011) with minor modifications of Kwiatek et al. (2013), using 25S (used for detection of 25-5.8-18S rDNA loci) and 5S rDNA (pTa794). The hybridization mixture (40 μl per slide) contained 90 ng of each probe in the presence of salmon sperm DNA, 50 % formamide, 2×SSC, 10 % dextran sul- phate, and was denatured at 75 °C for 10 min and stored on ice for 10 min. Chromosomal DNAwas denatured in the pres- ence of the hybridization mixture at 75 °C for 5 min and allowed to hybridize overnight at 37 °C. For detection of the hybridization signals, anti-digoxigenin conjugated with FITC (Roche) was used. After documentation of the FISH sites, the slides were washed according to Heslop-Harrison (2000) (2× 45 min in 4×SSC Tween, 2×5 min in 2×SSC, at room temperature). PCR amplification of powdery mildew resistance gene marker The identification of particular chromosomes were made by comparing the signal pattern of 5S rDNA, 25S rDNA, pSc119.2 and pAs1 probes according previous study (Kwiatek et al. 2013) and similar cytogenetic analysis (Cuadrado and Jouve 1994; Schneider et al. 2003, Evaluation of the powdery mildew infection During the vegetation period, the level of powdery mildew natural infection was evaluated according to COBORU (Cultivated Varieties National Research Centre) recommenda- tions on a 9° scale, where 9 is the most favourable state for agriculture (Fig. 1b, c). The means of powdery mildew ex- pression scores in BC1F1, BC2F1, BC2F2 hybrids, Ae. variabilis × S. cereale ampiploids and triticale ‘Lamberto’ were compared each year to the results of PCR amplification of Pm13 marker using ANOVA calculations and Tukey’s HSD test. Probe labelling Total genomic DNA was extracted from fresh leaves of Ae. umbellulata (UU), Ae. longissima (SlSl) and triticale ‘Lamberto’ (AABBRR) using GeneMATRIX Plant & Funghi DNA Purification Kit (EURx Ltd.). Genomic DNA from Ae. umbellulata and Ae. longissima was labelled by nick translation (using NickTranslation Kit, Roche, Mannheim, Germany) with digoxigenin-11-dUTP (Roche) or tetramethyl-5-dUTP-rhodamine (Roche), respectively. Blocking DNA from triticale was sheared to fragments of 5– 10 kb by boiling for 30–45 min and used at a ratio of 1:50 (probe:block). The 5S rDNA probe was amplified from the 332 M. Kwiatek et al. Pm13 marker analysis and powdery mildew reaction in parental forms The amplification products of 517 bp in size were found in DNA extracts of Ae. longissima (PI 604112), Ae. variabilis and 20 plants of Ae. variabilis × S. cereale, which were used in 333 Triticale distant hybrids with powdery mildew resistance Fig. 1 a Amplification products (517 bp) of PCR with primers specific to Pm 13 gene marker. Lane 1 - 100 bp ladder (GeneRuler, Thermo Fischer Scientific Inc.), lane 2—A. longissima, lane 3—Ae. variabilis, lane 4— Ae. variabilis × S. cereale, lane 5—Ae. umbellulata, lane 6—S. cereale ‘Strzękęcińskie,’ lane 7 - triticale ‘Lamberto’; b leaf of (Ae. variabilis × S. cereale) × triticale infected by B. graminis; c no symptoms of B. graminis infection pecific to o Fischer lane 4— cereale ‘Strzękęcińskie,’ lane 7 - triticale ‘Lamberto’; b leaf of (Ae. variabilis × S. cereale) × triticale infected by B. graminis; c no symptoms of B. graminis infection ‘Strzękęcińskie,’ lane 7 - triticale ‘Lamberto’; b leaf of (Ae. variabilis × S. cereale) × triticale infected by B. graminis; c no symptoms of B. graminis infection Fig. 1 a Amplification products (517 bp) of PCR with primers specific to Pm 13 gene marker. Lane 1 - 100 bp ladder (GeneRuler, Thermo Fischer Scientific Inc.), lane 2—A. longissima, lane 3—Ae. variabilis, lane 4— Ae. variabilis × S. cereale, lane 5—Ae. umbellulata, lane 6—S. cereale further crosses with triticale. The bands of all samples gave clear and strong fluorescence after separation (Fig. 1a). The marker for Pm 13 (517 bp) was not identified in rye ‘Strzekęcińskie’ (used for production of Ae. variabilis × S. cereale ampihiploids, Wojciechowska and Pudelska 1999) and triticale ‘Lamberto.’ The powdery mildew expression mean scores in Ae. variabilis were made in three subsequent years of experiments and ranged between 8.05 and 8.25 (Table 3). The observations of the infection symptoms con- ducted on triticale ‘Lamberto’ showed much lower tolerance to powdery mildew. The mean scores of infection ranged be- tween 2.85 and 2.95 (Table 3). FISH) with pSc 119.2 and pAs1 probes resulted in specific patterns on chromosomes of triticale ‘Lamberto’ and Ae. variabilis. The chromosomes of A-genome of triticale carried only pAs1 signals, mainly on the distant and pericentromeric regions (Fig. 2b). The most distinguishable chromosome was 7A with strong pAs1 signal on the short arm. Pm13 marker analysis and powdery mildew reaction in parental forms The pSc 119.2 and pAs1 signal locations on chromosomes of B-genome of triticale were more diversified and appeared also in interstitial regions. R-genome chromosomes of triticale had strong pSc119.2 sites and weak, dispersed pAs1 signals. The loca- tions of pSc119.2 sites on 2R and 3R chromosomes were similar, but the difference of chromosome arms length allowed to distinguish those two. The chromosomes Uv-ge- nome of Ae. variabilis (Figs. 2e and 3) carried both the pSc119.2 sites and the pAs1 sites. The strongest pSc119.2 signal was observed in the telomeric region of 3Uv chromo- some. The pAs1 sites were located both on distal and intersti- tial chromosomes. The most characteristic pattern was ob- served on 6Uv chromosome. The pSc119.2 and pAs1 probes hybridized also with Sv-genome chromosomes (Fig. 3). The pSc119.2 sites were located on the telomeric regions of chro- mosomes with an exception of long arm of 5Sv. The strongest signals were observed on the long arms of 3Sv and 7Sv chro- mosomes. The pAs1 sites were mostly dispersed. Distal re- gions of chromosome 4Sv and short arm of chromosome 7Sv carried the most visible signals of pAs1. Identification of particular mitotic chromosomes of parental forms The chromosome composition of Ae. variabilis (UvUvSvSv) and triticale ‘Lamberto’ (AABBRR), used as parental forms in presented distant crossing were studied (Fig. 2). The analysis were made using 5S rDNA, 25S rDNA (Fig. 2a, d), pSc119.2 and pAs1 probes (Fig. 2b, e) and multicolour GISH with total genomic DNA used as a probe (Fig. 2c, f). Identification of particular chromosomes of A- and B-genome, R-genome, Uu- genome and Sl-genome was made basing on previous reports of Cuadrado and Jouve (2002), Schneider et al. (2003, 2005) and Badaeva et al. 1996a, b and 2004, respectively and chro- mosome arms ratio. The rDNA-FISH experiment on chromo- somes of triticale ‘Lamberto’ (2n=6x=42 chromosomes, AABBRR) resulted in 12 signals of 5S rDNA (on chromo- somes 1A, 5A, 1B, 5B, 1R and 5R) and 6 signals of 25S rDNA (on chromosomes: 1B, 6B and 1R; Fig. 2a). By con- trast, rDNA-FISH on Ae. variabilis (UvUvSvSv) chromosomes showed 8 signals of 5S rDNA in 1Uv, 5Uv, 1Sv and 5Sv chro- mosomes and 8 signals of 25S rDNA in 1Uv, 5Uv, 5Sv (weak) and 6Sv (weak) chromosomes (Fig. 2d). The same locations of rDNA signals appeared on chromosomes of Ae. variabilis × S. cereale amphiploid. The repetitive sequence FISH (seq- Evaluation of crossing efficiency 106 flowers of triticale ‘Lamberto’ were pollinated by the pollen of Ae. variabilis × S. cereale forms (Table 1). 19 F1 seeds were obtained, that indicates 18 % of crossing efficiency (CE). Six F1 plants were germinated and evaluated using GISH analysis. Backcrossing of 68 flowers of F1 hybrids with the triticale ‘Lamberto’ pollen resulted in obtaining of 17 seeds of BC1F1 hybrid generation (CE=25 %). Five BC1F1 plants were chosen on the basis of molecular marker (Pm13) 334 M. Kwiatek et al. Fig. 2 Fluorescence in situ hybridization (FISH) using 5S and 25S rDNA (a, d); pAs1 and pSc119.2 (b, e) repetitive DNA probes, and genomic in situ hybridization (GISH) on mitotic chromosomes of triticale (× Triticosecale Wittm.) ‘Lamberto’ (a, b, c) and Ae. variabilis Eig. (d, e, f). On the GISH images: c the R-genome is visualized in red, the A-genome in green and the B-genome in blue; f the Uv-genome is visualized in red and the Sv-genome in green. Scale bars: 10 μm Fig. 2 Fluorescence in situ hybridization (FISH) using 5S and 25S rDNA (a, d); pAs1 and pSc119.2 (b, e) repetitive DNA probes, and genomic in situ hybridization (GISH) on mitotic chromosomes of triticale (× Triticosecale Wittm.) ‘Lamberto’ (a, b, c) and Ae. variabilis Eig. (d, e, f). On the GISH images: c the R-genome is visualized in red, the A-genome in green and the B-genome in blue; f the Uv-genome is visualized in red and the Sv-genome in green. Scale bars: 10 μm Evaluation of introgression of Ae. variabilis chromatin in triticale hybrids test and cytogenetic analysis of mitotic chromosomes of root meristems for further crossing with triticale. After crossing of 330 flowers with triticale pollen, 25 seeds of BC2F1 genera- tion were obtained. Thereafter, 15 plants were chosen for fur- ther hybridizations. 329 flowers of BC2F1 hybrids were self- pollinated, that resulted in 50 seeds of BC2F2 generation. The correct establishing of the introgression of Ae. variabilis chromatin carrying the resistance to powdery mildew was assured by combining the GISH and FISH methods with mo- lecular marker (Pm13) analysis and the results of infection scoring. The chromosome constitution of six F1 (Ae. variabilis × S. cereale) × triticale hybrids consist of 28 chromosomes of triticale (14 chromosomes of A- and B-genomes and 14 R- genome chromosomes), seven Uv-genome chromosomes and seven Sv-genome chromosomes, which were detected by probing with Uu- and Sl-genomic DNA and blocking with total DNA of triticale (AABBRR) (Table 2, Fig. 4a). FISH experiment with 4 kinds of probes allowed to distinguish chromosomes from each group (group-1 to group-7). Fig. 3 Representative karyotype of Ae. variabilis metaphase chromosomes after fluorescence in situ hybridization with signals originating pAs1 (red) and pSc119.2 (green) Afterwards, five of 17 plants of the BC1F1 generation car- ried Pm13 marker, which was correlated with the infection scores that ranged from 6 to 8, whereas the another 12 plants were more infected, which was comparable with the infection level of triticale ‘Lamberto’ (Table 3). In those 5 hybrids (with Pm13 marker) the total number of chromosomes varied from Fig. 3 Representative karyotype of Ae. variabilis metaphase chromosomes after fluorescence in situ hybridization with signals originating pAs1 (red) and pSc119.2 (green) Triticale distant hybrids with powdery mildew resistance 335 Table 2 Cytogenetic analysis of F1 to BC2F2 hybrids of triticale ‘Lamberto’ × (Ae. variabilis × S. cereale) carrying Ae. variabilis chromatin with Pm13 marker Generation Number of plants Chromosome composition Total number of chromosomes F1 6 14″+1′1Uv+1′2Uv+1′3Uv+1′4Uv+1′5Uv+1′6Uv+1′7Uv+1′1Sv+ 1′2Sv+1′3Sv+1′4Sv+1′5Sv+1′6Sv+1′7Sv 42 BC1F1 1 16″+1′3B+1′2Uv+1′3Uv+1′4Uv+1′6Uv+1′2Sv+1′3Sv+1′4Sv 40 1 16″+1′3Uv+1′4Uv+1′2Sv+1′3Sv+1′4Sv 37 1 17″+1′2B+1′2Uv+1′3Uv+1′4Uv+1′2Sv+1′3Sv+1′4Sv 41 1 17″+1′2Uv+1′3Uv+1′4Uv+1′6Uv+1′7Uv+1′2Sv+1′3Sv+1′4Sv+1′7Sv 43 1 17″+1′2Uv+1′3Uv+1′4Uv+1′6Uv+1′2Sv+1′3Sv+1v4Sv+1′7Sv 42 BC2F1 3 20″+1′3Sv/3B 41 4 21″+1′3S′ 43 6 20″+1″3Sv/3B 42 1 20″+1′3Sv/3B+1′2Sv 43 1 20″+1′2B+1″3Sv/3B+1′2Sv 44 BC2F2 9 21″+1′3Sv 43 10 20″+1″3Sv/3B 42 7 21″+1″3Sv 44 xx″- number of pairs of triticale chromosomes, 1″xy- one pair of y-genome chromosomes of group-x; 1′xy- a singular group-x chromosome of y- genome; 1″xy/xz- substitution pair of chromosomes. Evaluation of introgression of Ae. variabilis chromatin in triticale hybrids The nomenclature and abbreviation of the genetic stocks of hybrids were described according Raupp et al. 1995 (http://wheat.pw.usda.gov/ggpages/nomenclature.html) xx″- number of pairs of triticale chromosomes, 1″xy- one pair of y-genome chromosomes of group-x; 1′xy- a singular group-x chromosome of y- genome; 1″xy/xz- substitution pair of chromosomes. The nomenclature and abbreviation of the genetic stocks of hybrids were described according Raupp et al. 1995 (http://wheat.pw.usda.gov/ggpages/nomenclature.html) 37 to 43 (Table 2). The number of Uv-genome chromosomes was between 2 and 5, the number of Sv chromosomes was 3– 4, the number of R-genome chromosomes was 14 in each plant and the A and B-genome chromosomes number varied from 18 to 21 (Fig. 4b). The 12 other plants, without Pm13 marker, had large number of intergeneric translocations. The GISH analysis showed the chromosomes of A- and B-genome with the translocations of S-genome chromosome segments (Fig. 4c). Selected five BC1F1 hybrids (with Pm13 marker) were backcrossed with triticale pollen. The molecular analysis showed that the 3 of 5 BC1F1 plants reproduced 15 descen- dants (BC2F1) with the Pm13 marker (Table 1). The infection scores of those group of hybrids were significantly different in comparison with hybrids without Pm13 marker and triticale ‘Lamberto.’ triticale chromosomes was the same as in the previous gener- ation. FISH experiments allowed to distinguish 9 plants with one, additional chromosome 3Sv, 10 plants with a substitution pair of 3Sv/3B chromosomes and 7 plants with an additional pair of 3Sv chromosomes. Pm13 marker was identified only in plants with introgression of Aegilops chromatin, which was correlated with the powdery mildew infection scores (Table 3). Chromosome pairing behaviour in BC2F1 and BC2F2 of (Ae. variabilis × S. cereale) × triticale hybrids Chromosome pairing behaviour in BC2F1 and BC2F2 of (Ae. variabilis × S. cereale) × triticale hybrids variabilis and 21 chromosomes of triticale with introgression of Sv-genome chromatin. d BC2F1 hybrid with 3 chromosomes from of Sv-genome of Ae. variabilis. Scale bars: 10 μm of ly he ts 8, 72 he ts B o- nt o- o- ti- of rod bivalents, ring bivalents and univalents, where mean chromosome configuration for five analysed BC2F2 plants (2n=42 chromosomes) with a substitution pair of 3Sv/3B chromosomes was 4.60 I+18.70 II (12.56 rod+6.14 ring). The mean of rod and ring Sv-genome bivalents was approxi- mate (0.22 and 0.46; respectively). The comparison of ANOVA results of chromosome configuration between BC2F1 and respective BC2F2 progeny hybrids shows that the differences in means are not significant. Considering the Sv- genome univalents, the mean in BC2F2 plants (Table 5) was lower than in BC2F1 plants (Table 4). Five of six hybrids of BC2F1 (42 chromosomes each), which carried a substitution pair of 3Sv/3B chromosomes were evaluated (Table 2). All of them were the progeny of the most fertile hybrid line no. 4 (Table 1). of rod bivalents, ring bivalents and univalents, where mean chromosome configuration for five analysed BC2F2 plants (2n=42 chromosomes) with a substitution pair of 3Sv/3B chromosomes was 4.60 I+18.70 II (12.56 rod+6.14 ring). The mean of rod and ring Sv-genome bivalents was approxi- mate (0.22 and 0.46; respectively). The comparison of ANOVA results of chromosome configuration between BC2F1 and respective BC2F2 progeny hybrids shows that the differences in means are not significant. Considering the Sv- genome univalents, the mean in BC2F2 plants (Table 5) was lower than in BC2F1 plants (Table 4). Five of six hybrids of BC2F1 (42 chromosomes each), which carried a substitution pair of 3Sv/3B chromosomes were evaluated (Table 2). All of them were the progeny of the most fertile hybrid line no. 4 (Table 1). bivalents (12.22; 5.80; respectively). Similarly, the mean of rod bivalents of A-, B- and R-genome was considerably higher than ring bivalents of those genomes. Considering the Sv-genome bivalents, the mean number of Sv/Sv rod bivalents and Sv/Sv ring bivalents was almost equal (0.30 and 0.38, respectively). The mean of Sv-genome univalents was 0.72 and the number of univalents ranged between 0 and 2. The mean chromosome configuration for five analysed plants (2n=42 chromosomes) with a substitution pair of 3Sv/3B chromosomes was 5.96 I+18.02 II (12.22 rod+5.80 ring). Chromosome pairing behaviour in BC2F1 and BC2F2 of (Ae. variabilis × S. cereale) × triticale hybrids The multicolour GISH allowed to distinguish the Sv-genome chromosomes (green) and the triticale chromosomes (Fig. 5a– d). Chromosome configuration means at MI of meiosis in PMCs were examined in selected hybrid plants of BC2F1 with total number of chromosomes amounting 42, that carried a substitution pair of 3Sv/3B chromosomes (Table 4) and in BC2F2 hybrids divided in two groups. First group consisted of plants with 42 chromosomes, having a substitution pair of 3Sv/3B chromosomes (Table 5), while second group associat- ed the plants with 43 chromosomes having an additional 3Sv chromosome (Table 6). The Uv-genome chromosomes were not identified in all of 15 plants of BC2F1 generation, but 1 to 3 chromosomes of Sv - genome appeared in those plants (Fig. 4d). FISH analysis showed that 3 plants carried 41 chromosomes with one chro- mosome 3Sv and the lack of 3B chromosome pair. Another 4 plants possessed additional chromosome 3Sv. The 6 other plants carried substitution pair of 3Sv/3B chromosomes. Moreover, one of BC2F1 hybrids had a substitution pair of 3Sv/3B chromosomes and one additional chromosome 2Sv. The other singular plant carried: a substitution pair of 3Sv/ 3B chromosomes, an one additional 2Sv chromosome and one chromosome 2B (Table 2). The variance analysis of the chromosome configurations in BC2F1 plants with 42 chromosomes, that carried a substitution pair of 3Sv/3B chromosomes showed that the differences be- tween the means of chromosome configurations were not sig- nificant (Table 4). The mean of total number of bivalents were 18.02. Bivalents ranged from 9 to 20 per cell. The mean of rod bivalents was nearly two times higher than the mean of ring In the BC2F2 generation the Sv -genome chromosomes were eliminated in 24 plants, however in 26 hybrids 1–2 chro- mosomes of Sv -genome were identified and the range of 336 M. Kwiatek et al. Fig. 4 Genomic in situ hybridization (GISH) on mitotic chromosomes of (Ae. variabilis × S. cereale) × triticale ‘Lamberto’ hybrids. On the GISH images, the R-genome is visualized in blue, the A-genome and the B-genome in grey; the Uv-genome is visualized in red and the Sv- genome in green. a F1 hybrid with 14 chromosomes of Ae. variabilis (7 chromosomes of Uv-genome and 7 chromosomes of Sv- genome). b BC1F1 hybrid with 7 chromosomes of Ae. variabilis (4 chromosomes of Uv-genome and 3 chromosomes of Sv-genome). c BC1F1 hybrid with 2 chromosomes from Uv-genome of Ae. Chromosome pairing behaviour in BC2F1 and BC2F2 of (Ae. variabilis × S. cereale) × triticale hybrids The ANOVA test for BC2F2 hybrids with the same chro- mosome constitution (20′″+3Sv′) obtained from different BC1F1 plants, carrying a substitution pair of 3Sv/3B chromo- somes showed that the differences between means of the chro- mosome configurations of particular hybrids were not statisti- cally significant. The mean (and the range) of bivalents per PMC was 18.7 (9–20) and was similar to the results in BC2F1 hybrids. The same situation appeared considering the means Chromosome configuration means at MI of meiosis in PMCs were also examined in four BC2F2 hybrid plants (2n=43 chromosomes) carrying additional chromosome 3Sv. Triticale distant hybrids with powdery mildew resistance 337 Table 3 Evaluation of the natural infection level caused by B. graminis on the BC1F1, BC2F1 and BC2F2 hybrids of (Ae. variabilis × S. cereale) × triticale ‘Lamberto’ that carried or did not carry the Pm13 marker Generation Number of plants Means (range) of infection scores With Pm13 marker Without Pm13 marker With Pm13 marker Without Pm13 marker 1 2 3 4 Ae. variabilis hybrids triticale ‘Lamberto’ hybrids Ae. variabilis hybrids triticale ‘Lamberto’ hybrids BC1F1 20 5 20 12 8.25 (7–9) 7.40 (6–8) 2.90 (2–4) 3.50 (2–4) BC2F1 20 15 20 10 8.10 (7–9) 6.80 (6–8) 2.95 (2–4) 2.90 (2–4) BC2F2 20 26 20 24 8.05 (7–9) 6.62 (6–8) 2.85 (2–4) 2.92 (2–4) Tukey’s Honest Significant Difference (HSD) test Generation HSD level 1 vs 2 1 vs 3 1 vs 4 2 vs 3 2 vs 4 3 vs 4 HSD0.05 HSD0.01 BC1F1 0.81 1.00 P<0.05 P<0.01 P<0.01 P<0.01 P<0.01 n/s BC2F1 0.68 0.83 P<0.01 P<0.01 P<0.01 P<0.01 P<0.01 n/s BC2F2 0.59 0.72 P<0.01 P<0.01 P<0.01 P<0.01 P<0.01 n/s The mean chromosome configuration for this group was 4.65 I+19.18 II (9.9 rod+9.28 ring). The ANOVA and Tukey’s HSD test showed that the differences of chromosomes config- uration means between plants with the same chromosome constitution (21′″+3Sv′) obtained from different BC2F1 plants (4/6 and 4/10) were significant. The differences affected the means of A-genome, B-genome and R-genome rod and ring bivalents and also means of univalents of A- and B-genome (Table 6). Discussion Considering the growing tendency in brakeage of triticale re- sistance to fungal diseases, especially powdery mildew, and from the other hand, the narrow genetic diversity of triticale could lead to the conclusion that it is necessary to utilize the wild Triticeae relatives to enrich the genetic pool of cultivated triticale. The gene order in Poaceae species is generally con- served (Chantret et al. 2008) and the synteny facilitates com- parative genomics analyses in grass families (Abrouk et al. 2010). Therefore, it could be expected that the region of chro- mosome 3Sl of A. longissima that is responsible for powdery mildew resistance could be collinear with the same region in the chromosome 3Sv of Ae. variabilis (2n=4x=28, UvUvSvSv). Nonetheless, there are discrepant reports concerning the powdery mildew resistance of Ae. variabilis. From the one side, Spetsov and Iliev (1991) obtained a diso- mic addition line (2n=44) by crossing wheat cv. ‘Roussallka’ with Ae. variabilis, that manifested a high powdery mildew resistance in seedling and in adult plant stage. From the other side, Cenci et al. (2003) reported that disomic line of wheat cv. ‘Chinese Spring’ 3Sv (K-2) and the derived ditelosomic 3SvS (K-2/SvS) addition lines from Ae. variabilis (Yang et al. 1996) were susceptible, with strong powdery mildew symptoms and abundant sporulation. However, the assumption of a possible synteny between the S-genome chromosomes became mean- ingful, considering the verification of available powdery mil- dew STS markers made by Stępień et al. (2001), which showed that Pm13 marker was present in Ae. speltoides (ac- cessions 2056, 2067, d10, d42, d50) that also carry S-genome chromosomes. In presented study, the Ae. variabilis and the Ae. variabilis × S. cereale amphiploids carrying Pm13 marker manifested a low powdery mildew reaction, confirmed by infection scores made on 20 plants each year of the experi- ment (Fig. 1c; Table 3). In comparison, triticale ‘Lamberto’ was much more infected, which was confirmed by Tukey’s HSD test (Fig. 1c; Table 3). Moreover, 1402 Polish isolates of B. graminis are reported to be 100 % virulent to triticale ‘Lamberto’ in three subsequent years of experiment (2008– 2010) carried out by Czembor et al. (2014). Furthermore, the molecular analysis showed the Pm13 marker was not present in triticale ‘Lamberto’ (Table 3). The Pm13 marker is located 338 M. Kwiatek et al. Fig. 5 Chromosome associations at meiosis of pollen mother cells of BC2F2 (Ae. variabilis × S. Discussion cereale) × triticale ‘Lamberto’ hybrids. GISH images created using Sv-genome genomic DNA as a probe (green), with blocking genomic DNA of triticale. Chromosomes were counterstained with propidium iodide (a) or DAPI (b, c, d). a One 3Sv/ 3Sv bivalent in 3Sv/3B substitution line (2n=42) at metaphase I of meiosis. b One 3Sv univalent in 3Sv addition line (2n=43) at b metaphase I, c anaphase I and d telophase I of meiosis. Scale bars: 10 μm Fig. 5 Chromosome associations at meiosis of pollen mother cells of BC2F2 (Ae. variabilis × S. cereale) × triticale ‘Lamberto’ hybrids. GISH images created using Sv-genome genomic DNA as a probe (green), with blocking genomic DNA of triticale. Chromosomes were counterstained with propidium iodide (a) or DAPI (b, c, d). a One 3S 3Sv bivalent in 3Sv/3B substitution line (2n=42) at metaphase I meiosis. b One 3Sv univalent in 3Sv addition line (2n=43) at metaphase I, c anaphase I and d telophase I of meiosis. Scale bars: 10 counterstained with propidium iodide (a) or DAPI (b, c, d). a One 3Sv/ 3Sv bivalent in 3Sv/3B substitution line (2n=42) at metaphase I of meiosis. b One 3Sv univalent in 3Sv addition line (2n=43) at b metaphase I, c anaphase I and d telophase I of meiosis. Scale bars: 10 μm Fig. 5 Chromosome associations at meiosis of pollen mother cells of BC2F2 (Ae. variabilis × S. cereale) × triticale ‘Lamberto’ hybrids. GISH images created using Sv-genome genomic DNA as a probe (green), with blocking genomic DNA of triticale. Chromosomes were on the distal region of the short arm of chromosome 3Sl (Cenci et al. 2003). In purpose to identify the particular chromosomes of Ae. variabilis, the FISH experiment with repetitive se- quences as probes was carried out. The location of 25S rDNA and 5S rDNA signals in U- and S-genome chromo- somes of Ae. variabilis were similar like in the ancestor spe- cies, considering chromosomes 1Uu 5Uu and 5Sl and 6Sl of Ae. umbellulata and Ae. longissima, respectively (Badaeva et al. 1996b). However, the 25S rDNA signals on 1Sl, 3Sl and 6Uu chromosomes were not present on the homologue chromosomes of Ae. variabilis. There were also some differ- ences in pSc119.2 signals pattern between diploid ancestors (Badaeva et al. 1996a) and Ae. variabilis (Fig. 3). There were no signals in the telomeric regions of long arms of 2Uv, 3Uv, 5Uv and 6Uv chromosomes. Discussion variabilis Plant number (number of chromosomes) Number of PMC’s Mean and range of chromosome configurations at metaphase I Bivalents Univalents Rods Rings ∑ S AB R ∑ ∑ AB/AB R/R S/S ∑ AB/AB R/R S/S 4/3 (42) 10 12.1 (8–17) 6.8 (4–10) 5 (4–6) 0.3 (0–1) 5.2 (1–9) 3.4 (0–6) 1.5 (0–3) 0.3 (0–1) 17.3 (9–20) 0.8 (0–2) 5.4 (2–16) 1.2 (0–6) 7.4 (2-24) 4/5 (42) 10 11.6 (10–14) 6.8 (5–10) 4.3 (3–5) 0.5 (0–1) 6.5 (2–9) 3.8 (0–6) 2.2 (1–4) 0.5 (0–1) 18.1 (16–20) 0 4.8 (2–8) 1 (0–4) 5.8 (2-10) 4/6 (42) 10 12.3 (9–15) 7.5 (4–10) 4.6 (3–6) 0.2 (0–1) 5.9 (3–8) 3.1 (0–6) 2.4 (1–4) 0.4 (0–1) 18.2 (15–20) 1 (0–2) 4.4 (0–10) 0.2 (0–2) 5.6 (2-12) 4/10 (42) 10 11.2 (5–15) 6.4 (3–9) 4.6 (2–7) 0.2 (0–1) 6.7 (2–9) 4.2 (0–7) 2.2 (0–5) 0.3 (0–1) 17.9 (14–20) 1 (0–2) 4.8 (0–12) 0.4 (0–2) 6.2 (2-14) 4/11 (42) 10 13.9 (10–17) 8.2 (6–12) 5.4 (4–7) 0.3 (0–1) 4.7 (1–9) 2.8 (0–6) 1.5 (0–3) 0.4 (0–1) 18.6 (16–20) 0.8 (0–2) 3.8 (2–8) 0.2 (0–2) 4.8 (2-10) Mean 12.22 (5-17) 7.14 (3–12) 4.78 (2–7) 0.30 (0–1) 5.80 (1–9) 3.46 (0–7) 1.96 (0–5) 0.38 (0–1) 18.02 (9–20) 0.72 (0–2) 4.64 (0–16) 0.60 (0–6) 5.96 (2–24) ANOVA F 2.05 1.66 1.66 0.68 1.21 0.81 1.36 0.27 0.54 1.97 0.35 1.52 0.54 summary P 0.103368 0.175949 0.175949 0.609437 0.319804 0.525409 0.262852 0.895752 0.707101 0.115345 0.842649 0.212455 0.707101 Table 5 Analysis of chromosome configurations during metaphase I of meiosis of PMCs of five BC2F2 hybrids (2n=42) with an introgression of a 3Sv chromosome pair of Ae. Discussion variabilis Plant number (number of chromosomes) Number of PMC’s Mean and range of chromosome configurations at metaphase I Bivalents Univalents Rods Rings ∑ S AB R ∑ ∑ AB/AB R/R S/S ∑ AB/AB R/R S/S 4/6/1 (42) 10 12.4 (10–15) 7.5 (6–9) 4.7 (4–6) 0.2 (0–1) 6.8 (4–9) 4.2 (2–6) 2.1 (1–3) 0.5 (0–1) 19.2 (18–20) 0.6 (0–2) 2.6 (0–4) 0.4 (0–2) 3.6 (2–6) 4/6/3 (42) 10 12.2 (10–15) 7.4 (6–10) 4.7 (4–6) 0.1 (0–1) 6.9 (4–9) 4.0 (2–6) 2.2 (1–3) 0.7 (0–1) 19.1 (17–20) 0.6 (0–2) 3.0 (0–6) 0.2 (0–2) 3.8 (2–8) 4/10/5 (42) 10 12.0 (9–15) 7.1 (5–9) 4.8 (4–7) 0.1 (0–1) 6.8 (2–9) 4.3 (0–7) 1.9 (0–3) 0.6 (0–1) 18.8 (16–21) 0.6 (0–2) 3.2 (0–8) 0.6 (0–4) 4.4 (0–10) Table 5 Analysis of chromosome configurations during metaphase I of meiosis of PMCs of five BC2F2 hybrids (2n=42) with an introgression of a 3Sv chromosome pair of Ae. Discussion variabilis Plant number (number of chromosomes) Number of PMC’s Mean and range of chromosome configurations at metaphase I Bivalents Univalents Rods Rings ∑ S AB R ∑ ∑ AB/AB R/R S/S ∑ AB/AB R/R S/S 4/6/1 (42) 10 12.4 (10–15) 7.5 (6–9) 4.7 (4–6) 0.2 (0–1) 6.8 (4–9) 4.2 (2–6) 2.1 (1–3) 0.5 (0–1) 19.2 (18–20) 0.6 (0–2) 2.6 (0–4) 0.4 (0–2) 3.6 (2–6) 4/6/3 (42) 10 12.2 (10–15) 7.4 (6–10) 4.7 (4–6) 0.1 (0–1) 6.9 (4–9) 4.0 (2–6) 2.2 (1–3) 0.7 (0–1) 19.1 (17–20) 0.6 (0–2) 3.0 (0–6) 0.2 (0–2) 3.8 (2–8) 4/10/5 (42) 10 12.0 (9–15) 7.1 (5–9) 4.8 (4–7) 0.1 (0–1) 6.8 (2–9) 4.3 (0–7) 1.9 (0–3) 0.6 (0–1) 18.8 (16–21) 0.6 (0–2) 3.2 (0–8) 0.6 (0–4) 4.4 (0–10) 4/10/7 (42) 10 14.0 (10–17) 8.2 (6–12) 5.4 (4–7) 0.4 (0–1) 4.5 (1–9) 2.8 (0–6) 1.5 (0–3) 0.2 (0–1) 18.5 (16–20) 0.8 (0–2) 4.0 (2–8) 0.2 (0–2) 5.0 (2–10) 4/10/8 (42) 10 12.2 (8–17) 6.9 (4–10) 5.0 (4–6) 0.3 (0–1) 5.1 (1–9) 3.9 (0–6) 1.5 (0–3) 0.3 (0–1) 17.3 (9–20) 0.8 (0–2) 5.4 (2–16) 1.2 (0–6) 6.2 (2–12) mean 12.56 (8–17) 7.42 (4–12) 4.92 (4–6) 0.22 (0–1) 6.14 (1–9) 3.84 (0–7) 1.84 (0–3) 0.46 (0–1) 18.7 (9–20) 0.68 (0–2) 3.40 (0–16) 0.52 (0–6) 4.60 (0–12) ANOVA F 1.67 1.05 0.97 0.97 2.21 1.13 1 1.81 1.56 0.12 1.13 1.22 1.56 summary P 0.173585 0.392246 0.433323 0.433323 0.082983 0.354443 0.417531 0.143525 0.201351 0.974664 0.354443 0.315690 0.201351 Table 4 Analysis of chromosome configurations during metaphase I of meiosis of PMCs of five BC2F1 hybrids (2n=42) with an introgression of a 3Sv chromosome pair of Ae. Discussion When comparing pAs1 signals on the U-genome chromosomes, small, dispersed signals were observed on 1Uv, 3Uv and 5Uv chromosomes. Moreover, Badaeva et al. (1996a) did not observed the pAs1 signals on S-genome chromosomes of Ae. longissima, however chromo- somes of Ae. variabilis carried weak, scattered landmarks on both arm of each chromosome and strong site on distal region of long arm of 7Sv chromosome. The cytogenetic analysis of triticale ‘Lamberto’ chromosomes revealed also some novel data. The elimination of 25-5.8-18S rDNA was observed in 1A chromosome of triticale, comparing to 1A of wheat. The rDNA aberrations are probably connected with the changes in ploidy level, which commonly appear in hybrids (Shcherban et al. 2008). Knowing the cytogenetic markers distribution on the chro- mosomes of parental forms (Ae. variabilis × S. cereale amphi- ploids and triticale ‘Lamberto’), and the results of Pm13 mo- lecular marker analysis connected with the evaluation of nat- ural infection by B. graminis, the study of hybrid generations of (Ae. variabilis × S. cereale) × triticale ‘Lamberto’ were made. As expected, the F1 hybrids (2n=6x=42, UvSvABRR) carried 7 chromosomes of Uv-, Sv-, A- and B-genome and complete set of 14 chromosomes of R-genome. The chromo- some composition of F1 hybrids was anticipated on the basis of related studies, i.e. in the study of Aegilops biuncialis (2n= 4x=28, UUMM) × wheat (2n=6x=42, AABBDD) hybridiza- tions (Schneider et al. 2005), the chromosome set of F1 hy- brids were parallel (ABDUM, 2n=5x=35), with only one dif- ference, that in case of (Ae. variabilis × S. cereale) × triticale hybridizations, R-genome chromosomes can pair and behave Triticale distant hybrids with powdery mildew resistance 339 Table 4 Analysis of chromosome configurations during metaphase I of meiosis of PMCs of five BC2F1 hybrids (2n=42) with an introgression of a 3Sv chromosome pair of Ae. Discussion variabilis Plant number (number of chromosomes) Number of PMC’s Mean and range of chromosome configurations at metaphase I Bivalents Univalents Rods Rings ∑ S AB R ∑ ∑ AB/AB R/R S/S ∑ AB/AB R/R S/S 4/3 (42) 10 12.1 (8–17) 6.8 (4–10) 5 (4–6) 0.3 (0–1) 5.2 (1–9) 3.4 (0–6) 1.5 (0–3) 0.3 (0–1) 17.3 (9–20) 0.8 (0–2) 5.4 (2–16) 1.2 (0–6) 7.4 (2-24) 4/5 (42) 10 11.6 (10–14) 6.8 (5–10) 4.3 (3–5) 0.5 (0–1) 6.5 (2–9) 3.8 (0–6) 2.2 (1–4) 0.5 (0–1) 18.1 (16–20) 0 4.8 (2–8) 1 (0–4) 5.8 (2-10) 4/6 (42) 10 12.3 (9–15) 7.5 (4–10) 4.6 (3–6) 0.2 (0–1) 5.9 (3–8) 3.1 (0–6) 2.4 (1–4) 0.4 (0–1) 18.2 (15–20) 1 (0–2) 4.4 (0–10) 0.2 (0–2) 5.6 (2-12) 4/10 (42) 10 11.2 (5–15) 6.4 (3–9) 4.6 (2–7) 0.2 (0–1) 6.7 (2–9) 4.2 (0–7) 2.2 (0–5) 0.3 (0–1) 17.9 (14–20) 1 (0–2) 4.8 (0–12) 0.4 (0–2) 6.2 (2-14) 4/11 (42) 10 13.9 (10–17) 8.2 (6–12) 5.4 (4–7) 0.3 (0–1) 4.7 (1–9) 2.8 (0–6) 1.5 (0–3) 0.4 (0–1) 18.6 (16–20) 0.8 (0–2) 3.8 (2–8) 0.2 (0–2) 4.8 (2-10) Mean 12.22 (5-17) 7.14 (3–12) 4.78 (2–7) 0.30 (0–1) 5.80 (1–9) 3.46 (0–7) 1.96 (0–5) 0.38 (0–1) 18.02 (9–20) 0.72 (0–2) 4.64 (0–16) 0.60 (0–6) 5.96 (2–24) ANOVA F 2.05 1.66 1.66 0.68 1.21 0.81 1.36 0.27 0.54 1.97 0.35 1.52 0.54 summary P 0.103368 0.175949 0.175949 0.609437 0.319804 0.525409 0.262852 0.895752 0.707101 0.115345 0.842649 0.212455 0.707101 340 M. Kwiatek et al. Table 6 Analysis of chromosome configurations during metaphase I of meiosis of PMCs of four BC2F2 hybrids (2n=43) with additional 3Sv chromosome of Ae. Discussion The further backcrossing of selected BC1F1 hybrids with triticale pollen resulted in elimination of Aegilops chromosomes. There was lack of Aegilops chromatin in 9 BC2F1 plants. On the other hand, FISH/GISH analysis allowed to distinguish chromo- some(s) 3Sv in each of 15 BC2F1 plants and in addition, one chromosome 2v in 2 plants, where also Pm13 marker was identified. Moreover, the intensity of the level of powdery mildew infection on those plants was lower, when comparing with triticale ‘Lamberto’ and hybrids without Pm13 marker. Two subsequent backcrosses resulted in the elimination of unneeded Aegilops chromosomes and allow to select the plants with the S-genome chromosomes carrying the resis- tance. Therefore, the self-fertilization of BC2F1 was carried out to maintain the S-genome chromosome in BC2F2 hybrids. 26 of 50 hybrids had singular or a pair of 3Sv chromosomes, that carried Pm13 marker and were more tolerant for B. graminis infection. It cannot be omitted, that the HSD test of the means of infection scores of hybrids with Pm13 marker compared with the mean of infection scores of amphiploids (Ae. variabilis × S. cereale) shows the significant differences (Table 3), that points the tolerance for powdery mildew is a little bit lower in hybrids than in amphiploids, however is much higher than in triticale ‘Lamberto’ and hybrids without Pm13 marker. It can be supposed that triticale ‘Lamberto’ carry a virulence factors, that have an influence on Pm13 gene expression. Notwithstanding, the tolerance for powdery mil- dew was markedly improved in hybrids with Pm13 marker. Afterwards, the genomic in situ hybridization was employed to study the 3Sv chromosome(s) behaviour in PMC’s of se- lected BC2F1 and BC2F2 hybrids of (Ae. variabilis × S. cereale) × triticale ‘Lamberto.’ There were no intergenomic chromosome configurations observed in the plant carrying 2Sv and/or 3Sv chromosomes, which is opposite to other published studies concerning intergenomic hybridiza- tions between cultivated cereals and Aegilops species. For example, Molnár and Molnár-Láng (2010) reported the intergenomic rod and ring bivalents and trivalent between 2 M, 3 M, 3U and 7 M chromosomes of Ae. biuncialis and wheat (Chinese Spring ph1b) chromo- 1958). The Chinese Spring ph1b (CSph1b) mutant ge- notype (Sears 1977), which lacks the Ph1 locus, has been successfully used for the introgression of alien genetic material into the wheat genome by the induction of homoeologous pairing (Lukaszewski 2000). Discussion variabilis Plant number (number of chromosomes) Number of PMC’s Mean and range of chromosome configurations at metaphase I Bivalents Univalents Rods Rings ∑ S AB R ∑ ∑ AB/AB R/R S/S ∑ AB/AB R/R S/S 4/6/2 (43) 10 12.2 (10–15) 7.4 (6–9) 4.8 (4–6) 0 6.3 (3–8) 4.3 (2–6) 2 (1–3) 0 18.5 (17–20) 1 4.6 (2–6) 0.4 (0–2) 6 (3–9) 4/10/2 (43) 10 8.6 (7–10) 5.3 (4–6) 3.3 (2–5) 0 11.1 (9–14) 7.6 (6–9) 3.5 (1–5) 0 19.7 (19–21) 1 2.2 (0–4) 0.4 (0–2) 3.6 (1–5) 4/10/3 (43) 10 9.2 (7–11) 6.1 (4–8) 3.1 (1–5) 0 9.9 (6–13) 6.1 (2–8) 3.8 (1–6) 0 19.1 (17–21) 1 3.6 (0–8) 0.2 (0–2) 4.8 (1–9) 4/10/4 (43) 10 9.6 (7–13) 6.7 (6–8) 2.9 (1–5) 0 9.8 (7–13) 6.5 (5–10) 3.3 (2–4) 0 19.4 (18–21) 1 2.2 (0–4) 1 (0–4) 4.2 (1–7) Mean 9.9 (7–15) 6.38 (4–9) 3.53 (1–6) 0 9.28 (3–14) 6.13 (2–10) 3.15 (1–6) 0 19.18 (17–21) 1 3.15 (0–8) 0.5 (0–4) 4.65 (1–9) ANOVA F 12.26 8.07 5.45 0 14.34 8.93 4.91 0 2.49 0 4.48 1.26 2.49 Summary P <0.0001 0.000307 0.003408 1 <0.0001 0.000148 0.005818 1 0.075836 1 0.008997 0.302638 0.075836 Tukey’s HSD test HSD0.05 1.73 1.2 1.41 n/a 2.08 1.75 1.37 n/a n/a n/a 2.11 n/a n/a HSD0.01 2.14 1.49 1.75 n/a 2.59 2.17 1.69 n/a n/a n/a 2.62 n/a n/a 4/6/2 vs 4/10/2 P<0.05 P<0.01 P<0.05 n/a P<0.01 P<0.01 P<0.05 n/a n/a n/a P<0.05 n/a n/a 4/6/2 vs 4/10/3 P<0.05 P<0.05 P<0.05 n/a P<0.01 P<0.05 P<0.05 n/a n/a n/a n/s n/a n/a 4/6/2 vs 4/10/4 P<0.05 n/s P<0.01 n/a P<0.01 P<0.01 n/s n/a n/a n/a P<0.05 n/a n/a 4/10/2 vs 4/10/3 n/s n/s n/s n/a n/s n/s n/s n/a n/a n/a n/s n/a n/a 4/10/2 vs 4/10/4 n/s P<0.05 n/s n/a n/s n/s n/s n/a n/a n/a n/s n/a n/a 4/10/3 vs 4/10/4 n/s n/s n/s n/a n/s n/s n/s n/a n/a n/a n/s n/a n/a Triticale distant hybrids with powdery mildew resistance 341 in diploid manner. The crossing of F1 hybrids with triticale pollen had an influence on reduction of the Aegilops chromo- somes in one group of BC1F1 plants and appearing of Sv/AB translocations in the latter group of BC1F1 plants. Marker analysis showed that plants with Aegilops chromosomes car- ried also Pm13 marker. Moreover, those plants were much more tolerant for B. graminis infection (Table 3). Discussion variabilis × S. cereale) shows the significant differences (Table 3), that points the tolerance for powdery mildew is a little bit lower in hybrids than in amphiploids, however is much higher than in triticale ‘Lamberto’ and hybrids without Pm13 marker. It can be supposed that triticale ‘Lamberto’ carry a virulence factors, that have an influence on Pm13 gene expression. Notwithstanding, the tolerance for powdery mil- dew was markedly improved in hybrids with Pm13 marker. Afterwards, the genomic in situ hybridization was employed to study the 3Sv chromosome(s) behaviour in PMC’s of se- lected BC2F1 and BC2F2 hybrids of (Ae. variabilis × S. cereale) × triticale ‘Lamberto.’ There were no intergenomic chromosome configurations observed in the plant carrying 2Sv and/or 3Sv chromosomes, which is opposite to other published studies concerning intergenomic hybridiza- tions between cultivated cereals and Aegilops species. For example, Molnár and Molnár-Láng (2010) reported the intergenomic rod and ring bivalents and trivalent between 2 M, 3 M, 3U and 7 M chromosomes of Ae. biuncialis and wheat (Chinese Spring ph1b) chromo- somes. It is assumed, that triticale has the same control- ling system of homologue chromosome pairing as wheat, that hampers the pairing of the chromosomes from different genomes. In wheat, homoeologous chro- mosome pairing and consequent recombination is sup- pressed by the function of the Ph1 locus, localized on the long arm of chromosome 5B (Riley and Chapman In conclusion, our study showed that molecular cytogenet- ics and marker-assisted selection combined with evaluation of powdery mildew infection constitute a useful tool for the re- sistance breeding. Using these methods we have obtained 26 plants carrying 3Sv chromosome(s) with the powdery mildew resistance, which can be used in the triticale breeding programmes. On the other hand, these genetic stocks could be used for sequencing the specific region of 3Sv chromo- some, responsible for powdery mildew tolerance and for com- parative studies with the Pm13 gene sequence originated from Ae. longissima. Acknowledgments We would like gratefully acknowledge the techni- cal assistance of Mrs. Grażyna Cicha and Mrs. Joanna Maszner. We also thank Prof. Barbara Apolinarska for her priceless suggestions and clues. This work was financed by the National Science Centre (DEC-2012/05/ N/NZ9/01563 and DEC-2013/11/D/NZ9/02719). Author contribution statements M.K. and H.W. initiated the project and designed the study. M.K., M.M. and J.B. performed the research. M.K. wrote the paper. Discussion From this reason the intergenomic bivalent and trivalent appeared in Molnár and Molnár-Láng (2010) study. Considering presented study, FISH experiments showed that the pair of chromosomes 5B was present in all hybrids of each generation and probably is responsible for diploid-like pairing of chromosomes during meiosis, which was con- firmed by ANOVA tests (Tables 4, 5 and 6) that dem- onstrated no differences in means of chromosome con- figurations between hybrid plants. However, Tukey’s HSD test showed the differences in means of bivalent configurations between BC2F2 progeny obtained from 4/6 plant compared with the progeny of 4/10 hybrid (Table 6). It can be supposed that S-genome chromatin has no influence on chromosome pairing of triticale chromosomes. In other words, the way of triticale chro- mosomes behaviour during first metaphase of meiosis of PMCs seems to be individual regarding to parental form. Furthermore, the way of 3Sv chromosome pairing and transmission to next generation is independent, dip- loid-like. in diploid manner. The crossing of F1 hybrids with triticale pollen had an influence on reduction of the Aegilops chromo- somes in one group of BC1F1 plants and appearing of Sv/AB translocations in the latter group of BC1F1 plants. Marker analysis showed that plants with Aegilops chromosomes car- ried also Pm13 marker. Moreover, those plants were much more tolerant for B. graminis infection (Table 3). The further backcrossing of selected BC1F1 hybrids with triticale pollen resulted in elimination of Aegilops chromosomes. There was lack of Aegilops chromatin in 9 BC2F1 plants. On the other hand, FISH/GISH analysis allowed to distinguish chromo- some(s) 3Sv in each of 15 BC2F1 plants and in addition, one chromosome 2v in 2 plants, where also Pm13 marker was identified. Moreover, the intensity of the level of powdery mildew infection on those plants was lower, when comparing with triticale ‘Lamberto’ and hybrids without Pm13 marker. Two subsequent backcrosses resulted in the elimination of unneeded Aegilops chromosomes and allow to select the plants with the S-genome chromosomes carrying the resis- tance. Therefore, the self-fertilization of BC2F1 was carried out to maintain the S-genome chromosome in BC2F2 hybrids. 26 of 50 hybrids had singular or a pair of 3Sv chromosomes, that carried Pm13 marker and were more tolerant for B. graminis infection. 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https://openalex.org/W2125139025	https://www.ajol.info/index.php/pamj/article/download/71083/60058	English	null	Stress among Mansoura (Egypt) baccalaureate nursing students	The Pan African medical journal	2,011	cc-by	4,613	Abstract Background: Over the last years, details regarding levels of stress and sources of stress have emerged in studies of nursing students in Western population To date, there only few similar reports on clinical stress, anxiety, depression among the Arab population .This study was conducted to examine the level of perceived stress among baccalaureate Mansoura nursing students and to highlight the possible predicting factors. Methods: In this cross- sectional study, Data were obtained from 373 students using a self-administered questionnaire, including questions on sociodemographics, list of possible stressors, perceived stress, physical wellbeing factors, anxiety and depressive symptoms. Results: Prevalence of high stress level, anxiety and depression were 40.2%, 46.6% and 27.9%, respectively. On average each student reported a mean of 4.6 stressors and academic pressures were the most frequent stressors .In regression analysis the number of stressors and global sickness index score were predictors of high stress level. Conclusion: These findings call for introduction of stress management programs and psychiatric care into nursing health services of the University. Open Access Received: 13/01/2011 - Accepted: 06/03/2011 - Published: 16/03/2011 Received: 13/01/2011 - Accepted: 06/03/2011 - Published: 16/03/2011 Received: 13/01/2011 - Accepted: 06/03/2011 - Published: 16/03/2011 Pan African Medical Journal – ISSN: 1937- 8688 (www.panafrican-med-journal.com) Published in partnership with the African Field Epidemiology Network (AFENET). (www.afenet.net) AMostafa Amr1,&, Abdel-Hady El-Gilany1, Hanan El-Moafee2, Lamea Salama2, Cristóbal Jimenez3 1Departments of Psychiatry and Community Medicine, College of Medicine, Mansoura University, Mansoura, Egypt, 2Department of Community Nursing, College of Nursing, Mansoura University, Mansoura Egypt, 3Department of Nursing, School of Nursing University of Córdoba, Andalucía, Spain &Corresponding author: Mostafa Amr, Associate professor of Psychiatry, College of Medicine, Mansoura University, Egypt - Department of Clinical Neurosciences College of Medicine, KFU, KSA Key words: Nursing students, stress, Depression, Anxiety, Arab culture, Egypt Key words: Nursing students, stress, Depression, Anxiety, Arab culture, Egypt Methods This was a cross-sectional descriptive study. Setting and sample The study was conducted on nursing students of Mansoura College of Nursing during March (after mid-year vacation) of the academic year of 2008/2009 G. The study was conducted on nursing students of Mansoura College of Nursing during March (after mid-ye 2008/2009 G. After literature review, a specially designed questionnaire was in English was used as the tool for data collection. This was pilot tested on a sample of 40 students (10 from each educational year), over a one-week period (not included in the full-scale study). An interview was conducted after approval of the students. The questionnaire was modified accordingly in its final form e.g. rephrasing of some question and adding explanatory notes. This pilot study revealed that about 35% of students suffer severe stress. Finally the questionnaire was approved by the college authority as there is no formal research ethics committee. Sample size was calculated using Epiinfo® version 6.02. According to students´ affairs administration, the total number of registered nursing students was 1627 (all females)in the four years. From the pilot study it is expected that at least 35% of students suffer severe stress. With the worst acceptable level 32%, the sample needed for the study was estimated to be at least 350 students at 95% confidence level. First students were stratified into the different academic years (first to fourth). From each year Students were selected through systemic sampling technique (every 4th student) using the master list of students’ academic numbers. A total of 402 questionnaires were distributed and 381 were returned.Of these 8 were excluded due to incomplete or missed data. Thus 373 questionnaires were analyzed with a response rate of 92.8%. Background Stress has been identified as a 20th century disease and has been viewed as a complex and dynamic transaction between individuals and their environments [1]. Stressors can be broadly defined as situations or events that have the potential to affect health outcomes [2]. Cognitive appraisal, defined as the individual interpretation of a potential stressor, as a mediating process, can influence the impact of stress on health [3]. People who view the events as threatening (i.e., cannot employ adaptive coping skills) are more prone to cognitive deficits (for example, attention and concentration difficulties), physical illness, decreased life satisfaction [4-7], neuroticism [8], poor health behaviors and impaired academic performance [9]. The faculty of nursing, Mansoura University was founded in 2000. The 4-year undergraduate curriculum in Mansoura is designed to ensure that the theoretical and clinical practice requirements identified by the Nursing sector Committee of the Supreme Council of Egyptian Universities are met at appropriate levels throughout the 4 years of the program. During the clinical practicum, students are assigned to various clinical specialties in hospitals and medical centers of the university to gain clinical experience. Their clinical knowledge, skills, problem solving ability, and professional attitudes have to be assessed in each clinical practicum course [10]. Levels of stress and sources of stress have been reported in studies of nursing students in Western population [1,11-14] However, there has been limited research on clinical stress, anxiety, depression among the Arab population [15,16]. No study has investigated the perception of stress among Egyptian baccalaureate nursing students. This study was conducted to examine such students´ stress. The research questions of the study were the following: 1) what is the level of stress perceived by baccalaureate nursing students? 2) What type of stressors are commonly experienced by the students? 3) What is the level of anxiety and depression perceived by students? 4) What factors are associated with the frequency of stress experienced by the students? Pan African Medical Journal. 2011; 8:26 This article is available online at: http://www.panafrican-med-journal.com/content/article/8/26/full/ © Mostafa Amr et al. The Pan African Medical Journal - ISSN 1937-8688. 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. Page number not for citation purposes 1 Pan African Medical Journal – ISSN: 1937- 8688 (www.panafrican-med-journal.com) Published in partnership with the African Field Epidemiology Network (AFENET). (www.afenet.net) Page number not for citation purposes 1 Procedure The questionnaire used in the study was administered in classrooms under the guidance of the researchers. Students were briefed about the study, encouraged to participate and motivated to express their experiences. The students give fully informed verbal consent to participate. It was emphasized that all data collected was strictly confidential. Efforts were made to minimize under-reporting, strongly emphasizing to the student that the questionnaire was anonymous and that the data would be used for scientific purposes only. The questionnaires were distributed and recollected in the same setting. Data collection Participants completed an anonymous self-administered questionnaire two months before examination to avoid its possible direct stressful effect. The questionnaire covered socio-demographic factors, grade of the previous year, presence of stressors if any that had occurred during the past twelve months; Perceived Stress Scale (PSS), assessment of physical well-being factors, hospital anxiety and depression scale, neuroticism and extraversion subscales of Eysenck personality questionnaire, including fifteen potential sources of stress (stressors) were included. Students were asked to indicate the specific stressors, if any, affecting them. Perceived stress was measured by a previously validated 14-item Perceived Stress Scale (PSS) likert type,.The PSS has internal consistency of 0.85 (Cronbach ∝ coefficient) and test-retest reliability during a short retest interval (several days) of 0.85 [17]. The Arabic version was tested among a sample of US Arab immigrants [18]. The PSS does is not linked to appraisal of a particular situation as it is sensitive to the non-occurrence of events as well as to ongoing life circumstances. High scores on the PSS mean greater stress levels.The stress score was stratified into “no”, “mild”, “moderate” (less than the first, second and third quartiles, respectively) stress (All merged under “low level” stress, or severe (equal to or above the fourth quartile) labeled as high level stress. A self-report questionnaire for assessing physical well-being factors, designed by Hojat et al.[19] included 15 health problems: a) somatic symptoms of stress including questions about skin rash, back pain, allergies, infectious diseases, frequent colds and generalized body pain; b) agitation symptoms e.g. sleep problems, headache, nausea, lack of appetite; c) eating/drinking and smoking problems; and d) chronic illness and health problems interfering with daily activities. The global sickness index was based on an average score obtained from all health problems listed in the questionnaire. The Cronbach ∝ coefficients for the four physical well-being factors were in the 0.90s. The hospital anxiety and depression scale (HAD) [20] was used to measure subjective anxiety and depression where a score of 12 or more for either the anxiety or the depression components denotes possible anxiety or depression. This cut off point has a sensitivity of 0.89 and specificity of 0.75 [21]. The Arabic version of the HAD scale was validated by El-Rufaie and Absood [22]. The overall Cronbach ∝ measures of internal consistency were 0.78 and 0.87 for anxiety and depression, respectively. Page number not for citation purposes 2 Results The study included 273 Baccalaureate Nursing Students. Their age ranged from 17-22 years with a mean of 18.8±1.2, 68 % of the sample was from rural areas. Family income was reported to be satisfactory in 78.2 % of students and a family size of more than 5 persons was reported by 55.7% of students. 77.5% of fathers and 75.9 % of mothers of the respondents were of secondary and above secondary education level. More than two thirds of fathers were working as professionals (63.8%) and 59.2% of mothers were housewives. Mild to moderate stress level (Low stress) and severe stress (High stress) were encountered in 59.8 and 40.2% of the students respectively. Clinical anxiety was reported in nearly half of the sample (46.6%) and depression in 104 students (27.9%). Stressors were reported by 97.3% of students and the number of stressors reported by students ranged from 0 to 13, with a mean of 4.6±2.5. The reported rate for the occurrence of stressful events ranged from a low of (9%) for the death of a family member to a high of 82.6% for "fear of future". The five most frequently reported stressors were fear of future, self reported anxiety and depression, increased class workload, accommodation problems and congested classrooms (Table 1). Regarding the PSS, the mean overall score was 28.6±6.7(15-89), low and high stress groups were 27±4.9(7-32) and 38 ±4.6(33.55). Table 2 Shows that the prevalence of high level of stress, anxiety and depression were 40.2%, 46.6% and 27.9%; respectively. The overall mean of global sickness index was 28.6±6.7(15-89). There were significant differences in the sociodemographic data of those with high and low stress level such as family residence (p= 0.016), father´s education (p=0.015), father´s work (p=0.022) and grade of previous year (p= 0.031).The high stress group had a significant trend for living in rural areas, their fathers were less frequently professional, had lower school education and grade in the previous year (Table 3). Multivariate logistic regression analysis (Table 4) showed that that the number of stressors and Global Sickness Index score (GSI) were independent predictors of high stress level (p=0.000 and p=0.04 respectively). Data Processing and analysis Data was analyzed using SPSS (Statistical Package for Social Sciences) version 11. In quantitative data unpaired student´s t-test was used for group comparison. In categorical data Chi-squared test was used for comparison between groups. Odds ratio and 95% confidence interval was calculated. Significant factors predicting stress on univariate analysis were entered into multivariate logistic regression analysis. P<0.05 was considered statistically significant. Discussion Early marriage is no longer the standard it once was in Arab countries: the average age at marriage for both men and women is generally rising, and more Arab women particularly who are college graduates are staying single longer or not marrying at all [29]. Finally, the poor reputation of the nursing profession in Egypt because of the involvement of the nurse (female) and the doctor (male) on the one hand or the nurse and the patient on the other hand [27]. Stressors identified by the students in this study such as self reported anxiety and depression, increased class workload and, financial and relationship problems are similar to what been described in previous studies of similar student groups [19,30,31]. Despite some similarity in the types of stressors identified, perceived stressors such as worry of students towards their future, accommodation problems and overcrowded classrooms appear unique to this setting. Having to cope in a crammed classes and hostels would be extremely stressful in a course with a heavy workload requiring increased study time. The observations made by the students have been substantiated by college authorities who carried a program to motivate students’ active learning through applying problem-based learning in the third year of an undergraduate nursing program among some groups of students who enrolled in nursing administration course. The students in the PBL group gained more knowledge and were more motivated for learning than those in the lecture group [32]. On logistic regression analysis, High Stress was positively associated with number of stressors, and the global sickness index. A meta-analysis of 40 students on psychological distress among U.S. and Canadian college students explored the relationship between level of perceived stress and student distress [33]. Okasha et al. [34] concluded that most cases of psychological distress among Egyptian college students were reactions to either maturational or environmental stresses rather than endogenous factors. It is possible that medical students find medical education stressful with a high stress level reported along with a higher level of related psychosomatic activity and increased mood disturbances [17]. Liu and colleagues [35] showed that poor health status, test pressure, conflict with classmates and personality trait of introversion were independently associated with the presence of anxiety. Cohen and Williamson [36] added that stress as measured by the PSS would be moderately correlated with the number of stressors. Discussion Our study showed that 40.2% of nursing students who reported high stress (40.2%) which was higher than other studies using different distress measures in both developed and developing countries. Papazisis et al. [14] reported that 71.8% of nursing students in Greece perceived stress, most of them in mild levels (31.8%). About 12.4% reported very high levels of stress. Pryjmachuk and Richards [13] found that that the overall General Health Questionnaire caseness in pre-registration nursing students was around one-third. Arafa et al. [23] revealed that 21.67% of nurses recorded moderate to severe psychological distress on the General Health questionnaire (GHQ-30 items). This higher prevelance of stress in our sample could be interpreted as follows: Firstly, In Egypt, the current education policy allows an increasing number of admitted college students.The performance and quality of higher education is severely compromised. Traditionally, most students spend the majority of their school careers in passive learning environments in which faculty are disseminators of information, and students are required to memorize information. The system doesn´t foster the development of synthesising, problem solving and creative thinking abilities that impair the clinical skill training [24]. Second, in Egypt there are three types of nurses: college graduates, technical institute graduates and nursing school graduates, also known as diploma nurses. The first two types of nurses comprise four and two per cent of the Egyptian nursing staff respectively, while diploma nurses make up the remaining 94 per cent [25]. Recent reforms in the health sector eliminated the high school nursing by 2009, allowing a gradual replacement of diploma nurses with baccalaureate graduates,thus bringing about a change in the already established role of college graduates as staff nurses having the leadership and prestigious positions in the hospital environment to assistant practical nurses who only obey the orders of the junior doctors and senior diploma nurses. Third, these future nurses are expecting financial hardships after graduation due to low salaries which might reflect on their future social and family life [26]. Nurses are often blamed for errors, and are generally treated as scapegoats by other members of the medical profession [27]. Page number not for citation purposes 3 Fourth, families in the Arab world are undergoing major changes as new patterns of marriage emerge across the region [28]. Competing interests The authors declared they have no competing interests. Also there are no sources of funding. Conclusion It is clear that the Egyptian student nurses surveyed were exposed to a variety of academic, personal and environmental stressors. Academic pressures and the effect on social life and means for sustenance are key areas for intervention. The importance of small classes, active method of learning provision of equipment for practical work, improving accommodation should be emphasized. Given the detrimental effects of stress on health and academic performance, college administrators should consider incorporating stress management training into orientation activities for nursing students. Other approaches may be the use of stress management, assertiveness skills, time management and counseling sessions, may be effective in reducing stress experienced by nursing students. More studies need to be considered at a multi-center level using more informative sociodemographic, psychosocial and institutional variables in order to confirm the present findings and to enlighten corrective interventions. Discussion The limitations of this study are that the findings are based on self-reported information provided by students and thus some potential for reporting bias may have occurred because of respondents´ interpretation of the questions or desire to report their emotions in a certain way or simply because of inaccuracies of responses. The study takes place at one point in time which will limit the ability to generalize the findings to other time periods; this is referred to as a threat to temporal validity. In addition, the study took place at only one college, which will affect the generalizability to other institutions. Authors contribution MA: Study concept, design, Statistical analysis, data discussion manuscript writing, and editing. AG: Study concept, design of the questionnaires, Statistical analysis, manuscript writing and review. HM, LS: Female researchers who made awareness of the students, Data collection and manuscript writing and review. CJ: helped in manuscript review and editing Acknowledgments The authors would like to thank all the nursing students at the College of Nursing, Mansoura University for their sincere cooperation. References 1. Evans William, Kelly Billy. Pre-registration diploma student nurse stress and coping measures. Nurse Education Today. 2004; 24(6):473- 482. This article on PubMed 2. Barling Julian. Employment, stress and family functioning. New York: John Wiley & Sons; 1990 3. Cassidy Tony. Stress, healthiness and health behaviours: An exploration of the role of life events, daily hassles, cognitive appraisal and the coping process. Counseling Psychology Quarterly. 2000; 13(4):293-311 4. Bailey Roger C, Miller Christy. Life satisfaction and life demands in college students. 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El-Noshokaty Amira. (2004). The job of mercy. Al-Ahram Weekly. http://weekly.ahra.org/eg/print/2004/690/fe2.htm. A accessed 10 January 2010 28. Rashad Hoda,Osman Magued, Rouda-Fahimi Farzaneh (2005). Marriage in the Arab world. Population Reference Bureau at http:/www.prb.org. Accessed 11 January 2010 29. Elzanty Fatma, Way Ann(2003). Egypt Interim and Demographic Health Survey 2003(Cairo: Ministry of Health,2004) 30. Morrison Jill, Moffat K. More on medical student stress. Medical Education. 2001;35:617-618. This article on PubMed 31. Honkalampi Kirsi, Koivumaa-Honkanen Heli, Hintikka Junka, Antikainen Risto, Haatainen Kaisa, Tanskanen Antti, Viinamäki Heimo. 36. Cohen Sheldon, Willamson Gail. Perceived stress in a probability sample of the United State. In: Spacapan S, Oskam S. (Eds.). The social psychology of health. Newbury Park, CA: Sage, 1988 pp. 31-67 Page number not for citation purposes 6 Categories are not mutually exclusive Page number not for citation purposes 7 Categories are not mutually exclusive e.g. overcrowed accomodation, noisy livining environement, transportation problems. e.g. overcrowed accomodation, noisy livining environement, transportation problem References Do stressful life-events or sociodemographic variables associate with depression and alexithymia among a general population? A 3-year follow-up study. Comprehensive Psychiatry. 2004;45(4):254-60. This article on PubMed 32. Gabr Hala, Mohamed Nearmat. Effects of problem-based learning on undergraduate nursing students enrolled in nursing administration course. International Journal of Academic research. 2010; 3(1): 154-164 33. Dyrbye Liselotte N, Thomas Matthew R, Shanafelt Tait D. Systematic review of depression, anxiety and other indicators of psychological distress among U S and Canadian Medical students. Academic Medicine. 2006; 81(4):354-373. This article on PubMed 34. Okasha Ahmed, Kamel Mostafa, Sadek Adel, Lotaif ZB. Psychiatric Morbidity Among University Students in Egypt. British Journal of Psychiatry. 1977;131:149-54. This article on PubMed 35. Liu XC, Oda S, Peng X, Asai K. Life events and anxiety in Chinese medical students. Soc Psychiatry Psychiatr Epidemiol. 1997 Feb;32(2):63-7. This article on PubMed 36. Cohen Sheldon, Willamson Gail. Perceived stress in a probability sample of the United State. In: Spacapan S, Oskam S. (Eds.). The social psychology of health. Newbury Park, CA: Sage, 1988 pp. 31-67 Page number not for citation purposes 6 Table 1 : Prevalence of different stressors among Mansoura (Egypt) baccalaureate nursing students (n=373) N (%) No stressors 10(2.7) Number of stressors X± SD (Min – Max) 4.6 ± 2.5 (0-13) Stressors* Relationship issues Relationship problems with parents 54(14.5) Problems with the opposite gender 85(22.8) Trouble with coursemates 80(21.4) Personal troubles Personal illness or injury 56(15.0) Death of a family member 37(9.0) Change of a family member's health 83(22.3) Financial problems 115(30.8) Self-reported anxiety and/or depression 237(63.5) Academic pressures Congested classroms 174(46.6) Increased class workload 223(59.8) Inconsiderate and insensitive instructors. 72(19.3) Fear of future 308(82.6) Environmental problems Accomodation problems** 203(54.4) Close contact with serious diseases and illness 80(21.4) Time limitation for sports and hoppies 156(41.8) Categories are not mutually exclusive e.g. overcrowed accomodation, noisy livining environement, transportation problems. e.g. overcrowed accomodation, noisy livining environement, transportation problems. Page number not for citation purposes References Table 2: Overall prevalence of stress, anxiety and depression stressors among Mansoura (Egypt) baccalaureate nursing students N (%) 95% CI High stress 150(40.2) 35.4-45.3 Anxiety 174(46.6) 41.6-51.7 Depression 104(27.9) 23.6-32.6 CI = Confidence interval verall prevalence of stress, anxiety and depression stressors among Mansoura (Egypt) baccalaureate t Table 3: Level of stress according to sociodemographic and academic characteristics of stressors of Mansoura (Egypt) baccalaureate nursing students Stress level Significance Low N (%) High N (%) Total N (%) Overall 223(59.8) 150(40.2) 373(100) Family residence Urban 63(52.9) 56(47.1) 119(31.9) c2=8.2, P=0.016 Rural 160(63.0) 94(37.0) 254(68) Student's residence With family 174(59.8) 117(40.2) 291(78) Away from the family 49(59.8) 33(40.2) 82(22) Family income Unsatisfactory 42(51.9) 39(48.1) 81(21.8) c2=2.7, P=0.1 Satisfactory 181(62.0) 111(38.0) 292(78.2) Family size Up to 5 104(62.7) 62(37.3) 166(44.5) c2=1.02, P=0.3 > 5 119(57.5) 88(42.5) 207(55.4) Father's education Less than secondary 54(64.3) 30(35.7) 84(22.5) c2=8.4, P=0.015 Secondary 87(51.8) 81(48.2) 168(45) Above secondary 82(67.8) 39(32.2) 121(32.5) Father's work Unemployed 19(50.0) 19(50.0) 38(10.1) c2=7.6, P=0.022 Professional/semiprof 135(56.7) 103(43.3) 238(63.8) Others 69(71.1) 28(28.9) 97(25.3) Mother's education Less than secondary 57(63.3) 33(36.7) 90(24.1) c2=1.5, P=0.5 Secondary 118(57.0) 89(43.0) 207(55.5) Above secondary 48(63.2) 28(36.8) 76(20.4) Mother's work Working 87(57.6) 64(42.4) 151(40.8) c2=0.5, P=0.5 Housewives 136(61.3) 86(38.7) 222(59.2) Academic year First 55(55.0) 45(45.0) 100(26.8) c2=3.7, P=0.3 Second 64(56.1) 50(43.9) 114(30.6) Third 59(65.6) 31(34.4) 90(24.1) Four 45(65.2) 24(34.8) 69(18.5) Grade of previous year Excellent 91(54.8) 75(45.2) 166(44.5) c2=8.9, P=0.031 Very good 98(69.0) 44(31.0) 142(38.1) Good 11(45.8) 13(54.2) 24(6.4) Others ** 23(56.1) 18(43.9) 41(11.0) Others include : private business, farmers, manual workers, trades *Others include : pass and failed Page number not for citation purposes Table 4: Multivariate analysis using a stepwise logistic regression model Predictor High stress level b P OR(95% CI) Global sickness index:(continuous) 0.04 0.04 1.01 (1.01-1.1) Number of stressors: (continuous) 0.2 0.000 1.2 (1.1-1.3) Constant -2.4 Percent correctly predicted 64.3% Model c² 41.7, P=0.000 OR= Odds ratio, CI= Confidence Interval, r=reference group Table 4: Multivariate analysis using a stepwise logistic regression model Page number not for citation purposes
https://openalex.org/W4224255867	https://www.intechopen.com/citation-pdf-url/80462	English	null	Postharvest Preservation Technology of Cereals and Legumes	IntechOpen eBooks	2,022	cc-by	10,385	Abstract Cereals and legumes are prone to perishability and have very short shelf-life if not given proper treatment. During different handling and marketing operations, there is a huge postharvest loss of agricultural produce. The qualitative and quan- titative losses incurred in cereals and legumes commodities between harvest and consumption are huge. Qualitative losses such as loss inedibility, nutritional quality, calorific value, and consumer acceptability of fresh produce are much more diffi- cult to assess than are quantitative losses. The major cause of postharvest loss (PHL) is the availability of poor infrastructure for postharvest technology (PHT) and processing of commodities. These losses can only be minimized by proper handling, marketing, and processing of the agricultural commodities; as well as the use of modern preservation technologies such as irradiation, radio frequency heating, etc. The sufficient knowledge of pre-and post-harvest preservation technologies and the provision of adequate and sufficient storage facilities for cereals and legumes handling and distribution would help to mitigate the incidence of postharvest deterioration and therefore improve the availability of cereals and legumes in the market and subsequent reduction in malnutrition for increased food security. Postharvest preservation technology of cereals and legumes is very fundamental in reducing postharvest losses and increasing food security. Keywords: postharvest, physiology, deterioration, losses, postharvest technology Postharvest Preservation Technology of Cereals and Legumes Theophilus M. Ikegwu, Clement C. Ezegbe, Chioke A. Okolo and Chigozie E. Ofoedu 1. Introduction Both testa and pericarp are called the bran. Conversely, legumes are flowering plants (dicotyledons) in the Leguminosae family and were derived from the latin word legere (to gather) and legumen (seeds harvested in pods) during the mid-17th Century. It includes chickpea, black gram, mung bean, and pigeon pea which have an estimated 16,000–19,000 species in 750 genera. Asia ranks first both in area harvested and in production capacity. India, on the other hand, accounts for 75 and 96% of the total global production of chickpea and pigeon pea, respectively [4]. The expression food legumes usually mean the immature pods and seeds as well as mature dry seeds used as food by humans. Based on Food and Agricultural Organization (FAO) practice, the term legume is used for all leguminous plants. Legumes such as French bean, lima bean, alfafa, or others that contain a small amount of fat are termed pulses, and legumes that contain a higher amount of fat, such as soybean and peanuts, are termed leguminous oilseeds. Legumes represent an important source of food in developing countries. Soybean, groundnut, dry bean, pea, broad bean, chickpea, and lentil are the common legumes in most countries. In some countries, depending on the climatic condition and food habits, other legumes are grown. Legumes are next to cereals in terms of their economic and nutritional importance as human food sources [3]. They are cultivated not only for their protein and carbohydrate content but also because of the oil content of oilseed legumes such as soybeans. g y Legumes are sources of protein and are relatively costlier economically compared to cereals with great food value; and are reasonable nutrients for the maintenance of the body, e.g., vitamins and minerals. The legume has almost the same energy value per unit weight compared to the cereal grains (4.2 kcal), albeit, they provide more calcium, iron, thiamine, riboflavin, pantothenic acid, among others than cereals. The utilization of legumes is highest in India and Latin America owing to religious restrictions and food attitudes. Legumes also contain some anti-nutritional factors, such as trypsin and chymotrypsin, phytate, lectins, polyphenols, flatulence-provoking and cyanogenic compounds, lathyrogens, estrogens, goitrogens, saponins, anti-vitamins, and allergens. However, heat treatment is known to destroy the anti-nutrients, such as protease inhibitors and lectins, although it also destroys vitamins and amino acids. 1. Introduction The cereals are monocotyledons while the legumes are dicots. The cereal belongs to the grass family with more than 300,000 species. Furthermore, more than 190,000 species are angiosperms that are economically viable horticultural plants; and there are approximately 50 different types cultivated throughout the world in which about 51 species are grown. However, cereal’s contribution to human nutrition cannot be overemphasized as it had been estimated that nearly 18 spe- cies of cereals cultivated provide more than 91% of the food supply to the world population. The cereals cover about 74% of the total tilled land surface. It had been estimated that more than 50% of the protein needs of the world population are provided by cereals [1, 2]. Currently, France ranks first in the Export of cereals such 1 Postharvest Technology - Recent Advances, New Perspectives and Applications as wheat, rice, maize, and barley in Europe but 5th in the production of wheat in the world [1, 2]. Other cereals include millet, sorghum, rye, oats, etc. The major grains such as wheat, rice, and corn add up to make three-quarters of the world- wide production of grain [1, 2]. Therefore, cereal grains remain the main source of dietary carbohydrates for the supply of vital food energy to the diet [1, 2]. Although cereal grains, such as maize, rice, millet, and wheat are mostly in higher demand for energy provision, other cereals also provide very important food uses while there are more researches to explore the underutilized ones [3]. When cereal crops are grown for the edibility of their fruits, they are referred to as grains (botanically called caryopsis). y p Structurally, the cereal seed is composed mainly of two components; the endo- sperm and the embryo (germ). The endosperm (more than 90% 0f the bulk seed) provides the energy. The pericarp (outer wall) develops from the ovary wall and encloses the endosperm. Beneath the pericarp is the testa (a selectively permeable layer) that borders the embryo which is a product of the inner reproductive gland (ovary wall). The permeability of testa to water is high and aids in seed germination but in the presence of salt, the testa may lose its vigor which would consequently lead to nongermination of seeds planted in soils with dissolved salts. The aleurone layer (with thick-walled cells) is free of starch and is the third important layer of cereal grain. Postharvest Preservation Technology of Cereals and Legumes DOI: http://dx.doi.org/10.5772/intechopen.102739 Healthy cereal grains and legumes are the demanding enterprises of the recent era for the production of high yield in the next season. The cereal grains and legumes must be properly stored for the maintenance of a high-yielding crop. Losses of high magnitude are encountered during storage that is due to biological and non-biological agents. The incidence of high losses of cereals and legumes after harvest in many countries of the world could account for the food security issues such as malnutrition, diabetes, and hunger which are counterproductive to miti- gating efforts towards the improvement of food security. The effect of low yield, poor quality of produce, and the prevalence of chemical toxicants and mycotoxin contamination are significant problems that militate against the genuine and con- certed efforts to improve postharvest losses (PHLs), provide appropriate handling and processing technologies for improved postharvest opportunities. In an attempt to maintain high-quality crops during postharvest operations (PHOs), care must be taken during harvesting to minimize damage and ensure appropriate postharvest handling techniques. Reliable methods for the assessment of postharvest losses should be developed while the use of the appropriate techniques to minimize loss and ensure the quality and safety of crops that meet quality standards are desired. In developing countries, Nigeria inclusive, cereals and legumes produced mainly by small-scale farmers are produced and stored on farms [4]. Biological and non-bio- logical agents have been implicated in the postharvest losses of cereals and legumes (Figure 1) [5, 6]. g There is a direct correlation between plentiful harvest and postharvest spoilage. In countries with huge harvests, postharvest losses are higher than in countries with less bumper harvests which may be a consequence of a lack of care arising from a short supply of laborers to preserve the excess grains. Consequently, farmers may be forced to sell their grains at a less reasonable price during the harvesting season to prevent possible postharvest losses. The glut in the price of cereals and legumes could lead to short supply leading to increased losses arising from insect pest attacks (Prostephanus truncates). However, the effect of bumper harvests on losses had not been measured, and overall; the effect would be minimal compared with the losses resulting from an unfavorable climate at harvest. Certainly, farmers are often supplied with sufficient storage capacity in developed countries so that at least Figure 1. Considerations for postharvest preservation technologies. Figure 1. 1. Introduction Legumes are a good source of dietary fiber; the crude fiber, protein, and lipid components have a hypocholesterolemic effect. 2 Postharvest Preservation Technology of Cereals and Legumes DOI: http://dx.doi.org/10.5772/intechopen.102739 Postharvest Preservation Technology of Cereals and Legumes DOI: http://dx.doi.org/10.5772/intechopen.102739 Postharvest Preservation Technology of Cereals and Legumes DOI: http://dx.doi.org/10.5772/intechopen.102739 Considerations for postharvest preservation technologies. Figure 1. Considerations for postharvest preservation technologies. Figure 1. d 3 Postharvest Technology - Recent Advances, New Perspectives and Applications good harvests can be accommodated in fixed stores; unlike in developing countries where less attention is paid to farming and facilities for storage are lacking. In such instances, farmers are content to store surplus cereals and legumes in sacks in their houses. In most cases, especially, in locations where subsistence farming is com- mon, the use of bag storage rather than traditional structures is practiced. It was strongly believed by the 1970s that postharvest losses (PHLs) at the farm level were high due to traditional practices. However, traditional practices are unlikely culprits as farmers have survived more difficult conditions over long periods by adapting their practices to the situational challenges [7]. Nonetheless, compelling losses do sometimes occur that could be due to agricultural develop- ments for which the farmer is not versed due to nonavailability of extension agents. Among these agricultural developments is the introduction of high yielding variet- ies that are more susceptible to pest damage, additional cropping seasons that result in the need for harvesting and drying when weather is damp or cloudy or farmers producing significant surplus grains, and because it is to be marketed rather than consumed by the household, the farmer failed to provide the necessary storage facilities for the preservation of the surplus grains. 3. Postharvest pest management Pests pose a very big challenge during the postharvest storage of grain legumes, transportation as well as during distribution. The quality and quantity of grains are reduced by pests if not properly controlled. Pest infestation is a big source of worry for both farmers and food processors because of the losses in investment and profit depletion that come with it. Some of the grain pest control techniques conven- tionally adopted are fumigation and controlled atmosphere of CO2 and N2. Novel techniques have also been developed to take care of some of the shortcomings of conventional pest management practices like fumigation that make use of chemi- cals. Examples of some of the emerging technologies which have found use in pest management include irradiation, radio frequency, infrared, and microwaves [7]. Methyl bromide application and treatment with hot air on grain legumes storage facilities or systems is also a common practice for disinfection in the grain storage industry [4]. Postharvest Preservation Technology of Cereals and Legumes DOI: http://dx.doi.org/10.5772/intechopen.102739 vi. Controlled atmosphere storage – in controlled atmosphere storage, the food products’ environment is modified to prevent spoilage. Other methods are the application of high-frequency currents, irradiation, etc. Additionally, other technologies such as pyrolysis, gasification, combustion, and chemical and biochemical processing are used for the conversion of cereals and legumes by-products to chemicals, energy, and other value-added products in the food value chain. 3.1 Irradiation (IR) Food irradiation is a food preservation technique during which ionizing radia- tion (0.1–50 KGy) is used to destroy target microorganisms in order to extend the shelf life of foods. During irradiation, microbial inactivation is achieved through free radical development which disrupts DNA and cell membrane integrity [7]. It has shown to be effective in sprout inhibition, elimination of parasites and insects, destruction of spoilage, and pathogenic microorganisms [8]. Radiation treatment at low and moderate doses has been recommended for the disinfestation of legumes [8]. The treatment has also been found to be effective for the reduction of flatulence-causing oligosaccharides as well as trypsin and chy- motrypsin inhibitors. With these effects of irradiation on anti-nutritional factors in legumes, the nutritive quality of irradiated beans is thereby improved. Stored produces, especially grains have been successfully decontaminated with ionizing radiation as it affects the internal structure [8, 9]. Irradiation technology has been very effective in controlling the Aspergillus, Penicillium, Rhizopus, and Fusarium fungi infection in many grains and prolonging the shelf life over 6 months [10]. The source of radiation that is usually utilized is Co-60 and selenium. 2. Preservation Theoretically, any method of food preservation should prevent all the above three (microbial, enzymatic, and proteolytic) types of spoilage. However, current industrial innovation methods have failed to meet these expectations as a whole. Most importantly, microbial spoilage must be prevented at all costs in whichever preservation method was employed, but the effectiveness of thermo-bacteriological treatment for microbial destruction varies to different degrees in the prevention of enzyme activities, proteolytic reactions, and the destruction of different micro- organisms. Recent innovative preservation technologies such as ohmic heating, irradiation, infrared, pulse electric field, edible coating, radio frequency, and encapsulation lack the ability to forestall all the concerns posed by spoilage effects completely. These industrial methods employ distinct preservation principles aimed at arresting and or preventing food spoilage. In a nutshell, the industrial method of food preservation makes use of the following principles; i. The ability to remove moisture through the use of drying/dehydration, evaporation/concentration, etc. ii. The ability to remove heat from food products by lyophilization/freeze concentration, refrigeration/cold storage, freezing, etc. iii. Heat addition - heat could be added to food products to destroy microorgan- isms or inactivate their activities by canning, sterilization, pasteurization, thermization, etc. iv. Addition of chemicals/preservatives – some chemicals called preservatives may be added to processed food to prevent contamination by the micro- organism or forestall enzymatic/browning reactions. Examples of such chemical additives are sorbates, benzoates, etc. v. Fermentation - during fermentation, secondary metabolites are produced by microorganism which preserves the food product. v. Fermentation - during fermentation, secondary metabolites are produced by microorganism which preserves the food product. 4 4 Postharvest Preservation Technology of Cereals and Legumes DOI: http://dx.doi.org/10.5772/intechopen.102739 3.2 Radio frequency (RF) heating Radio frequencies (RF) are electromagnetic waves that are able to penetrate dielectric materials. They usually are characterized by a wavelength of about 11 m and with a frequency range of 1 to 300 MHz [11]. With this ability to penetrate dielectric materials like food grains, they are able to produce heat volumetrically. They are able to do this through ionic polarization or dipole rotation. With the 5 Postharvest Technology - Recent Advances, New Perspectives and Applications higher moisture content in food grains, their ability to act as dielectric materials is increased, allowing them to act as electric capacitors and resistors and useful in the storage and conversion and electrical to thermal energy. This can be possible within an electromagnetic field [11]. In comparison, the higher moisture content in insects and the consequent higher electrical conductivity would make them require higher lethal temperatures and higher lethal time. At a lethal temperature and time of 50°C for 29 minutes or 54°C for 5 minutes, it would be possible to completely destroy a wide range of insects. This process of higher heating rates and its application finds use in the disinfection of grains on an industrial scale [9, 11]. When insects feed directly on grains, they produce webs and feces on stored pulses thereby reducing grain quality and this represents a huge challenge during the storage, transportation, and distribution of grains. To mitigate this huge chal- lenge, RF heating has been used in the disinfection of dried cereals and legumes. This was demonstrated using a 27 MHz and 6 kW RF unit where the RF proved superior to forced hot air with respect to heating time required (5–7 minutes as against 275 minutes) to heat 3 kg of legumes to 60°C. Good quality product and uni- formity in temperature distribution across the surface and interior of the legumes was achieved in the legume samples by a combination of RF heating followed by a movement of forced hot air as grains move through conveyors at 0.56metres per minute. The final interior temperatures of the containments used were above 55.8°C while 57.3°C was recorded for the surfaces of all legumes tested with resultant low index values for uniformity of 0.014–0.016 (ratio of standard deviation to the aver- age temperature rise) for the distribution of interior temperature and 0.061–0.078 for the distribution of surface temperatures. 3.2 Radio frequency (RF) heating Legumes treatment with RF in com- bination with forced hot air (60°C) to retain the needed treatment temperature for 10 min followed by the rapid cooling of the air through a 1 cm product layer yielded products with high quality. There were no significant differences in weight, moisture, color, and germination when samples used for control were compared to treated ones [12]. 3.5 Fumigation Fumigation is a very active pest control technique. Phosphine gas for example is used to kill grain pests at every growth level of their life cycle; this is inclusive of pests with high resistance ability. Nonetheless, the phosphine gas application level needs to be up to 300 parts per million (ppm) and sustained at this level for a minimum of one week at 25°C or more. Alternatively, at a temperature of 25°C or less, a 200 ppm concentration of phosphine gas should be maintained for 10 days for effective and efficient destruction of pests that destroy legumes. Phosphine application exists in two forms; they include bag chains and tablets. There are also a number of ways with which each choice can be adopted effectively in a gas-proof secured silo. Bag chains are also considered a very safe system that assures one of not having any fumigant residue on the grain nor having the operator harmed in whatever way. The next form that phosphine exists in tablet form and is the most widely used and accepted. There exists a third approach in phosphine application which involves the use of a phosphine blanket and is mostly used for very large storages of above 600 tones. The application of phosphine and the concentrations to be used depend on the silo (which should be gas-tight and sealable) volume used for the fumigation. The phosphine concentration to be used is strictly determined based on the volume of the silo rather than the quantity of grain in the silo [13]. q y g An airtight-covered silo especially one that passes the half-life pressure test must have to remain sealed through the entire fumigation period in other to attain a perfect fumigation result with the use of phosphine tablets and/or bag chains. In an airtight-sealed silo, fumigation is expected to last for 7 days with a temperature of above 25°C, and 10 days if the temperature falls between 15 and 25°C. Nonetheless, if the temperature in the silo is less than 15°C, pests particularly insects will be inac- tive and phosphine is not usually effective at such low temperatures. Based on the ineffectiveness of phosphine at temperatures lower than 15°C, phosphine applica- tion is not advisable at temperatures lower than 15°C. 3.4 Microwaves Microwaves are electromagnetic radiations with short-wavelength; which has an excellent microbial destruction potential when compared to other conventional chemical methods. Microwave technology is now a highly adopted process by most grain industries for disinfection [8, 13]. They provide protection on grains from insects [10], storage fungi, and field fungi [12]. However, treatment with the use of microwave can induce several adverse effects on seed germination and can affect grain quality. These adverse effects of microwaves are due to variations in heating caused by the difference in cold and hot spot temperature [9]. 3.3 Infrared Infrared is a segment in the electromagnetic spectrum found in between the microwave region and the visible spectrum area characterized by a wavelength of about 0.5 to 100 μm [9]. The absorption of infrared rays produces vibrations in the molecules of water, with consequent heat generation. Infrared-based technologies have been found to be energy-efficient and eco-friendly when compared with other conventional methods. Infrared technology also has many other merits like short process duration, uniform effect on food material, low energy requirement, high rate of heat transfer, and enhanced quality of products [9]. As a result of some of the above-listed characteristics, infrared-based technologies have been used in very many food operations like boiling, heating, drying, peeling, recovery of polyphe- nols and antioxidants, freeze-drying, roasting, microbiological inactivation, grains sterilization, juice and bread production, and cooking. The idea of the usage of infrared rays to disinfect/sanitize grains was established in the early 1960s and 1970s. Based on its exceptionally effective microbial inactivation characteristics, grain industries usually adopt it as a preferred operation for grain disinfection against various chemical methods. Infrared operations involve three different mechanisms in destroying micro-organisms namely thermal inactivation, induction heating, and the distortion of DNA integrity. As documented by [9], the Infrared treatment of mung bean for 5 minutes at an intensity and temperature of 0.29 kWm and 70°C respectively resulted in the total inactivation of fungal growth. Since the 6 Postharvest Preservation Technology of Cereals and Legumes DOI: http://dx.doi.org/10.5772/intechopen.102739 Postharvest Preservation Technology of Cereals and Legumes DOI: http://dx.doi.org/10.5772/intechopen.102739 penetration rate of infrared is low, its effectiveness gets reduced with an increase in the depth of food. It is therefore recommended more for food surfaces sterilization than other processes. Catalytic-infrared emitters have also been developed and used for the control of weevils in rice, merchant grain beetle, and saw-toothed grain beetle. Generally, a little exposure of about 60 seconds is adequate to destroy insects that strive externally or internally in the grains kept in storage facilities [9]. 3.6 Controlled atmosphere In spite of the fact that phosphine is the common most used gas fumigant, there exist other gas fumigants for controlling pests in stored grain. These alternatives are however more expensive than phosphine and still require a gas-tight, seal- able silo but they offer other options for resistant pest species. Nitrogen (N2) and carbon dioxide (CO2) have the advantage of being nonchemical control alternatives. Because nitrogen and CO2 methods of control change the balance of natural atmo- spheric gases to produce a toxic atmosphere, they are hence referred to as controlled atmosphere (CA) [13]. 3.6.1 Carbon dioxide (CO2) Treatment with CO2 involves displacing the air inside a gas-tight silo with CO2 at concentrations high enough to be toxic to grain pests. This requires a seal imperme- able to gases, measured by a half-life pressure test of no less than five minutes. In order to eliminate all life stages of the main grain pests, CO2 must be retained at a minimum concentration of 35% for 15 days [14]. To achieve a 35% concentration level of CO2 for 15 days, 30 kg (size G) cylinder per 15 tones of storage capacity is required. CO2 is an odorless, colorless, non-flammable gas that is approximately one and a half times heavier than air. Food grade CO2 comes in form of a liquid in pressurized cylinders and when released from the cylinder, changes to a gas. Carbon dioxide is less effective at temperatures below 20°C. This is because insects are less active at this temperature, so the CO2 concentration must be maintained for an extended period. 3.5 Fumigation The silo must remain closed when fumigation is on and should only be accessed by personnel with suitable personal protective equipment (PPE) as it is dangerous for the operator. Constant opening of the silo is also detrimental to the effectiveness of the fumigation process considering the fact that the phosphine gas concentration and absorption rate would have been reduced below the lethal level recommended for pests’ destruc- tion. Recommendations for the phosphine label came to be as a result of detailed 7 Postharvest Technology - Recent Advances, New Perspectives and Applications testing by the industry, in other words, making use of phosphine as indicated on the label will ensure perfect results [13]. Phosphine is rated high as a very reliable fumigant for the control of pests in grain storage facilities and other production enterprises [13]. Nevertheless, there has been a continuous misuse of fumigants with a resultant effect of poor pest control and the development of resistance in certain species of pests. More so, just as the continuous use of herbicides that has the same principle of action advances weeds being resistant, continuous use of phosphine could lead to grain pest resistance. Nonetheless, in the case of herbicides, the development of resistance by pests can yearly be circumvented by alternating the chemicals used. The same cannot be said for stored grain fumigation as options are limited and where available, they are not cost-effective [13]. In other words, it is best to avoid the resistance of phosphine by using it as instructed. p p y g Other fumigants and a controlled atmosphere may be used for stored grain pests but they are often high in price. However, to prevent resistance of stored grain pests, phosphine sealed in a silo that is impermeable to gas should be used. 4. Drying technologies Scientists from all over the world continuously search for new and effective means and use of renewable sources of energy as a result of the continuous increase in the price of fossil fuels and increased levels of greenhouse gas emissions. The world’s energy intake is doubled every 20 years and this increase in energy consump- tion, has resulted in fossil fuels causing many environmental problems and pollu- tion [15]. Drying is a processing technique used for food product preservation and reducing food spoilage. About 3.62% of the world’s energy is used for the drying of agricultural products [16]. Presently, the requirement for new drying technology that promotes the higher quality product and efficient drying in shorter periods is the current need. And as a result, hybrid drying systems have emerged as an excellent technique for their versatile drying outcomes, with lower energy requirements and minimum envi- ronmental impact. Lately, various hybrid solar dryers which are more efficient in conjunction with other sources for heating the air, hence reducing drying cost and energy consumption have been developed [17, 18]. gy p p Grain legumes are usually dried after harvesting before storage in storage facilities [17]. Drying grain legumes to a recommended safe moisture level is fundamental in achieving safe storage of grain legumes. However, too rapid dry- ing of nuts can lead to hardening of the grain core with poor interior while very slow drying may result in microbial growth which will lead to quality deteriora- tion. Recirculation of the solar drying air is thus employed to make efficient use of the heated air by giving a drying rate that provides acceptable product quality. Drying of pulses is essential because they contain high moisture content of about 18–25% at the time of harvest and, for safe storage, the optimum moisture content need to be in the range of 9–12% to avoid mycotoxin produc- tion. It is essential that the grain is dried to a safe moisture level as quickly as possible to avoid deterioration regardless of the drying system employed. There are several techniques of non-natural open-sun drying of grains with hot air. Some of these forms of drying include spouted-bed drying, fixed bed drying, moving bed drying, fluidized-bed drying, and thin-layer drying [19]. Apart from some of these specialized dryers used for grain drying, all-purpose grain drying systems can as well be used in the drying of grain legumes. 3.6.2 Nitrogen The method of application entails purging the silo to its base with gas majorly composed of nitrogen. This is done in order to force out from the silo the oxygen-rich air through the top of the silo. Several hours of operation are required for PSA to build up about 99.5% pure nitrogen and before the air composition reduces to 2% oxygen. It is difficult for adult insects to thrive in 2% concentration of oxygen, provided this concentration is maintained for 21 days at 25°C or above for the temperature of the grain [14]. The inhibition of the different stages of the life cycle of insects (eggs, larvae, and pupae) will be difficult below these recommended temperatures and the number of days for grain storage. For grain temperatures below 25°C, this treatment duration should further be extended to a 28-day period. Additional purging of the silo may be needed to get rid of oxygen that has diffused from the grains and it must be re-evaluated 24 hours after fumigation in order to achieve effective and efficient pest control. 3.6.2 Nitrogen Grains stored in a nitrogen saturated environment ensure the control of insects and preserve product quality without the use of chemicals [13]. Nitrogen-based storage systems maintain the quality of canola and pulses through the inhibition of the respiration process that causes oxidation, which may result in the increase in free fatty acids, loss of color, and seed deterioration [13]. Grain treatment with nitrogen (for the purpose of pest control) is safe, environmentally friendly, and involves the usage of electricity for its major operations. Nitrogen produces no residues when used, so grains can be sold instantaneously whenever decided as against what is practiced for chemical fumigants which have recommendation period for 8 Postharvest Preservation Technology of Cereals and Legumes DOI: http://dx.doi.org/10.5772/intechopen.102739 Postharvest Preservation Technology of Cereals and Legumes DOI: http://dx.doi.org/10.5772/intechopen.102739 withholding after fumigation [13]. The use of nitrogen as an insect control technique involves the use of Pressure Swinging Adsorption (PSA) technology in adjusting the atmospheric composition of the grain storage system to expel other gases other than nitrogen, thus depriving the pests of the needed oxygen. The method of application entails purging the silo to its base with gas majorly composed of nitrogen. This is done in order to force out from the silo the oxygen-rich air through the top of the silo. Several hours of operation are required for PSA to build up about 99.5% pure nitrogen and before the air composition reduces to 2% oxygen. It is difficult for adult insects to thrive in 2% concentration of oxygen, provided this concentration is maintained for 21 days at 25°C or above for the temperature of the grain [14]. The inhibition of the different stages of the life cycle of insects (eggs, larvae, and pupae) will be difficult below these recommended temperatures and the number of days for grain storage. For grain temperatures below 25°C, this treatment duration should further be extended to a 28-day period. Additional purging of the silo may be needed to get rid of oxygen that has diffused from the grains and it must be re-evaluated 24 hours after fumigation in order to achieve effective and efficient pest control. withholding after fumigation [13]. The use of nitrogen as an insect control technique involves the use of Pressure Swinging Adsorption (PSA) technology in adjusting the atmospheric composition of the grain storage system to expel other gases other than nitrogen, thus depriving the pests of the needed oxygen. 4.1 Classification of solar dryer The three major types of solar dryers with various sizes, capacities, and designs are: i. Direct solar dryers i. Direct solar dryers ii. Indirect solar dryers iii. Mixed-mode solar dryers iii. Mixed-mode solar dryers 4. Drying technologies Generally, as 9 Postharvest Technology - Recent Advances, New Perspectives and Applications documented by [20], dryers or drying systems are categorized depending on the following: documented by [20], dryers or drying systems are categorized depending on the following: a. The flow of grain wherein the dryers are denoted as - batch, recirculating and continuous dryers, b. The relative motion of the grains and the circulating air used for drying. Concurrent, counter-current, cross/mixed flow dryers are found in this category. c. The source of heat: solar, propane, and electrical dryers are examples of dryers in this category. c. The source of heat: solar, propane, and electrical dryers are examples of dryers in this category. Regardless of the type of dryer used in drying grains, the concurrent heat energy transfer and moisture loss principle/process is the same for the drying of grain legumes and equally for other grains [19]. The process of drying grains involves the loss of free moisture which involves the drying of the grain until its equilibrium moisture content is attained. The equilibrium moisture content of the grain implies the final moisture content attained by the grain at a pre-determined relative humidity and temperature. The cardinal factors that influence the drying rate of grain legumes are temperature, grain moisture content, relative humidity, and air velocity [19]. p g y y The use of solar dryers is also another medium for drying legumes. A lot of solar drying systems exist for grain drying such as direct, non-direct, and solar. Solar dryers have the problem of the dehydration process being stopped as a result of an absence of solar radiation and absence of radiation at night or low insulation, which decreases the quality of the grains. So far, there have been efforts to proffer solutions to the problems of solar systems, some of which include – the addition of thermal storage materials, phase change materials, and adding a variety of heating modes either direct or indirect [21]. This has led to the evolution of several types of solar dryers. Thermal storage materials have the ability to store thermal energy when there is solar radiation and then make use of this thermal energy when the sun is not available. Three main forms of solar dryers exist with varying sizes, designs, and magnitude [22]. 4.1.1 Direct type solar dryer This is a form of the solar dryer where the radiation from the sun is used directly incident on the grains to be drained. The dryers are quite simple in structure, less expensive, little or no maintenance needed, and also simple to use. It can be fabri- cated with a wooden box with a glass cover and some holes for air entrance and exit also. After the usage of the direct type of solar dryers, the food products are usually 10 Postharvest Preservation Technology of Cereals and Legumes DOI: http://dx.doi.org/10.5772/intechopen.102739 Postharvest Preservation Technology of Cereals and Legumes DOI: http://dx.doi.org/10.5772/intechopen.102739 Postharvest Preservation Technology of Cereals and Legumes DOI: http://dx.doi.org/10.5772/intechopen.102739 not very nice in appearance, color, texture, and with a reduced nutritional quality. In direct-type solar drying, produces to be dried are spread on the ground or mats exposed directly to the sun to absorb solar radiation. As noted by [23], sun-dried grains are prone to high crop losses due to: i. Non-uniform moisture loss i. Non-uniform moisture loss ii. Attack by insects and rodents iii. Inability to attain moisture levels that are safe for the safe storage of grains iv. Proliferation of micro-organisms and possible toxin production. These challenges have led to the development of other drying techniques like solar drying to overcome the aforementioned challenges. Solar dryers have faster rates, better efficiency, more hygienic with less crop losses when compared to sun drying [23]. 4.1.1.1 Open sun drying Here, food products are placed right under the sun, below solar radiation to get rid of their moisture properties. The difference in density in the air from the atmosphere allows for air movement. In other words, to get a product dried, they are usually spread in a large area under solar radiation. It is usually time-consuming till it is dry to a required level. All that is needed is a large surface ground done with concrete or a suitable soil area with products laid on them between ten to thirty days depending on a favorable weather situation. This form of drying technique consumes a lengthy time of sun subjection which can sometimes bring down the nutrition level of the products, like sapping off their vitamins. p pp g Open solar drying is a good choice for food drying but comes with a lot of problems such as reduced product quality, adverse effects of rain, moist, wind, animal consumption, and dust [24]. The use of industrial drying comes in as another option which is very expensive. It would need a lot of fossil fuel which will result in air pollution. Nonetheless, the spread and adoption of solar energy is likely to take prominence in the coming years and is without negative environ- mental effective factors [24]. 4.1.1.2 Cabinet type solar dryer The cabinet form of the dryer is advantageous for preserving smaller food products such as vegetables, pepper, and fruits. It has a roof that is transparent with covers that could be either single or two, made using a black-colored plate cover that serves as an absorbing entity for the storage of energy from the sun. Suitable perforated holes allow for the free flow of air and the removal of moisture. 4.1.2 Indirect mode solar dryer When it comes to moisture removal and heat transfer, indirect sun dryers differ from direct solar dryers. This style of drier is utilized for quick drying. The atmo- spheric air is heated in a solar air collector in this dryer, and then this hot air moves towards the drying cabin, where products are kept to dry, and the hot air absorbs some moisture from the drying products before exiting through the chimney. 11 Postharvest Technology - Recent Advances, New Perspectives and Applications 4.1.3.1.1 Natural convection greenhouse dryer Incident sun energy is passed through the canopy and used to heat the crops in a natural convection greenhouse dryer. The temperature of the crop rises as a result of solar radiation absorption. The thermosyphic effect is used to operate the natural con- vection greenhouse drier. Humid air is vented through the dryer’s chimney or evacuated through an outlet on the top, while warm air is pumped through the crop by buoyancy forces. Natural convection mode refers to this type of airflow within the drying cham- ber, and a natural convection greenhouse drier is one that works in this manner [25]. 4.1.3 Mixed mode solar dryer The term “mixed mode solar dryer” refers to a solar dryer that uses both direct and indirect heating methods. The inlet air is heated at the solar air collector before enter- ing the drying chamber in a mixed mode solar dryer. Some of the drying chamber’s sides are composed of glass, which adds to the drying chamber’s overall warmth. The product is dried using a combination of hot air and direct sunlight in this procedure. In comparison to direct and indirect solar dryers, mixed mode solar dryers require less drying time. Biomass has been used in hybrid sun dryers as an auxiliary heat source to keep drying going all night. Cashews, for example, have been dried in these dryers [23]. 4.1.3.1 Greenhouse solar dryer Tent dryers are similar to greenhouse sun dryers. They have vent sizes that control airflow. Board glazing is used on all sides of this type of drying system. The greenhouse drying system provides a higher degree of control when used in con- junction with the appropriate settings. The main benefit of a greenhouse solar dryer is that it can provide alternate heating with charcoal or briquette burners during inclement weather and can also be used at night. g Greenhouse solar dryers are a type of solar dryer that was developed to address some of the issues that open solar dryers face. The greenhouse solar dryer might be created out of polycarbonate sheets in parabolic shapes, with direct current blowers to help with airflow in the dryer, which has a floor area made out of concrete [24]. Solar radiation intensity was observed between 390 to 820 W/m2. Greenhouse drying is one of the world’s oldest methods of crop preservation. It entails the phenomena of heat and mass transmission. The product’s thermal energy is used in two stages. The temperature of the product rises in the first step due to sensible heat, and the moisture in the product vaporizes in the second step due to the provision of latent heat of vaporization [25]. The greenhouse dryer provides a regulated environment in terms of relative humidity and temperature, which is better for crop drying and hence reduces drying time. The essential processes in the construction of a greenhouse system include vaporization. The greenhouse drier provides a regulated environment in terms of relative humidity and temperature, making crop drying more efficient [25]. a. Natural convection greenhouse dryer b. Forced convection greenhouse dryer 4.1.3.1.1 Natural convection greenhouse dryer 5. Storage of grain legumes Cleaning of grains to remove extraneous materials and contaminants is very fundamental in achieving good and safe storage. It established that cleaning before storage of grains influences the quality of the grain [26]. Cleaning involves the removal of unwanted extraneous material (straws, sand, stone, etc.) from the grain. The storage of grain legumes is a very cardinal stage in the postharvest handling of legumes. Its importance is based on the fact that if the optimal conditions for their safe storage are not maintained a high level of postharvest losses could be incurred. Different microorganisms and pests have the ability to destroy grain legumes after their harvest, during storage, or transportation to various locations of interest. Depending on the prevailing intrinsic and external factors, postharvest losses of grain legumes are estimated to be about 9% for USA and 40–50% for many develop- ing countries [27]. g The rapid decline in color, oil quality and ability to germinate, and many other changes in the quality characteristics of grain legumes can be caused by increase in temperature and moisture. High moisture content and elevated temperature of grains can lead to the development of molds in the category of Aspergillus species, Fusarium species, and Penicillium species, and the production of some mycotoxins such as aflatoxins, ocharatoxin A, and patulin produced by molds. High moisture content and temperature above optimal levels also aid the infestation of different varieties of insects (granari weevil, grain borer, grain moth, grain beetle, etc.) which feed directly on the grains with a resultant effect of the decline in grain quality and quantity. Infestation of grains by fungi results in reduced nutritional quality, reduction in the quality of proteins that synthesize gluten, and the ability of grains to germinate. Other effects include free fatty acid elevation, lowered starch content, increase in total soluble solids, the decline of non-reducing disaccharides and oligosaccharides. The grains can also be charred due to hot spot development and the formation of mycotoxins may occur as a result of fungal contamination creating very big public health issues [7, 17]. Globally produced grains of about 25% are contaminated by toxins from molds – mycotoxins [28]. The aflatoxins with the greatest intoxicating effect, genotoxic and carcinogenic characteristics of greatest concern are B1, B2, G1, G2, and M1 aflatoxins (Table 1) [31]. During storage, grain legume pests are capable of destroying up to 33–50% of global produces [27]. 4.1.3.1.2 Forced convection greenhouse dryer The forced convection greenhouse dryer was born out of a desire for increased air circulation and drying rates. To adjust temperature and moisture evaporation 12 Postharvest Preservation Technology of Cereals and Legumes DOI: http://dx.doi.org/10.5772/intechopen.102739 Postharvest Preservation Technology of Cereals and Legumes DOI: http://dx.doi.org/10.5772/intechopen.102739 according to the weather conditions, an optimal airflow should be given in the greenhouse drier during the drying process. An exhaust fan on the upper half of the west wall is used to evacuate humid air. Forced convection greenhouse dryers employ a fan or blower to control airflow [25]. The mixed mode solar dryer outperforms other types of solar dryers in terms of drying efficiency, drying time, and thermal efficiency. It has been discovered that a mixed mode solar dryer with a Phase-Change-Material is the best for drying grains with higher efficiency and shorter drying times, as well as being smaller, having fewer moving parts, and requiring less maintenance [19]. In a mixed mode solar dryers with 1.5 m/s air velocity, beans with up to 60% moisture content can be reduced down to 6% within six hours of drying [19]. The time required for drying depends on factors like solar radiation, ambient condition, and relative humidity while the solar collector efficiencies can be as high as 61.82% [21]. 5. Storage of grain legumes This gives an insight on the seriousness of pest infestation and attack on grains if proper control measures are not put in place. The quality degradation which results in loss of the quantity of leguminous grains globally during storage can get up to 60% in some instances [27]. These losses are primarily 13 Postharvest Technology - Recent Advances, New Perspectives and Applications Postharvest Technology - Recent Advances, New Perspectives and Applications Chemical fumigants (phosphine tablets and methyl bromide) Compound/Mechanism Effect References Chemical Fumigant Phosphine Tablets and methyl bromide Toxic to living organisms and humans [17] Sensor-based vacuum hermetic fumigation and storage Hermetic contaminant, very low oxygen concentration No harmful effect to humans [29] Irradiation Ionizing irradiation (0.1–50 kGy) Effective in fungal destruction Grain disinfection [8] Radio frequency 1–300 MHz up to 11 M wavelength, penetrate dielectric material and produce heat volumetrically Destroy insects and disinfect dry grains [30] Infrared 0.5–100 μm Vibrations in molecules of water with heat generation [30] Table 1. Postharvest preservation technologies. Chemical fumigants (phosphine tablets and methyl bromide) Compound/Mechanism Effect References Chemical Fumigant Phosphine Tablets and methyl bromide Toxic to living organisms and humans [17] Sensor-based vacuum hermetic fumigation and storage Hermetic contaminant, very low oxygen concentration No harmful effect to humans [29] Irradiation Ionizing irradiation (0.1–50 kGy) Effective in fungal destruction Grain disinfection [8] Radio frequency 1–300 MHz up to 11 M wavelength, penetrate dielectric material and produce heat volumetrically Destroy insects and disinfect dry grains [30] Infrared 0.5–100 μm Vibrations in molecules of water with heat generation [30] Table 1. Postharvest preservation technologies. Table 1. Postharvest preservation technologies. as a result of insect infestation, rodents attack, micro-organisms like mold as well as the breakdown in the normal physiology of grains. It’s a well-known fact that pathogenic micro-organisms, insects, rodents, and unwanted contaminants are capable of posing health hazards in grains when consumed. In storing grains from leguminous crops, the usage of suitable packaging and packaging materials is very crucial in achieving good results in postharvest management of leguminous grains. Packaging also serves a very key role during distribution and marketing (to main- tain quality) [27]. q y In village areas of developing and even developed nations, grains including pulses are still kept in traditional storage facilities which are fabricated with natural materials or woven threads. 5. Storage of grain legumes Typical examples of some of the traditional storage structures used include underground pits, thatched roof storage, plastic containers, and basket silos. Though these local structures have a low construction and mainte- nance cost, they are not very durable, easily invaded by insects and pests resulting in grain legume quality deterioration. Developing nations are currently adopting warehouse storage structures for storing their grains in very large quantities [17]. g g g y g q The materials used for the construction of storage facilities and structures have a direct influence on the moisture content and temperatures in the storage structures [17]. Wooden sticks, concrete blocks, cement, bamboo, and metals (aluminum or steel) are some of the very common materials used for the fabrication of storage structures for grains. 5.1 Silo Silos are currently very common storage facilities for storing grain in many countries and constitute about 79% of all on-farm grain storage facilities in Australia. Silos are very ideal storage alternative for grain legumes (pulses) espe- cially the cone-based variant which makes for very easy grain unloading/discharge with very low seed damage possibilities [15]. For long-term storage of above three 14 Postharvest Preservation Technology of Cereals and Legumes DOI: http://dx.doi.org/10.5772/intechopen.102739 Postharvest Preservation Technology of Cereals and Legumes DOI: http://dx.doi.org/10.5772/intechopen.102739 months duration, there is a need for the incorporation of aeration cooling systems and the use of gas-tight sealable storage which are recommended for efficient and effective fumigation regimes in managing and achieving best quality control. Metal silos are fabricated by incorporating augers and ventilators for grain aeration in order to reduce the formation of hot spots. Metal silos with ventilators and augers are considered advanced grain storage systems as they have the ability to extend the shelf-life of grain legumes through controlled respiration and the development of unfavorable conditions for all sorts of grain legume pests [7, 17]. It is advisable to always fill and empty silos from apertures provided at the center of the silos. This is especially important with grains as most grains have a high bulk density and loading or unloading outside the central opening at the center will put an uneven load on the structure which may cause the silo to collapse [14]. Metal silos of different sorts, fabricated with galvanized iron or recycled oil drums have been developed as an economic, effective, and efficient containers- storage option. These silos are suitable for a long duration of storage of cereals and grain legumes in a water-resistant and hermetically controlled environment. Grains stored in metal silos provide protection from rodents, insects, and water, and are thus very good storage systems for pulses [32]. However, there is a need to protect or shield silos from direct sun rays and other heating sources capable of increasing the temperature of the grains contained therein to avoid condensation. As an alterna- tive, silos can be situated in well-ventilated areas with shade to avoid elevating the temperature of the silos [32]. It is worthy of note that metal silos are very efficient and effective for grain storage but they are also expensive [33]. 5.1 Silo g g y p If there is direct exposure of silos to sunlight or the external air is lower than that in the silos which contain the grains, there may be a formation of currents of convectional flow. As a result of the convectional air currents generated, the moist air is being blown pass through the grains. As the moist air travels and meets cooler surfaces like the silo walls, condensation of the moisture will take place and the grain within that area will get dampened. This dampening occurrence is a cardinal problem associated with grains stored in silos made of steel and particularly utilized for storage in hot areas with daily clear sky [28]. High day temperatures and cool night temperatures are a result of a clear sky. The problem of elevated temperatures can be mitigated in small silos by providing a shield in form of a roof or a hat, to prevent direct contact of sun rays with the surface of the silos. Solutions for larger silos may involve grain silo ventilation or transferring of the grains from the silo with a high temperature to another one that has a cool condition. Grain movement during the transfer of grains to another cool silo has the tendency to provide grains with more homogenous moisture content. In a case where the moisture content is too high, then there will be a need to dry the grains again [28]. 5.2 Hermetic bags/cocoon It’s still possible for foreign pests like Callosobruchus maculatus and Callosobruchus chinensis to be located in grain legumes storage systems during storage if appropri- ate pest management regimes are not strictly adhered to. Grain legumes storage in hermetic bags/Cocoons has to a large extent aided farmers in many countries in storing and extending the shelf life of their produces as they await periods with bet- ter produce value and pricing. This has resulted in better financial gains for farmers that make use of Hermetic bags/cocoons storage in extending the shelf life of their produces with the target of a better sales period [33]. The technique of using her- metically sealed polyethylene silo bags is an effective alternative for the protection of stored grain legumes in commercial storage systems and is presently gaining more prominence for both on-farm sites and off-farm sites [34]. 15 Postharvest Technology - Recent Advances, New Perspectives and Applications 6. Conclusion Legumes are very important food crops that supply good amounts of plant source protein to our meals. Postharvest losses are incurred if grain legumes are not properly handled, prepared, and stored. Some of the notable postharvest handling practices adopted to preserve and extend the shelf life of legumes include drying, pest control, and storage. Pest control in harvested grains can be achieved through emerging technologies like irradiation, radio frequency ionization, infra-red, and microwave technology. Pest management can also be done through the age-long chemical means of fumiga- tion as well as controlled atmosphere technology as an alternative. The drying of grain legumes through the traditional means openly spreading in the sun yields poor drying results. Drying of grain legumes is better done through artificial means with hot air dryers or solar dryers of different sorts. Solar dryers have evolved greatly as a result of the need to reduce the level of greenhouse gases emitted by non-solar dryers, high fuel prices to run non-solar dryers, and the need for a renewable type of energy, unlike the non-solar dryers. yp gy y Storage of grain legumes for bulk commercial purposes is done in silos while hermetic bag storages are utilized for small-scale storage in other to achieve a fairly optimal storage condition for grain legumes. Author details Theophilus M. Ikegwu1, Clement C. Ezegbe1, Chioke A. Okolo1 and Chigozie E. Ofoedu2 1 Faculty of Agriculture, Department of Food Science and Technology, Nnamdi Azikiwe University, Awka, Anambra State, Nigeria 2 School of Engineering and Engineering Technology, Department of Food Science and Technology, Federal University of Technology Owerri, Imo State, Nigeria Address all correspondence to: tm.ikegwu@unizik.edu.ng © 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Author details 1 Faculty of Agriculture, Department of Food Science and Technology, Nnamdi Azikiwe University, Awka, Anambra State, Nigeria © 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 16 Postharvest Preservation Technology of Cereals and Legumes DOI: http://dx.doi.org/10.5772/intechopen.102739 Postharvest Preservation Technology of Cereals and Legumes DOI: http://dx.doi.org/10.5772/intechopen.102739 References Food Science and Nutrition. 2020; 60(15):2622-2642. DOI: 10.1080/ 10408398.2019.1651690 [1] FAO. World Food Situation. Netherlands: Food and Agriculture Organization of the United Nations; 2021. Available from: https://www.fao. org/worldfoodsituation/csdb/en/ [2] Beverly RL. Safety of food and beverages: Cereals and derive products. Encyclopedia of Food Safety. 2014; 3:309-314 [3] Stoskopf NC. Cereal grain crops. Reston: Reston Publishing Company, Inc.; 1985 [4] Yousaf Z, Saleh N, Ramazan A, Aftab A. 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https://openalex.org/W4387100249	https://www.mdpi.com/2309-608X/9/10/971/pdf?version=1695794443	English	null	Effects of MAT1-2 Spore Ratios on Fruiting Body Formation and Degeneration in the Heterothallic Fungus Cordyceps militaris	Journal of Fungi	2,023	cc-by	7,800	Citation: Vu, T.X.; Thai, H.-D.; Dinh, B.-H.T.; Nguyen, H.T.; Tran, H.T.P.; Bui, K.-L.T.; Tran, T.B.; Pham, H.T.; Mai, L.T.D.; Le, D.H.; et al. Effects of MAT1-2 Spore Ratios on Fruiting Body Formation and Degeneration in the Heterothallic Fungus Cordyceps militaris. J. Fungi 2023, 9, 971. https://doi.org/10.3390/jof9100971 Keywords: Cordyceps militaris; degeneration; fruiting body formation; heterokaryotic strains; heterothallic fungus; mating-type loci; sexual reproduction; spore ratios; successive culturing generations Academic Editor: Paloma Sánchez-Torres Received: 25 August 2023 Revised: 25 September 2023 Accepted: 25 September 2023 Published: 27 September 2023 Fungi Journal of Fungi Journal of Fungi Journal of Effects of MAT1-2 Spore Ratios on Fruiting Body Formation and Degeneration in the Heterothallic Fungus Cordyceps militaris Tao Xuan Vu 1,2, Hanh-Dung Thai 1, Bich-Hang Thi Dinh 1, Huong Thi Nguyen 1, Huyen Thi Phuong Tran 1, Khanh-Linh Thi Bui 1, Tram Bao Tran 2, Hien Thanh Pham 3, Linh Thi Dam Mai 3, Diep Hong Le 3, Huy Quang Nguyen 1,3 and Van-Tuan Tran 1,3,* 1 National Key Laboratory of Enzyme and Protein Technology, University of Science, Vietnam National University, Hanoi (VNU), 334 Nguyen Trai, Thanh Xuan, Hanoi 100000, Vietnam; taovx.tsa@gmail.com (T.X.V.); hanhdungthai@gmail.com (H.-D.T.) g ( ); g g ( ) 2 Center for Experimental Biology, National Center for Technological Progress, Ministry of Science an Technology, C6 Thanh Xuan Bac, Thanh Xuan, Hanoi 100000, Vietnam gy 3 Faculty of Biology, University of Science, Vietnam National University, Hanoi (VNU), 334 Nguyen Tra Thanh Xuan, Hanoi 100000, Vietnam * Correspondence: tuantran@vnu.edu.vn Abstract: The medicinal mushroom Cordyceps militaris is widely exploited in traditional medicine and nutraceuticals in Asian countries. However, fruiting body production in C. militaris is facing degeneration through cultivation batches, and the molecular mechanism of this phenomenon remains unclear. This study showed that fruiting body formation in three different C. militaris strains, namely G12, B12, and HQ1, severely declined after three successive culturing generations using the spore isolation method. PCR analyses revealed that these strains exist as heterokaryons and possess both the mating-type loci, MAT1-1 and MAT1-2. Further, monokaryotic isolates carrying MAT1-1 or MAT1- 2 were successfully separated from the fruiting bodies of all three heterokaryotic strains. A spore combination of the MAT1-1 monokaryotic isolate and the MAT1-2 monokaryotic isolate promoted fruiting body formation, while the single monokaryotic isolates could not do that themselves. Notably, we found that changes in ratios of the MAT1-2 spores strongly inﬂuenced fruiting body formation in these strains. When the ratios of the MAT1-2 spores increased to more than 15 times compared to the MAT1-1 spores, the fruiting body formation decreased sharply. In contrast, when MAT1-1 spores were increased proportionally, fruiting body formation was only slightly reduced. Our study also proposes a new solution to mitigate the degeneration in the heterokaryotic C. militaris strains caused by successive culturing generations. 1. Introduction Cordyceps species are entomopathogenic ascomycete fungi. The Cordyceps genus comprises over 400 species that mainly parasitize in arthropods. Many species of this fungal genus are able to produce abundant bioactive substances with beneﬁcial effects on human health [1–4]. Among Cordyceps species, Cordyceps militaris is cultivated artiﬁcially at a large scale for fruiting bodies. Fruiting bodies of C. militaris contain valuable bioactive ingredients such as cordycepin, adenosine, pentostatin, polysaccharides, and digestive enzymes. Therefore, sexual fruiting bodies of this fungus have been widely consumed as edible and medicinal ingredients in Asian countries, including Vietnam [5–8]. 2.2. Media for Fungal Cultivation The potato dextrose broth (PDB) medium comprised the infusion broth with 200 g peeled potato, 20 g glucose, and distilled water to 1000 mL. To this medium, 2% agar was added, and it was named potato dextrose agar (PDA). Brown rice (BR) medium was used for the fruiting body formation of C. militaris strains. This medium, including 28 g brown rice and 60 mL of a nutrient solution, was added to a 600 mL round transparent plastic box for fungal inoculation. The nutrient solution comprised the infusion broth from 200 g peeled potato, 15 g glucose, 15 g sucrose, 1 g MgSO4, 1 g KH2PO4, 1 g peptone, 100 g ﬁnely ground silkworm pupae biomass, and distilled water to 1000 mL. All media were autoclaved at 121 ◦C for 20 min before use. 2.1. Fungal Strains Five C. militaris strains, namely G12, H8, M5, B12, and HQ1, were provided by the National Key Laboratory of Enzyme and Protein Technology, University of Science, Vietnam National University, Hanoi. These strains were preserved in 20% glycerol at 4 ◦C. 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/). g g C. militaris is a heterothallic fungus whose sexual reproduction to form fruiting bodies requires both opposite mating-type partners, MAT1-1 and MAT1-2. A heterokaryotic strain https://www.mdpi.com/journal/jof J. Fungi 2023, 9, 971. https://doi.org/10.3390/jof9100971 J. Fungi 2023, 9, 971 2 of 11 (a dikaryon/diploid) harbors both MAT1-1 and MAT1-2 nuclei, and single-ascospore isola- tion can help to obtain haploidic MAT1-1 and MAT1-2 isolates (monokaryons/haploids) from the parental heterokaryotic strain [9,10]. The MAT1-1 and MAT1-2 loci contain tran- scription factor genes required for mating and sexual development. Structural analysis of these mating-type loci in C. militaris revealed that the MAT1-1 locus contains the MAT1-1-1 and MAT1-1-2 genes, while the MAT1-2 locus carries only the MAT1-2-1 gene. The accurate functioning of this mating system results in the formation of complete fruiting bodies as perithecial stromata [9,11]. Deletion of MAT1-1 or MAT1-2 resulted in impairment of the fruiting body formation in C. militaris [9]. Strikingly, some single MAT1-1 mating-type strains of C. militaris, Cordyceps takaomontana, and Cordyceps cardinalis can also form fruit- ing bodies, but these stromata are sterile, without perithecia or ascospores [11–13]. The molecular mechanism for this phenomenon is still not known yet. p y Successive subculturing of mycelia or spores for several generations in ﬁlamentous fungi and mushrooms causes degeneration [12–18]. Strain degeneration in C. militaris is characterized by the reduction in fruiting body formation, mycelial growth rate, pig- mentation, and production of bioactive metabolites [19,20]. Degeneration of fruiting body formation can occur after two to ten consecutive subculturing generations via mycelial plug passages, depending on the tested strains [21–23]. However, the mechanism of degen- eration in C. militaris is complex and remains unclear. It may involve environmental factors, genetic variations, and changes in gene expression [21,23,24]. In this study, we showed that the heterokaryotic C. militaris strains, including G12, B12, and HQ1, degenerated after three consecutive culturing generations by using the spore isolation method and effects of the MAT1-2 mating-type spores on the degeneration of fruiting body formation in these strains. 2.3. Isolation of Ascospores from Fruiting Bodies A single fruiting body of each heterokaryotic C. militaris strain was mounted on the inside lid of a Petri dish with sterile sticky tape as previously reported [25]. The Petri dish used for the fruiting body mounting contained the PDA medium supplemented with 100 mg/L chloramphenicol. The plate was incubated at 25 ◦C for 4 days under continuous illumination by ﬂuorescent lamps with a 250–300 lux light intensity. Ascospores (sexual spores) from the fruiting body dropped on the surface of the PDA medium and formed colonies. Single colonies were separately transferred to new PDA plates and grown at 25 ◦C in the dark for 4 days. The plates were then exposed to continuous lighting for 2 days to promote conidia (asexual spores). Conidia were collected and washed with sterile distilled water after ﬁltration through Miracloth (Calbiochem, Darmstadt, Germany) and by centrifugation at 6000 rpm (SIGMA 3K30, Sartorius AG, Göttingen, Germany) for 10 min J. Fungi 2023, 9, 971 3 of 11 at 4 ◦C. The concentration of the spore suspension was adjusted to 106 spores/mL and stored at 4 ◦C for further experiments. 2.4. Total DNA Extraction Fruiting body samples obtained from the artiﬁcial cultivation of ﬁve C. militaris strains (G12, H8, M5, B12, and HQ1) were chopped and used directly as the starting material for DNA extraction. Total DNA was also extracted from fungal mycelium prepared: one milliliter of the spore suspension (106 spores/mL) was added to a 100 mL Erlenmeyer ﬂask containing 50 mL of the PDB medium. The ﬂask was incubated in an orbital shaker at 200 rpm and 25 ◦C for 3 days. The culture was ﬁltered through Miracloth to collect fungal mycelial biomass. y Total DNA extraction was adapted from our previous work with slight adjustments [26]. Brieﬂy, 200 mg of fungal biomass (fungal mycelium or fruiting bodies) was distributed to a 2 mL tube. The sample was well pounded with a clean metal rod or pulverized in liquid nitrogen. A volume of 600 µL of the extraction buffer (2.5% SDS, 200 mM Tris-HCl pH 8, 250 mM NaCl, 25 mM EDTA, 0.2% β-mercaptoethanol) was added to the tube. The tube was strongly vortexed and incubated at 60 ◦C for 30 min. Next, 300 µL of 3M sodium acetate (pH 5.2) was added, and the sample was mixed by inverting several times. The tube was centrifuged at 12,000 rpm (Mikro 185, Hettich, Tuttlingen, Germany) for 20 min at 4 ◦C. The supernatant phase was transferred to a new 1.5 mL tube. One equal volume of cold isopropanol was added to the tube to precipitate DNA. The tube was gently inverted several times and centrifuged at 12,000 rpm for 10 min at 4 ◦C. The obtained pellet was washed with 700 µL of 70% ethanol and centrifuged at 12,000 rpm for 5 min. The pellet was dried in the SpeedVac system (Thermo Scientiﬁc, Waltham, MA, USA) and dissolved in 30 µL of TE buffer (Tris-EDTA, pH 8). Then, 3 µL of RNase A (10 mg/mL) was added to the tube, which was incubated at 60 ◦C for 30 min to eliminate RNA. The DNA sample was quantiﬁed using NanoDrop (Thermo Scientiﬁc, Waltham, MA, USA) and stored at −20 ◦C for further experiments. 2.5. Identiﬁcation of the Mating-Type Genes by PCR Total DNA samples extracted from fungal materials of the C. militaris strains were used for PCR to detect the mating-type genes MAT1-1-1 (for the MAT1-1 locus) and MAT1-2-1 (for the MAT1-2 locus). PCR ampliﬁcations involved GoTaq® Green Mas- ter Mix (Promega, Fitchburg, WI, USA) and two speciﬁc primer pairs, MAT1-1-1-F (5′- ATGGAACACAGATCGAGCGACAC-3′)/MAT1-1-1-R (5′-ATATACCTTCGCGATCATT GCCCAG-3′) and MAT1-2-1-F (5′-TGTTTTGTCGCGATGGTTCTGG-3′)/MAT1-2-1-R (5′- CCTCTGGAGGTTCTGCATTCCA-3′) [25]. The steps of the PCR procedure included 94 ◦C (6 min); 35 cycles of 94 ◦C (30 s), 58 ◦C (30 s), 72 ◦C (40 s); and 72 ◦C (10 min). PCR products were analyzed on agarose gels with electrophoresis. 2.9. Statistical Analysis Data were expressed as means ± standard deviations and analyzed statistically using one-way analysis of variance (ANOVA). Analyses were performed with GraphPad Prism 8 (GraphPad Software, San Diego, CA, USA) using Tukey’s test. A statistical difference was considered when p < 0.05. 2.8. Maintaining Fruiting Body Formation in the Heterokaryotic Strains The MAT1-1 and MAT1-2 monokaryotic isolates derived from the heterokaryotic C. militaris strains (G12, B12, and HQ1) were cultured separately on the PDA medium for ﬁve successive generations using the spore isolation method. For each generation, the MAT1-1 isolate was mixed with the MAT1-2 isolate at the 1:1 ratio and grown in the PDB medium supplemented with 0.1% peptone (HiMedia Laboratories, Maharashtra, India). The mixed culture was spread on the BR medium to promote fruiting body formation as described above. The effectiveness of fruiting body formation was evaluated through height, weight, and number of fruiting bodies per culture box. 2.6. Assays for the Fruiting Body Formation through Successive Culturing Generations 2.6. Assays for the Fruiting Body Formation through Successive Culturing Generations Five C. militaris strains were consecutively grown on the PDA medium for ﬁve gen- erations. First, an original strain was cultivated on a PDA plate to collect new spores (1st generation). Next, the harvested spores were grown to harvest new spores (2nd generation). This cycle was repeated to have the next generations (3rd, 4th, 5th). All ﬁve generations of each C. militaris strain were used to evaluate the fruiting body formation ability. One milliliter of a spore suspension (106 spores/mL) from each generation was inoculated into a ﬂask containing the PDB medium supplemented with 0.1% peptone (HiMedia Laboratories, Maharashtra, India). The ﬂask was kept in a shaking incubator at 200 rpm and 25 ◦C in the dark for 3 days. A volume of 3 mL of the culture was spread evenly onto the surface of the brown rice (BR) medium in a 600 mL round transparent plastic box (Viet Nhat Plastic, Hanoi, Vietnam). After the inoculation, the box was incubated at 25 ◦C in the dark for 7 days to promote the colonization of fungal mycelium to the entire surface of the substrate. The box was transferred to culture conditions of a lighting cycle of 12 h under the light/12 h J. Fungi 2023, 9, 971 4 of 11 in the dark, a light intensity of 500–700 lux, a temperature of 22 ◦C, and a humidity of 85–90%. The box was maintained under these conditions for 45 days to promote fruiting body formation. 2.7. Assays for Effects of Spore Ratios on Fruiting Body Formation 2.7. Assays for Effects of Spore Ratios on Fruiting Body Formation Monokaryotic isolates carrying MAT1-1 or MAT1-2 derived from the heterokaryotic C. militaris strains (G12, B12, and HQ1) were mixed in pairwise combinations to examine fruiting body formation. Spores from each MAT1-1 isolate and each MAT1-2 isolate were combined with different ratios: 1:1, 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 5:1, 10:1, 15:1, 20:1, 25:1, and 30:1. Each mixed spore ratio was added to a conical ﬂask containing PDB medium and grown at 200 rpm and 25 ◦C for 3 days. Obtained cultures were inoculated to the BR medium to promote fruiting body formation as described above. Fruit bodies were harvested, and the parameters of height, weight, and number of fruit bodies from each culture box were documented for comparative analyses. 3. Results and Discussion 3.1. Heterokaryotic C. militaris Strains Carrying Both Mating-Type Loci Degenerate through Consecutive Culturing Generations 3.1. Heterokaryotic C. militaris Strains Carrying Both Mating-Type Loci Degenerate through Consecutive Culturing Generations We ﬁrst examined the mating-type loci (MAT1-1 and MAT1-2) in all ﬁve tested C. mil- itaris strains (G12, H8, M5, B12, and HQ1) with PCR using the primers speciﬁc for the MAT1-1-1 and MAT1-2-1 genes. Results showed that strains H8 and M5 carried only the MAT1-1 mating-type locus, while G12, B12, and HQ1 were conﬁrmed as heterokaryotic strains carrying both the mating-type loci, MAT1-1 and MAT1-2, in their genomes. Artiﬁcial cultivation of these C. militaris strains on the BR medium revealed that all ﬁve strains could form normal fruiting bodies (Figure 1A). These strains were further evaluated for their ability to form fruiting bodies through ﬁve successive generations. Results indicated that all these strains could form normal fruiting bodies in the two ﬁrst generations. However, only two monokaryotic strains (H8 and M5) carrying MAT1-1 could maintain the ability to produce fruiting bodies from the third to the ﬁfth generation. In contrast, the fruiting body formation in three heterokaryotic strains (G12, B12, and HQ1) was strongly decreased (Figure 1B). J. Fungi 2023, 9, 971 5 of 11 he third ruiting Figure 1. Fruiting body formation in different C. militaris strains. (A) The mating-type loci (MAT1-1 MAT1-2) in ﬁve tested C. militaris strains (G12, H8, M5, B12, and HQ1) were examined with PCR using the primer pairs speciﬁc for the MAT1-1-1 and MAT1-2-1 genes. PCR products were analyzed on 0.7% agarose gels. All ﬁve C. militaris strains were artiﬁcially cultivated to evaluate the fruiting body formation ability. (B) The C. militaris strains were cultured on the PDA medium for ﬁve successive generations (ﬁrst to ﬁfth) using the spore isolation method. All ﬁve generations were inoculated to the BR medium with the culture conditions of 22 ◦C, a humidity of 85–90%, and a lighting cycle o 12 h light/12 h dark for 45 days to promote fruiting body formation. Figure 1. Fruiting body formation in different C. militaris strains. (A) The mating-type loci (MAT1-1, MAT1-2) in ﬁve tested C. militaris strains (G12, H8, M5, B12, and HQ1) were examined with PCR using the primer pairs speciﬁc for the MAT1-1-1 and MAT1-2-1 genes. PCR products were analyzed on 0.7% agarose gels. All ﬁve C. militaris strains were artiﬁcially cultivated to evaluate the fruiting body formation ability. (B) The C. militaris strains were cultured on the PDA medium for ﬁve successive generations (ﬁrst to ﬁfth) using the spore isolation method. 3.1. Heterokaryotic C. militaris Strains Carrying Both Mating-Type Loci Degenerate through Consecutive Culturing Generations All ﬁve generations were inoculated to the BR medium with the culture conditions of 22 ◦C, a humidity of 85–90%, and a lighting cycle of 12 h light/12 h dark for 45 days to promote fruiting body formation. Although sexual reproduction in heterothallic Cordyceps species usually requires both MAT1-1 and MAT1-2 for fruiting body production [9,11–13], Zheng et al. (2011) also indicated that the sequenced C. militaris strain (Cm01) carrying only MAT1-1 without an opposite mating-type partner in its genome is still able to form fruiting bodies (stromata). However, these fruiting bodies are incomplete due to the lack of perithecia (the ﬂask- shaped fruiting structure) and ascospores [11]. This phenomenon was also discovered in some haploidic MAT1-1 strains of C. takaomontana and C. cardinalis [12,13]. In our study, J. Fungi 2023, 9, 971 6 of 11 monokaryotic strains H8 and M5 harboring the MAT1-1 mating-type locus were more stable for fruiting body formation than heterokaryotic strains G12, B12, and HQ1 through successive culturing generations (Figure 1). These strains appear to resemble the sequenced Cm01 strain. The monokaryotic strain Cm01 is culturally stable and commercialized in China [11]. In agreement with a previous study [23], our results also showed that the degeneration in all three heterokaryotic strains (G12, B12, and HQ1) of C. militaris began in the third generation and was more severe in the fourth generation. In the ﬁfth generation, fruiting body formation in these strains was completely impaired (Figure 1B). 3.2. Both the Mating-Type Loci, MAT1-1 and MAT1-2, Are Required for Fruiting Body Formation in the Heterokaryotic C. militaris Strains 3.2. Both the Mating-Type Loci, MAT1-1 and MAT1-2, Are Required for Fruiting Body Formation in the Heterokaryotic C. militaris Strains Using the fruiting body mounting method [25], we successfully isolated ascospores from the fruiting bodies of all three heterokaryotic C. militaris strains (G12, B12, and HQ1). Fungal colonies developed from ascospores were examined for the mating-type loci MAT1- 1 and MAT1-2. PCR results showed that some colonies carried only a single mating-type locus as MAT1-1 or MAT1-2. However, we also found that many colonies still existed as heterokaryons carrying both MAT1-1 and MAT1-2. Perhaps it is difﬁcult to separate single ascospores from each other by using the fruiting body mounting method. We selected six colonies as monokaryotic (haploid) isolates derived from the het- erokaryotic strains (G12, B12, and HQ1) for fruiting body formation assays. 3.1. Heterokaryotic C. militaris Strains Carrying Both Mating-Type Loci Degenerate through Consecutive Culturing Generations These monokary- otic isolates included G4 (MAT1-1) and G2 (MAT1-2) from strain G12, B5 (MAT1-1) and B6 (MAT1-2) from strain B12, and H3 (MAT1-1) and H2 (MAT1-2) from strain HQ1 (Figure 2A). Previously, Zheng et al. (2011) reported that the inoculation of MAT1-1 or MAT1-2 monokaryotic isolates obtained from the hybrid C. militaris Cm06 strain on caterpillar pupae resulted in the formation of sterile fruiting bodies lacking perithecia and ascospores [11]. However, no fruiting body could be formed in our study when the monokaryotic isolates derived from heterokaryotic strains G12, B12, and HQ1 were cultivated separately on the BR medium. Instead, a combination of a MAT1-1 isolate and a MAT1-2 isolate was required to induce the fruiting body formation on the BR medium (Figure 2B). Our results resemble the ones reported for the heterokaryotic C. militaris Cm09 strain [9]. In MAT1-1 and MAT1-2 mating-type loci structures, the most conserved mating-type genes are MAT1-1-1 and MAT1-2-1, respectively. These gene sequences were also em- ployed for phylogenetic analyses to identify species of Clavicipitaceae, including Cordyceps species [27]. Inactivation of MAT1-1 or MAT1-2 by deleting the respective genes resulted in the loss of fruiting body formation in C. militaris [9]. These results revealed the essential roles of both the mating-type loci in developing complete fruiting bodies in C. militaris. However, the effects of MAT1-1 and MAT1-2 proportions on fruiting body formation in C. militaris still need to be clariﬁed. 3.3. Excessive Increase in MAT1-2 Spore Ratios Caused the Degeneration in the Heterokaryotic Strains Sung et al. (2006) inspected the effect of different ratios of two mono-ascospore strains in pairwise combinations on fruiting body formation in C. militaris. The results indicated that fruiting bodies were similarly produced when two monokaryotic strains were combined at the ratios of 1:1, 1:2, 1:3, 1:4, 2:1, 3:1, and 4:1 [21]. Later, Zheng et al. (2011) also reported that the inoculation of the mixed spores of the MAT1-1 and the MAT1-2 monokaryotic isolates at the ratios of 1:1, 1:9, and 9:1 formed similarly sexual fruiting bodies (stromata) [11]. Based on these data, it seems that the combination of the opposite mating- type monokaryotic strains at different ratios is not necessary for stromata production. J. Fungi 2023, 9, 971 J. Fungi 2023, 9, x FO 7 of 11 7 of 12 Figure 2. Examining opposite mating-type ascospores from the heterokaryotic strains for fruiting body formation. (A) Isolation of ascospores from three heterokaryotic strains, G12, B12, and HQ1. Fungal colonies derived from ascospores were conﬁrmed for the mating-type loci (MAT1-1 and MAT1-2) by PCR. (B) Monokaryotic isolates from a representative heterokaryotic strain (G12) were cultured as separated or combined to evaluate fruiting body formation. Fruiting body formation assays were conducted on the BR medium for 45 days at 22 °C, with a humidity of 85–90% and a lighting cycle of 12 h light/12 h dark. Figure 2. Examining opposite mating-type ascospores from the heterokaryotic strains for fruiting body formation. (A) Isolation of ascospores from three heterokaryotic strains, G12, B12, and HQ1 Fungal colonies derived from ascospores were conﬁrmed for the mating-type loci (MAT1-1 and MAT1-2) by PCR. (B) Monokaryotic isolates from a representative heterokaryotic strain (G12) were cultured as separated or combined to evaluate fruiting body formation. Fruiting body formation assays were conducted on the BR medium for 45 days at 22 ◦C, with a humidity of 85–90% and a lighting cycle of 12 h light/12 h dark. In agreement with the previous works all three heterokaryotic strains in our study Figure 2. Examining opposite mating-type ascospores from the heterokaryotic strains for fruiting body formation. (A) Isolation of ascospores from three heterokaryotic strains, G12, B12, and HQ1. Fungal colonies derived from ascospores were conﬁrmed for the mating-type loci (MAT1-1 and MAT1-2) by PCR. (B) Monokaryotic isolates from a representative heterokaryotic strain (G12) were cultured as separated or combined to evaluate fruiting body formation. 3.3. Excessive Increase in MAT1-2 Spore Ratios Caused the Degeneration in the Heterokaryotic Strains Fruiting body formation assays were conducted on the BR medium for 45 days at 22 °C, with a humidity of 85–90% and a lighting cycle of 12 h light/12 h dark. Figure 2. Examining opposite mating-type ascospores from the heterokaryotic strains for fruiting body formation. (A) Isolation of ascospores from three heterokaryotic strains, G12, B12, and HQ1. Fungal colonies derived from ascospores were conﬁrmed for the mating-type loci (MAT1-1 and MAT1-2) by PCR. (B) Monokaryotic isolates from a representative heterokaryotic strain (G12) were cultured as separated or combined to evaluate fruiting body formation. Fruiting body formation assays were conducted on the BR medium for 45 days at 22 ◦C, with a humidity of 85–90% and a lighting cycle of 12 h light/12 h dark. In agreement with the previous works, all three heterokaryotic strains in our study could form similar fruiting bodies when two opposite mating-type monokaryotic isolates were combined (MAT1-1 × MAT1-2) at the spore ratios of 1:1, 1:5, 1:10, 5:1, 10:1, 15:1, 20:1, 25:1, and 30:1. However, it was astonishing that the formation of fruiting bodies strongly declined in the combinations at the ratios of 1:15, 1:20, 1:25, and 1:30. These results revealed J. Fungi 2023, 9, 971 8 of 11 that an excessive increase in the MAT1-2 spores (over 15 times) compared to the MAT1-1 spores led to the degeneration in the heterokaryotic strains of C. militaris. In contrast, an excessive increase in the MAT1-1 spores (even 30 times) of the combination only caused a slight reduction in fruiting body formation (Figure 3, Supplementary Materials Figure S1). EW 9 of 12 that an excessive increase in the MAT1-2 spores (over 15 times) compared to the MAT1-1 spores led to the degeneration in the heterokaryotic strains of C. militaris. In contrast, an excessive increase in the MAT1-1 spores (even 30 times) of the combination only caused a slight reduction in fruiting body formation (Figure 3, Supplementary Materials Figure S1). EW 9 of 12 Figure 3. Eﬀects of diﬀerent mating-type spore ratios in combinations on fruiting body formation in three heterokaryotic C. militaris strains. (A) Combinations of monokaryotic isolates G4 (MAT1-1) and G2 (MAT1-2) derived from strain G12. (B) Combinations of monokaryotic isolates B5 (MAT1-1) and B6 (MAT1-2) derived from strain B12. (C) Combinations of monokaryotic isolates H3 (MAT1-1) and H2 (MAT1-2) derived from HQ1. 3.3. Excessive Increase in MAT1-2 Spore Ratios Caused the Degeneration in the Heterokaryotic Strains (B) Combinations of monokaryotic isolates B5 (MAT1-1) and B6 (MAT1-2) derived from strain B12. (C) Combinations of monokaryotic isolates H3 (MAT1-1) and H2 (MAT1-2) derived from HQ1. Spore mixtures were inoculated on the BR medium for 45 days t 22 °C ith h idit f 85 90% d li hti l f 12 h li ht/12 h d k t t th Figure 3. Effects of different mating-type spore ratios in combinations on fruiting body formation in three heterokaryotic C. militaris strains. (A) Combinations of monokaryotic isolates G4 (MAT1-1) and G2 (MAT1-2) derived from strain G12. (B) Combinations of monokaryotic isolates B5 (MAT1-1) and B6 (MAT1-2) derived from strain B12. (C) Combinations of monokaryotic isolates H3 (MAT1-1) and H2 (MAT1 2) derived from HQ1 Spore mixtures were inoculated on the BR medium for 45 days Figure 3. Eﬀects of diﬀerent mating-type spore ratios in combinations on fruiting body formation in three heterokaryotic C. militaris strains. (A) Combinations of monokaryotic isolates G4 (MAT1-1) and G2 (MAT1-2) derived from strain G12. (B) Combinations of monokaryotic isolates B5 (MAT1-1) and B6 (MAT1-2) derived from strain B12. (C) Combinations of monokaryotic isolates H3 (MAT1-1) and H2 (MAT1-2) derived from HQ1. Spore mixtures were inoculated on the BR medium for 45 days at 22 °C, with a humidity of 85–90% and a lighting cycle of 12 h light/12 h dark to promote the formation of fruiting bodies. Figure 3. Effects of different mating-type spore ratios in combinations on fruiting body formation in three heterokaryotic C. militaris strains. (A) Combinations of monokaryotic isolates G4 (MAT1-1) and G2 (MAT1-2) derived from strain G12. (B) Combinations of monokaryotic isolates B5 (MAT1-1) and B6 (MAT1-2) derived from strain B12. (C) Combinations of monokaryotic isolates H3 (MAT1-1) and H2 (MAT1-2) derived from HQ1. Spore mixtures were inoculated on the BR medium for 45 days at 22 ◦C, with a humidity of 85–90% and a lighting cycle of 12 h light/12 h dark to promote the formation of fruiting bodies. g y p y f g g g in the Heterokaryotic Strains Caused by Successive Culturing Degeneration in heterokaryotic C. militaris strains occurs after a few generations of successive subculturing, and the formation of fruiting bodies can even be lost entirely [21– 23]. We cultured the MAT1-1 and MAT1-2 monokaryotic isolates for ﬁve consecutive gen- erations using the spore isolation method. 3.3. Excessive Increase in MAT1-2 Spore Ratios Caused the Degeneration in the Heterokaryotic Strains Spore mixtures were inoculated on the BR medium for 45 days at 22 °C, with a humidity of 85–90% and a lighting cycle of 12 h light/12 h dark to promote the formation of fruiting bodies. 3 4 P i th M k ti I l t S t l b f M ti Miti t th D ti Figure 3. Effects of different mating-type spore ratios in combinations on fruiting body formation in three heterokaryotic C. militaris strains. (A) Combinations of monokaryotic isolates G4 (MAT1-1) and G2 (MAT1-2) derived from strain G12. (B) Combinations of monokaryotic isolates B5 (MAT1-1) and B6 (MAT1-2) derived from strain B12. (C) Combinations of monokaryotic isolates H3 (MAT1-1) and H2 (MAT1-2) derived from HQ1. Spore mixtures were inoculated on the BR medium for 45 days at 22 ◦C, with a humidity of 85–90% and a lighting cycle of 12 h light/12 h dark to promote the formation of fruiting bodies. Figure 3. Eﬀects of diﬀerent mating-type spore ratios in combinations on fruiting body formation in three heterokaryotic C. militaris strains. (A) Combinations of monokaryotic isolates G4 (MAT1-1) and G2 (MAT1-2) derived from strain G12. (B) Combinations of monokaryotic isolates B5 (MAT1-1) and B6 (MAT1-2) derived from strain B12. (C) Combinations of monokaryotic isolates H3 (MAT1-1) and H2 (MAT1-2) derived from HQ1. Spore mixtures were inoculated on the BR medium for 45 days at 22 °C, with a humidity of 85–90% and a lighting cycle of 12 h light/12 h dark to promote the formation of fruiting bodies. 3.4. Preserving the Monokaryotic Isolates Separately before Mating Mitigates the Degeneration Figure 3. Effects of different mating-type spore ratios in combinations on fruiting body formation in three heterokaryotic C. militaris strains. (A) Combinations of monokaryotic isolates G4 (MAT1-1) and G2 (MAT1-2) derived from strain G12. (B) Combinations of monokaryotic isolates B5 (MAT1-1) and B6 (MAT1-2) derived from strain B12. (C) Combinations of monokaryotic isolates H3 (MAT1-1) and H2 (MAT1-2) derived from HQ1. Spore mixtures were inoculated on the BR medium for 45 days at 22 ◦C, with a humidity of 85–90% and a lighting cycle of 12 h light/12 h dark to promote the formation of fruiting bodies. Figure 3. Eﬀects of diﬀerent mating-type spore ratios in combinations on fruiting body formation in three heterokaryotic C. militaris strains. (A) Combinations of monokaryotic isolates G4 (MAT1-1) and G2 (MAT1-2) derived from strain G12. 3.3. Excessive Increase in MAT1-2 Spore Ratios Caused the Degeneration in the Heterokaryotic Strains The monokaryotic isolates from each genera- tion were then combined at a 1:1 spore ratio. Our results revealed that the formation of fruiting bodies in the hybrid strains was similarly maintained through all ﬁve generations (Figure 4). Previous studies reported that monokaryotic isolates derived from single asco- Zheng et al. (2011) showed that the MAT1-1/MAT1-2 heterokaryotic Cm06 strain produced normal stromata with perithecia and ascospores. PCR analysis of 30 single ascospore isolates derived from Cm06 revealed that 28 single ascospore isolates carried the MAT1-1 mating type, and only 2 isolates contained the MAT1-2 mating type [11]. This unequal distribution of the mating types corresponds to a 14:1 spore ratio. Both the MAT1-1 and MAT1-2 mating types are essential for fruiting body formation in the heterokaryotic strains [9,11]. However, a highly excessive number of MAT1-2 spores can cause degeneration (Figure 3). A recent comparative analysis with quantitative real-time g y p y f g g g in the Heterokaryotic Strains Caused by Successive Culturing Degeneration in heterokaryotic C. militaris strains occurs after a few generations of successive subculturing, and the formation of fruiting bodies can even be lost entirely [21– 23]. We cultured the MAT1-1 and MAT1-2 monokaryotic isolates for ﬁve consecutive gen- erations using the spore isolation method. The monokaryotic isolates from each genera- tion were then combined at a 1:1 spore ratio. Our results revealed that the formation of fruiting bodies in the hybrid strains was similarly maintained through all ﬁve generations (Figure 4). Previous studies reported that monokaryotic isolates derived from single asco- Zheng et al. (2011) showed that the MAT1-1/MAT1-2 heterokaryotic Cm06 strain produced normal stromata with perithecia and ascospores. PCR analysis of 30 single ascospore isolates derived from Cm06 revealed that 28 single ascospore isolates carried the MAT1-1 mating type, and only 2 isolates contained the MAT1-2 mating type [11]. This unequal distribution of the mating types corresponds to a 14:1 spore ratio. Both the MAT1-1 and MAT1-2 mating types are essential for fruiting body formation in the heterokaryotic strains [9,11]. However, a highly excessive number of MAT1-2 spores can cause degeneration (Figure 3). A recent comparative analysis with quantitative real-time J. Fungi 2023, 9, 971 9 of 11 9 of 11 PCR also indicated that the expression of the MAT1-2-1 gene in a degenerated C. militaris strain was signiﬁcantly higher than in its parental strain [28]. 3.3. Excessive Increase in MAT1-2 Spore Ratios Caused the Degeneration in the Heterokaryotic Strains Based on this information and our results, we suggest that the abundance of the MAT1-1 monokaryotic spores in the mating combinations can support the stability of the fruiting body formation in the heterokaryotic C. militaris strains. 3.4. Preserving the Monokaryotic Isolates Separately before Mating Mitigates the Degeneration in the Heterokaryotic Strains Caused by Successive Culturing 3.4. Preserving the Monokaryotic Isolates Separately before Mating Mitigates the Degeneration in the Heterokaryotic Strains Caused by Successive Culturing Degeneration in heterokaryotic C. militaris strains occurs after a few generations of suc- cessive subculturing, and the formation of fruiting bodies can even be lost entirely [21–23]. We cultured the MAT1-1 and MAT1-2 monokaryotic isolates for ﬁve consecutive genera- tions using the spore isolation method. The monokaryotic isolates from each generation were then combined at a 1:1 spore ratio. Our results revealed that the formation of fruiting bodies in the hybrid strains was similarly maintained through all ﬁve generations (Figure 4). Previous studies reported that monokaryotic isolates derived from single ascospores of heterokaryotic C. militaris strains were more stable for fruiting body production [10,21]. Our work further showed that the MAT1-1 and MAT1-2 monokaryotic isolates should be individually preserved as compatible partners for mating to mitigate the degeneration of fruiting body formation during cultivation. EW 10 of 12 Figure 4. Maintaining the fruiting body formation ability of the heterokaryotic strains. (A) Mono- karyotic isolates of the MAT1-1 mating type and the MAT1-2 mating type were preserved separately. These isolates were successively cultured for ﬁve generations. The opposite mating-type spores from each generation were mixed and cultivated on the BR medium for fruiting body formation. (B) Fruiting body formation in three heterokaryotic strains (G12, B12, and HQ1) after ﬁve successive culturing generations of the monokaryotic isolates and respective spore combinations by mating at 1 1 ti (C) Q tiﬁ ti f f iti b di f th bi ti Th t l Figure 4. Maintaining the fruiting body formation ability of the heterokaryotic strains. (A) Monokary- otic isolates of the MAT1-1 mating type and the MAT1-2 mating type were preserved separately. These isolates were successively cultured for five generations. The opposite mating-type spores from each generation were mixed and cultivated on the BR medium for fruiting body formation. Figure 4. Maintaining the fruiting body formation ability of the heterokaryotic strains. (A) Mono- karyotic isolates of the MAT1-1 mating type and the MAT1-2 mating type were preserved separately. 3.3. Excessive Increase in MAT1-2 Spore Ratios Caused the Degeneration in the Heterokaryotic Strains These isolates were successively cultured for ﬁve generations. The opposite mating-type spores from each generation were mixed and cultivated on the BR medium for fruiting body formation. (B) Fruiting body formation in three heterokaryotic strains (G12, B12, and HQ1) after ﬁve successive lt i ti f th k ti i l t d ti bi ti b ti t Figure 4. Maintaining the fruiting body formation ability of the heterokaryotic strains. (A) Monokary- otic isolates of the MAT1-1 mating type and the MAT1-2 mating type were preserved separately. These isolates were successively cultured for five generations. The opposite mating-type spores from each generation were mixed and cultivated on the BR medium for fruiting body formation. J. Fungi 2023, 9, 971 10 of 11 10 of 11 (B) Fruiting body formation in three heterokaryotic strains (G12, B12, and HQ1) after ﬁve successive culturing generations of the monokaryotic isolates and respective spore combinations by mating at a 1:1 ratio. (C) Quantiﬁcation of fruiting bodies from the combinations. Three parameters, namely number of fruiting bodies (FBs), height of FBs, and weight of FBs, were documented. Experiments were conducted in triplicate, and data are presented as means ± standard deviations. Error bars represent the standard deviations, and different lowercase letters indicate signiﬁcant differences (p < 0.05). 4. Conclusions The funder 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 This study showed that the sexual (fertile) fruiting body formation in the MAT1- 1/MAT1-2 heterokaryotic strains of C. militaris quickly degenerated after three successive generations via the asexual spore (conidia) isolation. In contrast, the sterile fruiting body formation in the MAT1-1 monokaryotic strains was more stable through consecutive cul- turing generations. We also discovered that an excessive increase in the MAT1-2 spores of over 15 times compared with the MAT1-1 spores led to the degeneration of fruiting body formation in all three examined heterokaryotic C. militaris strains (G12, B12, and HQ1). To mitigate the degeneration in the heterokaryotic strains through culturing generations, their monokaryotic isolates should be separately kept before being combined for fruiting body production. Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/jof9100971/s1, Figure S1: Quantiﬁcation of fruiting body formation for mating-type spore combinations at different ratios. Author Contributions: Conceptualization, V.-T.T.; methodology, V.-T.T., T.X.V., T.B.T. and D.H.L.; val- idation, H.-D.T., B.-H.T.D., H.T.N. and H.T.P.T.; formal analysis, T.X.V., V.-T.T. and H.Q.N.; investiga- tion, T.X.V., H.-D.T., B.-H.T.D., H.T.N., H.T.P.T., K.-L.T.B., T.B.T., H.T.P., L.T.D.M. and D.H.L.; resources, V.-T.T.; data curation, T.X.V.; writing—original draft preparation, V.-T.T. and T.X.V.; writing—review and editing, V.-T.T. and H.Q.N.; supervision, V.-T.T.; project administration, V.-T.T.; funding acquisi- tion, V.-T.T. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by Vietnam National University, Hanoi under grant number QG.20.19. Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: All data for this study are included in the manuscript. Acknowledgments: The authors thank Le Thi Ngoc Bich for excellent technical assistance and Thu V. Vuong (University of Toronto, Canada) for valuable comments. Acknowledgments: The authors thank Le Thi Ngoc Bich for excellent technical assistance and Thu V. Acknowledgments: The authors thank Le Thi Ngoc Bich for excellent technical assistance and Thu V. Vuong (University of Toronto, Canada) for valuable comments. Vuong (University of Toronto, Canada) for valuable comments. Conﬂicts of Interest: The authors declare no conﬂict of interest. 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https://openalex.org/W4230814467	https://europepmc.org/articles/pmc6755621?pdf=render	English	null	Correction to: 12th European Headache Federation Congress jointly with 32nd National Congress of the Italian Society for the Study of Headaches	The Journal of headache and pain	2,018	cc-by	10,191	© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. CORRECTION Open Access The Journal of Headache and Pain The Journal of Headache and Pain The Journal of Headache and Pain The Journal of Headache and Pain (2018) 19:19 https://doi.org/10.1186/s10194-018-0947-y The economic and humanistic burden of episodic and chronic migraine in Europe gy g y RESULTS: Analysis included 68 publications. Data from the World Health Organization indicated that in Europe, migraine burden weighed higher than that of epilepsy, multiple sclerosis, and Parkinson’s disease. Up to 57% of individuals with migraine report severe dis- ability, and many find treatments ineffective. Nausea and/or vomiting occurred in up to 74% of individuals with migraine. Depression and/or anxiety occur up to three times more often in individuals with migraine than in the general population. People with migraine have re- ported poorer health-related quality of life than those without migraine, which worsens with increasing mi- graine attack frequency. Europeans with migraine per- ceive that it has a negative impact on work (up to 76%), family situations, leisure time, studies, sexual life, social position, love, financial situation, career, and friendships. The prevalence of migraine is highest for men and women during their peak years of economic productivity (ages 25–55 years). In Europe, the estimated total annual cost was up to €111 billion (2008–2009), of which 72%– 98% was indirect costs (two-thirds of indirect costs due to reduced productivity). Annual direct costs for CM were up to four times higher than those for EM. RESULTS: Analysis included 68 publications. Data from the World Health Organization indicated that in Europe, migraine burden weighed higher than that of epilepsy, multiple sclerosis, and Parkinson’s disease. Up to 57% of individuals with migraine report severe dis- ability, and many find treatments ineffective. Nausea and/or vomiting occurred in up to 74% of individuals with migraine. Depression and/or anxiety occur up to three times more often in individuals with migraine than in the general population. People with migraine have re- ported poorer health-related quality of life than those without migraine, which worsens with increasing mi- graine attack frequency. Europeans with migraine per- ceive that it has a negative impact on work (up to 76%), family situations, leisure time, studies, sexual life, social position, love, financial situation, career, and friendships. The prevalence of migraine is highest for men and women during their peak years of economic productivity (ages 25–55 years). In Europe, the estimated total annual cost was up to €111 billion (2008–2009), of which 72%– 98% was indirect costs (two-thirds of indirect costs due to reduced productivity). Annual direct costs for CM were up to four times higher than those for EM. Correction to: 12th European Headache Federation Congress jointly with 32nd National Congress of the Italian Society for the Study of Headaches functioning), and utility outcomes (published 2007–Feb- ruary 1, 2018; geographical limitation: United Kingdom, France, Germany, Spain, Italy, the Netherlands, Poland, Denmark, Finland, Iceland, Norway and Sweden). Searches included: Embase, MEDLINE, and the Cochrane Library databases; specialty medicine associations; and health technology assessment agency websites. The economic and humanistic burden of episodic and chronic migraine in Europe Hicham Benhaddi1, Timothy Fitzgerald2, SophieMcCabe3, Ruth Zeidman3 Hicham Benhaddi1, Timothy Fitzgerald2, SophieMcCabe3, Ruth Zeidman3 1 Teva Pharmaceuticals, Wilrijk, Belgium 2 , j , g 2 Teva Pharmaceuticals, Frazer, Pennsylvania, USA 2 Teva Pharmaceuticals, Frazer, Pennsylvania, USA 2 Teva Pharmaceuticals, Frazer, Pennsylvania, USA 3 3 Covance Market Access, London, UK 3 Covance Market Access, London, UK * Correspondence: Hicham Benhaddi (Hicham. Benhaddi@tevaeu.com). * Correspondence: Hicham Benhaddi (Hicham. Benhaddi@tevaeu.com). The correspondence e-mail address contained an error. The correct e-mail address has now been in- cluded in this correction. BACKGROUND: Migraine is a debilitating neurological disorder characterized by attacks that may last 4–72 hours, with a high burden in Europeans. OBJECTIVE: This systematic literature review exam- ined the clinical, humanistic, and economic burden asso- ciated with chronic and episodic migraine (CM and EM, respectively) in Europe. OBJECTIVE: This systematic literature review exam- ined the clinical, humanistic, and economic burden asso- ciated with chronic and episodic migraine (CM and EM, respectively) in Europe. METHODS: Literature searches and evidence screening were structured according to the PICOS (population, intervention, comparators, outcomes, and study types) framework. Reviews and original observational studies in adults (≥18 years) with EM (<15 headache days per month) or CM (≥15 headache days with ≥8 migraine days per month) were included. Searches focused on resource utilization, treatment costs, productivity, quality of life (in- cluding generic and migraine-specific instruments and CONCLUSIONS: This research demonstrates that mi- graine has a substantial humanistic and economic bur- den on Europeans and affects all aspects of life. Correction After publication of this supplement [1], it was brought to our attention that e-mail errors were apparent in the follow- ing abstracts. This has now been included in this correction. O38 Impact of fremanezumab on response rates, acute medication use, and disability in patients with episodic migraine who have failed at least one prior migraine preventive medication Page 2 of 11 Page 2 of 11 The Journal of Headache and Pain (2018) 19:19 Paul K. Winner1, Rashmi B. Halker Singh2, Joshua M. Cohen3, Ronghua Yang3, Paul P. Yeung3, Verena Ramirez Campos4 Paul K. Winner1, Rashmi B. Halker Singh2, Joshua M. Cohen3, Ronghua Yang3, Paul P. Yeung3, Verena Ramirez Campos4 number of days of any acute headache medication use during the treatment period (quarterly [least-squares mean change ± standard error]: –3.1±0.5 days; monthly: –3.4±0.5 days) compared with placebo (–1.1±0.5 days; both, P<0.0001). Fremanezumab significantly improved disability from baseline, based on the change in MIDAS score during the treatment period (quarterly: –24.5±3.7, P=0.0006; monthly: –26.8±3.7, P<0.0001) compared with placebo (–11.1±3.4). 1 Premiere Research Institute, West Palm Beach, Florida, USA 2 Mayo Clinic, Phoenix, Arizona, USA 3 Teva Pharmaceuticals, Frazer, Pennsylvania, USA 3 Teva Pharmaceuticals, Frazer, Pennsylvania, USA 4 Teva Pharmaceuticals, Buenos Aires, Argentina 4 Teva Pharmaceuticals, Buenos Aires, Argentina * Correspondence: Paul K. Winner (pwinner777 @aol.com). * Correspondence: Paul K. Winner (pwinner777 @aol.com). CONCLUSIONS: Among EM patients who failed at least one prior preventive migraine medication, fremane- zumab treatment was efficacious, reduced acute headache medication use, and improved disability, with effect sizes greater than those seen in the overall trial population. The correspondence e-mail address contained an error. The correct e-mail address has now been in- cluded in this correction. TRIAL REGISTRATION: ClinicalTrials.gov, NCT0 2629861 BACKGROUND: Preventive medication is recom- mended for episodic migraine (EM) patients with ≥4 headache days per month. Fremanezumab, a fully hu- manized monoclonal antibody (IgG2a) that selectively targets calcitonin gene-related peptide (CGRP), is effica- cious in preventing EM, but its effectiveness in patients who failed previous preventive medications is unknown. Efficacy of fremanezumab in migraine patients who have failed at least one prior migraine preventive medication OBJECTIVE: To assess the effects of fremanezumab on response rates, acute headache medication use, and disability in EM patients who failed at least one prior preventive migraine medication. ETHICS APPROVAL The study was approved by relevant independent eth- ics committees or institutional review boards, according to national or local regulations. p g METHODS: Peter McAllister1, David W. Dodick2, Joshua M. Cohen3, Ronghua Yang3, Paul P. Yeung3, Verena Ramirez Campos4 In this Phase 3, multicenter, randomized, double-blind, placebocontrolled study, patients were randomized 1:1:1 to receive subcutaneous injections of fremanezu- mab quarterly (675 mg at baseline and placebo at Weeks 4 and 8), fremanezumab monthly (225 mg at baseline, Weeks 4 and 8), or placebo (at baseline, Weeks 4 and 8) over a 12-week treatment period. Analyses were performed in patients who failed at least one prior preventive migraine medication (due to lack of efficacy or intolerability) using Cochran– Mantel–Haenszel test or an analysis of covariance model. Endpoints included the proportion of patients with ≥50% reduction in the monthly average number of migraine days, mean change from baseline in the monthly average number of days of acute headache medication use, and mean change from baseline in the Migraine Disability Assessment (MIDAS) score during the 12-week treatment period. In this Phase 3, multicenter, randomized, double-blind, placebocontrolled study, patients were randomized 1:1:1 to receive subcutaneous injections of fremanezu- mab quarterly (675 mg at baseline and placebo at Weeks 4 and 8), fremanezumab monthly (225 mg at baseline, Weeks 4 and 8), or placebo (at baseline, Weeks 4 and 8) over a 12-week treatment period. Analyses were performed in patients who failed at least one prior preventive migraine medication (due to lack of efficacy or intolerability) using Cochran– Mantel–Haenszel test or an analysis of covariance model. Endpoints included the proportion of patients with ≥50% reduction in the monthly average number of migraine days, mean change from baseline in the monthly average number of days of acute headache medication use, and mean change from baseline in the Migraine Disability Assessment (MIDAS) score during the 12-week treatment period. 1 New England Institute for Neurology and Headache, Stamford, Connecticut, USA 1 New England Institute for Neurology and Headache, Stamford, Connecticut, USA 2 Department of Neurology, Mayo Clinic, Phoenix, Arizona, USA 2 Department of Neurology, Mayo Clinic, Phoenix, Arizona, USA 3 Teva Pharmaceuticals, Frazer, Pennsylvania, USA 4 Teva Pharmaceuticals, Buenos Aires, Argentina Correspondence: Peter McAllister (peter@neinh.com) The correspondence e-mail address contained an error. The correct e-mail address has now been in- cluded in this correction. BACKGROUND: Fremanezumab, a fully humanized monoclonal antibody (IgG2a) that selectively targets cal- citonin gene-related peptide (CGRP), has been shown to be effective in the prevention of chronic migraine (CM) and episodic migraine (EM). p g METHODS: Analyses were performed in the full analysis set (all randomized patients who re- ceived ≥1 dose of study drug and had ≥10 days of post-baseline efficacy assessments on the primary end- point). The data were analyzed using both the analysis of covariance approach, with baseline number of days with nausea or vomiting, or photophobia and phonophobia, and years since onset of migraines as covariates, and the Wilcoxon rank-sum test. CONCLUSIONS: Fremanezumab was efficacious in migraine patients who failed at least one prior migraine preventive medication, a potentially difficultto-treat population. Effect sizes in this subgroup were greater than those in the overall trial population. RESULTS: Fremanezumab treatment yielded greater reductions from baseline in the monthly number of days with nausea or vomiting during the 12-week treatment period (quarterly [least-squares mean ± standard error]: –1.9±0.19 days, P=0.0314; monthly: –2.1±0.19 days, P=0.0008) compared with placebo (–1.4±0.19 days). Re- ductions in nausea or vomiting were seen as early as Week 4 (quarterly: –1.7±0.21 days, P=0.0046; monthly: –1.9±0.21 days, P=0.0002) compared with placebo (–1.0 ±0.21 days). Fremanezumab treatment also yielded greater reductions from baseline in the number of days with photophobia and phonophobia during the 12-week treatment period (quarterly: –2.2±0.21 days, P=0.0038; monthly: –2.4±0.21 days, P=0.0001) compared with pla- cebo (–1.5±0.21 days). Significant reductions in days with photophobia and phonophobia were seen as early as Week 4 (quarterly: –2.0±0.23 days, P=0.0003; monthly: –2.2±0.23 days, P<0.0001) compared with pla- cebo (–1.0±0.23 days). RESULTS: Fremanezumab treatment yielded greater reductions from baseline in the monthly number of days with nausea or vomiting during the 12-week treatment period (quarterly [least-squares mean ± standard error]: –1.9±0.19 days, P=0.0314; monthly: –2.1±0.19 days, P=0.0008) compared with placebo (–1.4±0.19 days). Re- ductions in nausea or vomiting were seen as early as Week 4 (quarterly: –1.7±0.21 days, P=0.0046; monthly: –1.9±0.21 days, P=0.0002) compared with placebo (–1.0 ±0.21 days). Fremanezumab treatment also yielded greater reductions from baseline in the number of days with photophobia and phonophobia during the 12-week treatment period (quarterly: –2.2±0.21 days, P=0.0038; monthly: –2.4±0.21 days, P=0.0001) compared with pla- cebo (–1.5±0.21 days). Significant reductions in days with photophobia and phonophobia were seen as early as Week 4 (quarterly: –2.0±0.23 days, P=0.0003; monthly: –2.2±0.23 days, P<0.0001) compared with pla- cebo (–1.0±0.23 days). TRIAL REGISTRATION: ClinicalTrials.gov NCT02 621931 and NCT02629861 p g METHODS: p p p METHODS: In this multicenter, randomized, double- blind, placebocontrolled, Phase 3 study, patients with EM were randomized 1:1:1 to receive subcutaneous fre- manezumab quarterly (675 mg at baseline, placebo at Weeks 4 and 8), fremanezumab monthly (225 mg at baseline, Weeks 4 and 8), or placebo over a 12-week treatment period. Exploratory endpoints included mean change from baseline in the monthly average number of days with nausea or vomiting, and days with photopho- bia and/or phonophobia during the 12-week period after the first dose of study drug. Analyses were performed in the full analysis set (all randomized patients who re- ceived ≥1 dose of study drug and had ≥10 days of post-baseline efficacy assessments on the primary end- point). The data were analyzed using both the analysis of covariance approach, with baseline number of days with nausea or vomiting, or photophobia and phonophobia, and years since onset of migraines as covariates, and the Wilcoxon rank-sum test. g y RESULTS: In CM patients, fremanezumab yielded greater reductions in the monthly average number of headache days of at least moderate severity (quarterly [n=130] [least-squares mean change ± standard error]: –4.0±0.47, P<0.0001; monthly [n=141]: –4.6±0.46, P<0.0001) compared with pla- cebo (n=136; –1.9±0.49). There were similar reductions in the monthly average number of migraine days (quarterly: – 4.2±0.55, P=0.005; monthly: –4.8±0.53, P<0.0001) compared with placebo (–2.4±0.56). In EM patients, fremanezumab yielded greater reductions in the monthly average number of headache days of at least moderate severity (quarterly [n=58]: –3.0±0.51, P<0.0001; monthly [n=65]: –3.2±0.49, P<0.0001) compared with placebo (n=63; –0.7±0.47). There were similar reductions in the monthly average number of migraine days (quarterly: –3.3±0.61, P=0.0015; monthly: –3.8 ±0.59, P<0.0001) compared with placebo (–1.3±0.57). P-values stated are compared with placebo. METHODS: In this multicenter, randomized, double- blind, placebocontrolled, Phase 3 study, patients with EM were randomized 1:1:1 to receive subcutaneous fre- manezumab quarterly (675 mg at baseline, placebo at Weeks 4 and 8), fremanezumab monthly (225 mg at baseline, Weeks 4 and 8), or placebo over a 12-week treatment period. Exploratory endpoints included mean change from baseline in the monthly average number of days with nausea or vomiting, and days with photopho- bia and/or phonophobia during the 12-week period after the first dose of study drug. p g METHODS: BACKGROUND: Fremanezumab, a fully humanized monoclonal antibody (IgG2a) that selectively targets cal- citonin gene-related peptide (CGRP), has been shown to be effective in the prevention of chronic migraine (CM) and episodic migraine (EM). RESULTS: The subgroup who failed at least one prior migraine preventive therapy included 58 fremanezumab quarterly, 65 fremanezumab monthly, and 63 placebo patients. A greater proportion of patients who received fremanezumab had a ≥50% reduction in the monthly average number of migraine days during the treatment period (quarterly: 38%, P=0.0100; monthly: 43%, P=0.0010) compared with placebo (17%). Fremanezumab significantly reduced from baseline the monthly average OBJECTIVE: To assess the efficacy of fremanezumab in migraine patients who failed at least one prior pre- ventive migraine medication. METHODS: Fremanezumab was studied in two Phase 3, multicenter, randomized, double-blind, placebo-controlled, parallel-group trials. Patients with CM or EM (confirmed during a 28-day pre-treatment baseline period) received sub- cutaneous injections of fremanezumab quarterly (675 mg at Page 3 of 11 Page 3 of 11 The Journal of Headache and Pain (2018) 19:19 baseline and placebo at Weeks 4 and 8), monthly (CM: 675 mg at baseline and 225 mg at Weeks 4 and 8; EM: 225 mg at baseline and Weeks 4 and 8), or placebo (at baseline and Weeks 4 and 8) over a 12-week treatment period, with a final evaluation 4 weeks after the last dose of the study drug. Mean changes from baseline in the monthly average number of headache days of at least moderate severity or the monthly average number of migraine days during the 12-week treatment period were assessed in patients who failed at least one prior migraine preventive medication due to lack of efficacy or intolerability. Ana- lyses were performed in the intent-to-treat population using an analysis of covariance (ANCOVA) model. The correspondence e-mail address contained an error. The correct e-mail address has now been in- cluded in this correction. OBJECTIVES: Non–headache symptoms (nausea, vomiting, photophobia and phonophobia) are included in the International Classification of Headache Disor- ders, third edition (beta version) (ICHD-3 beta) criteria for migraine. Fremanezumab, a fully humanized mono- clonal antibody (IgG2a) that selectively targets calcitonin gene-related peptide (CGRP), reduced the number of migraine days in EM patients. We assessed the effect of fremanezumab on nausea or vomiting, and photophobia and phonophobia in EM patients. ETHICS APPROVAL The study was approved by all relevant independent ethics committees or institutional review boards, accord- ing to national or local regulations. p gy 2 Teva Pharmaceuticals, Frazer, Pennsylvania, USA * Correspondence: Jan L. Brandes (jbrandes1@msn. com) 1 Nashville Neuroscience Group, Vanderbilt University, Department of Neurology, Nashville, Tennessee, USA The impact of fremanezumab on symptoms associ- ated with migraine in patients with chronic migraine The impact of fremanezumab on symptoms associ- ated with migraine in patients with chronic migraine Peter McAllister1, Paul P. Yeung2, Ernesto Aycardi2, Ronghua Yang2, Yuju Ma2, Joshua M. Cohen2 Peter McAllister1, Paul P. Yeung2, Ernesto Aycardi2, Ronghua Yang2, Yuju Ma2, Joshua M. Cohen2 g g j 1 New England Institute for Neurology and Headache, Stamford, Connecticut, USA 1 New England Institute for Neurology and Headache, Stamford, Connecticut, USA 2 Teva Pharmaceuticals, Frazer, Pennsylvania, USA 2 Teva Pharmaceuticals, Frazer, Pennsylvania, USA CONCLUSIONS: Fremanezumab treatment rapidly improved non–head pain symptoms associated with mi- graine, including nausea or vomiting, and photophobia and phonophobia, in patients with CM. * Correspondence: Peter McAllister (peter@neinh.com) * Correspondence: Peter McAllister (peter@neinh.com) The correspondence e-mail address contained an error. The correct e-mail address has now been in- cluded in this correction. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02 638103 TRIAL REGISTRATION: ClinicalTrials.gov, NCT02 638103 ETHICS APPROVAL OBJECTIVES: The International Classification of Headache Disorders, third edition (beta version) (ICHD- 3 beta) criteria for migraine include nausea, vomiting, photophobia, and phonophobia symptoms. Fremanezu- mab, a fully humanized monoclonal antibody (IgG2a) that selectively targets calcitonin gene-related peptide (CGRP), reduced the frequency and severity of head- aches in patients with chronic migraine (CM). We assessed the effect of fremanezumab versus placebo on nausea or vomiting, and photophobia and phonophobia, in patients with CM. The study was approved by all relevant independent ethics committees or institutional review boards, accord- ing to national or local regulations. ETHICS APPROVAL The study was approved by all relevant independent ethics committees or institutional review boards, accord- ing to national or local regulations. The impact of fremanezumab on symptoms associated with migraine in patients with episodic migraine The impact of fremanezumab on symptoms associated with migraine in patients with episodic migraine Jan L. Brandes1, Paul P. Yeung2, Ernesto Aycardi2, Ronghua Yang2, Yuju Ma2, Joshua M. Cohen2 1 Nashville Neuroscience Group, Vanderbilt University, Department of Neurology, Nashville, Tennessee, USA 2 Teva Pharmaceuticals, Frazer, Pennsylvania, USA CONCLUSIONS: In patients with EM, fremanezumab treatment rapidly improved non–head pain symptoms Correspondence: Jan L. Brandes (jbrandes1@msn. com) CONCLUSIONS: In patients with EM, fremanezumab treatment rapidly improved non–head pain symptoms * Correspondence: Jan L. Brandes (jbrandes1@msn. com) Page 4 of 11 Page 4 of 11 Page 4 of 11 The Journal of Headache and Pain (2018) 19:19 associated with migraine, including nausea or vomiting, and photophobia and phonophobia. RESULTS: Fremanezumab treatment yielded greater reductions from baseline in the monthly number of days with nausea or vomiting during the 12-week treatment period (quarterly [least-squares mean ± standard error]: –3.3±0.29 days, P=0.0009; monthly: –3.2±0.28 days, P=0.0019) compared with placebo (–2.2±0.29 days). Sig- nificant reductions in nausea or vomiting were seen as early as Week 4 (quarterly: –3.2±0.30 days, P<0.0001; monthly: –2.9±0.29 days, P=0.0014) versus placebo (–1.9 ±0.29 days). Fremanezumab treatment also yielded greater reductions from baseline in the number of days with photophobia and phonophobia during the 12-week treatment period (quarterly: –3.5±0.32 days, P=0.0025; monthly: –3.7±0.32 days, P=0.0001) versus placebo (–2.4 ±0.32 days). Reductions in days with photophobia and phonophobia were seen as early as Week 4 (quarterly: – 3.5±0.33 days, P<0.0001; monthly: –3.5±0.32 days, P<0.0001) versus placebo (–2.1±0.33 days). TRIAL REGISTRATION: ClinicalTrials.gov, NCT026 29861 TRIAL REGISTRATION: ClinicalTrials.gov, NCT026 29861 Long-term impact of fremanezumab on response rates, acute headache medication use, and disability in patients with episodic migraine: interim results of a 1-year study Jan L. Brandes1, Paul P. Yeung2, Joshua M. Cohen2, Sanjay K. Gandhi2, Timothy Fitzgerald2, Ronghua Yang2, Yuju Ma2, Ernesto Aycardi2 Jan L. Brandes1, Paul P. Yeung2, Joshua M. Cohen2, Sanjay K. Gandhi2, Timothy Fitzgerald2, Ronghua Yang2, Yuju Ma2, Ernesto Aycardi2 METHODS: In this multicenter, randomized, double- blind, placebocontrolled, Phase 3 study, patients with CM were randomized 1:1:1 to receive subcutaneous in- jections of fremanezumab quarterly (675 mg at baseline, placebo at Weeks 4 and 8), fremanezumab monthly (675 mg at baseline, 225 mg at Weeks 4 and 8), or placebo (at baseline, Weeks 4 and 8) over a 12-week treatment period. Exploratory endpoints included the mean change from baseline in the monthly average number of days with nausea or vomiting, and days with photophobia and phonophobia during the 12-week period after the first dose of study drug. Analyses were performed in the full analysis set (all randomized patients who received ≥1 dose of study drug and had ≥10 days of post-baseline efficacy as- sessments on the primary endpoint) using analysis of covariance (with baseline number of days with the symptom, and years since onset of migraines as co- variates) and the Wilcoxon rank-sum test. 1 Nashville Neuroscience Group, Nashville, Tennessee 2 Teva Pharmaceuticals, Frazer, Pennsylvania, USA * Correspondence: Jan L. Brandes (jbrandes1@msn.com) * Correspondence: Jan L. Brandes (jbrandes1@msn.com) The correspondence e-mail address contained an error. The correct e-mail address has now been in- cluded in this correction. BACKGROUND: Fremanezumab, a fully humanized monoclonal antibody (IgG2a) that selectively targets calci- tonin gene-related peptide (CGRP), has demonstrated effi- cacy in preventing episodic migraine (EM) in 3-month studies; this analysis evaluates its long-term effects. OBJECTIVE: To investigate the long-term effect of fremanezumab on response, acute headache medication and disability in adults with EM. Page 5 of 11 The Journal of Headache and Pain (2018) 19:19 2 Teva Pharmaceuticals, Ulm, Germany; 3Teva Pharmaceuticals, Frazer, Pennsylvania, USA * Correspondence: Stephen D. Silberstein (Stephen.Silberstein@jefferson.edu) METHODS: This 52-week, multicenter, randomized, double-blind, parallel-group study evaluated the long-term safety, tolerability and efficacy of fremanezu- mab in adults with migraine; disability was assessed using the Migraine Disability Assessment (MIDAS). Most patients rolled over from a pivotal EM study, but some patients enrolled directly into this long-term study. Patients were assigned to one of two subcutaneous dose groups: (1) monthly dosing: 225 mg doses of fremanezu- mab every month, or (2) quarterly dosing: 675 mg doses of fremanezumab every 3 months. Long-term impact of fremanezumab on response rates, acute headache medication use, and disability in patients with episodic migraine: interim results of a 1-year study The change from baseline in the MIDAS disability score in patients with EM was similar in both treatment groups at Month 6; disability scores decreased by 27.1 and 27.3 at Month 6 in the monthly and quarterly treatment groups, respectively. For a subset of patients who completed the entire 12-month treatment period, data available at the cutoff date indicated that the response achieved at Month 6 was maintained throughout the treatment period. p g METHODS: Fremanezumab has been studied in four placebocontrolled studies in patients with migraine, in- cluding two Phase 2 and two Phase 3 studies. Each study was a 16-week, multicenter, randomized, double-blind, placebo-controlled, parallel-group study to compare the efficacy, safety, and tolerability of fremanezumab and placebo in adults with EM or CM. The studies evaluated fremanezumab at the proposed subcutaneous doses of 225 mg monthly (CM patients received a starting dose of 675 mg), 675 mg quarterly, and at two higher doses (675 mg monthly and 900 mg monthly) for 3 months. METHODS: Fremanezumab has been studied in four placebocontrolled studies in patients with migraine, in- cluding two Phase 2 and two Phase 3 studies. Each study was a 16-week, multicenter, randomized, double-blind, placebo-controlled, parallel-group study to compare the efficacy, safety, and tolerability of fremanezumab and placebo in adults with EM or CM. The studies evaluated fremanezumab at the proposed subcutaneous doses of 225 mg monthly (CM patients received a starting dose of 675 mg), 675 mg quarterly, and at two higher doses (675 mg monthly and 900 mg monthly) for 3 months. RESULTS: Most patients who received fremanezumab (N=1702) or placebo (N=861) were female (87%), with mean age of 41.4 years (range = 18 to 70 years), respect- ively. Serious adverse events (AEs) and AEs leading to discontinuation occurred infrequently, with similar inci- dences in patients who received fremanezumab (1% and 2%, respectively) versus patients who received placebo (2% for both subsets). The most common AEs in the placebo-controlled studies were injection-site reactions, including induration and erythema, which tended to be transient, mild and slightly more frequent in patients who received fremanezumab versus those given placebo. Upper respiratory tract infection and nasopharyngitis, both reported with similar incidence in patients who re- ceived either fremanezumab or placebo, were the next most frequently reported AEs. Cardiovascular AEs occurred infrequently and with a similar incidence in both fremanezumab and placebo groups. No signal for hepatoxicity was observed. Long-term impact of fremanezumab on response rates, acute headache medication use, and disability in patients with episodic migraine: interim results of a 1-year study No anaphylaxis or se- vere hypersensitivity occurred, and only three patients (two on placebo, and one on fremanezumab) had AEs of drug hypersensitivity of mild or moderate severity. None of these events was serious, and all resolved with steroid and/or antihistamine treatment. Incidence of antidrug antibody (ADA) formation was low, and CONCLUSION: Efficacy and disability data from this interim analysis indicated that the efficacy observed at Month 1 was maintained during the remainder of the study. TRIAL REGISTRATION: ClinicalTrials.gov, NCT026 38103 TRIAL REGISTRATION: ClinicalTrials.gov, NCT026 38103 Long-term impact of fremanezumab on response rates, acute headache medication use, and disability in patients with episodic migraine: interim results of a 1-year study Percentage of patients achieving ≥50% reduction in monthly average number of migraine days, the mean change from baseline in the monthly number of days of use of any acute headache medications, and the mean change from baseline in MIDAS score were assessed for both doses. The correspondence e-mail address contained an error. The correct e-mail address has now been included in this correction. BACKGROUND: Fremanezumab, a fully humanized monoclonal antibody (IgG2a) that selectively targets cal- citonin gene-related peptide (CGRP), has been shown to be effective in the prevention of episodic migraine (EM) or chronic migraine (CM). OBJECTIVE: To summarize the safety profile of fre- manezumab based on all placebo-controlled studies in patients with migraine. RESULTS: This study enrolled 780 EM patients. The mean change in monthly number of migraine days from baseline to Month 1 was – 4.6 days for the monthly treatment group and –4.9 days for the quarterly group. The proportion of patients achieving ≥50% reduction in monthly average number of migraine days at Month 6 was 61% with monthly dosing, and 65% with quarterly dosing. The mean change in monthly number of days of use of any acute headache medications from baseline to Month 6 in patients with EM was –4.1 days in the monthly group and –4.3 days in the quarterly group. The change from baseline in the MIDAS disability score in patients with EM was similar in both treatment groups at Month 6; disability scores decreased by 27.1 and 27.3 at Month 6 in the monthly and quarterly treatment groups, respectively. For a subset of patients who completed the entire 12-month treatment period, data available at the cutoff date indicated that the response achieved at Month 6 was maintained throughout the treatment period. RESULTS: This study enrolled 780 EM patients. The mean change in monthly number of migraine days from baseline to Month 1 was – 4.6 days for the monthly treatment group and –4.9 days for the quarterly group. The proportion of patients achieving ≥50% reduction in monthly average number of migraine days at Month 6 was 61% with monthly dosing, and 65% with quarterly dosing. The mean change in monthly number of days of use of any acute headache medications from baseline to Month 6 in patients with EM was –4.1 days in the monthly group and –4.3 days in the quarterly group. ETHICS APPROVAL The study was approved by all relevant independent ethics committees or institutional review boards, accord- ing to national or local regulations. 1 Thomas Jefferson University, Philadelphia, Pennsylvania, USA Disclosures: Disclosures: Joshua M. Cohen: Employee of Teva Pharmaceuticals. The correspondence e-mail address contained an error. The correct e-mail address has now been in- cluded in this correction. Kristen Bibeau: Former employee of Teva Pharma ceuticals. Maja Galic: Employee of Teva Pharmaceuticals. Michael J. Seminerio: Employee of Teva Pharmaceuticals. OBJECTIVE: To evaluate the effect of fremanezumab on reversion from chronic migraine (CM) to episodic migraine (EM). OBJECTIVE: To evaluate the effect of fremanezumab on reversion from chronic migraine (CM) to episodic migraine (EM). Verena Ramirez Campos: Employee of Teva Pharma- ceuticals. Rashmi B. Halker Singh: Received honoraria from Current Neurology and Neuroscience Reports, MedLink, and Amgen. BACKGROUND: CM and EM are clinically, function- ally, and anatomically differentiated, with evidence suggest- ing that they may be separate conditions. Furthermore, patients with CM usually have more comorbid conditions and more-frequent medication overuse, which complicates their clinical management. Fremanezumab, a fully human- ized monoclonal antibody (IgG2a) that selectively targets calcitonin gene-related peptide (CGRP), has demonstrated efficacy in migraine prevention. BACKGROUND: CM and EM are clinically, function- ally, and anatomically differentiated, with evidence suggest- ing that they may be separate conditions. Furthermore, patients with CM usually have more comorbid conditions and more-frequent medication overuse, which complicates their clinical management. Fremanezumab, a fully human- ized monoclonal antibody (IgG2a) that selectively targets calcitonin gene-related peptide (CGRP), has demonstrated efficacy in migraine prevention. Jessica Ailani: Received honoraria from Allergan (speaking/consulting), Avanir (speaking), Eli Lilly (speak- ing/advisory board), Teva Pharmaceuticals (advisory board), Promius (speaking), Current Pain and Headache Reports (section editor), Theranica (clinical trials). Reversion of patients with chronic migraine to an episodic migraine classification with fremanezumab treatment Joshua M. Cohen1; Kristen Bibeau1; Maja Galic2; Michael J. Seminerio1; Verena Ramirez Campos3; Rashmi B. Halker Singh4; Jessica Ailani5 CONCLUSIONS: Along with its efficacy as a migraine preventive treatment, fremanezumab demonstrated the potential benefit for reversion from CM to EM. g 1 Teva Pharmaceuticals, Frazer, Pennsylvania, USA 1 Teva Pharmaceuticals, Frazer, Pennsylvania, USA TRIAL REGISTRATION: ClinicalTrials.gov, NCT02 621931, NCT02629861 TRIAL REGISTRATION: ClinicalTrials.gov, NCT02 621931, NCT02629861 2 Teva Pharmaceuticals, Amsterdam, The Netherlands 2 Teva Pharmaceuticals, Amsterdam, The Netherlands ETHICS APPROVAL Overview of fremanezumab pooled safety data from placebocontrolled phase 2 and 3 studies Overview of fremanezumab pooled safety data from placebocontrolled phase 2 and 3 studies Stephen D. Silberstein1, Nicola Faulhaber2, Xiaoping Ning3, Paul P. Yeung3, Jimmy Schiemann3, Ronghua Yang3, Yuju Ma3, Ernesto Aycardi3 1 Thomas Jefferson University, Philadelphia, Pennsylvania, USA 1 Thomas Jefferson University, Philadelphia, Pennsylvania, USA Page 6 of 11 Page 6 of 11 The Journal of Headache and Pain (2018) 19:19 there were no AEs related to ADA or neutralizing antibody development. there were no AEs related to ADA or neutralizing antibody development. placebo over a 12-week treatment period. Post hoc ana- lyses evaluated the proportion of patients who reverted from CM to EM, defined as patients who had ≥15 head- ache days per month at baseline (28-day pre-treatment period) and then had <15 headache days per month in all 3 months of the treatment period. placebo over a 12-week treatment period. Post hoc ana- lyses evaluated the proportion of patients who reverted from CM to EM, defined as patients who had ≥15 head- ache days per month at baseline (28-day pre-treatment period) and then had <15 headache days per month in all 3 months of the treatment period. CONCLUSION: Four placebo-controlled studies dem- onstrate that fremanezumab, at the proposed monthly and quarterly dose regimens, is an efficacious and gener- ally safe and well-tolerated preventive therapy. TRIAL REGISTRATION: ClinicalTrials.gov, NCT026 21931, NCT02629861, NCT02021773, NCT02025556 ETHICS APPROVAL RESULTS: In an analysis of the 1130 CM patients ran- domized in this trial (quarterly, N=376; monthly, N=379; placebo, N=375), significantly more fremanezumab- treated patients reverted from having ≥15 headache days per month at baseline to <15 headache days per month in Months 1, 2, and 3 (quarterly: 121 patients [32%]; monthly: 133 patients [35%]) than those who received placebo (86 patients [23%]; both, P≤0.002). On average, these fremanezumab-treated patients had 18–19 head- ache days per month at baseline and showed reductions to 6–9 headache days during any month in the treat- ment period, representing up to an approximately 70% reduction in headache days. The study was approved by all relevant independent ethics committees or institutional review boards, accord- ing to national or local regulations. ETHICS APPROVAL 4 Mayo Clinic, Phoenix, Arizona, USA 4 Mayo Clinic, Phoenix, Arizona, USA 5 Medstar Georgetown University Hospital, Washington, District of Columbia, USA The study was approved by all relevant independent ethics committees or institutional review boards, accord- ing to national or local regulations. 5 Medstar Georgetown University Hospital, Washington, District of Columbia, USA * Correspondence: Joshua M. Cohen (Joshua.Cohen 05@tevapharm.com) * Correspondence: Joshua M. Cohen (Joshua.Cohen 05@tevapharm.com) Disclosures: Peter Goadsby: Personal fees from Teva Pharmaceuticals during the conduct of the study. He receives personal fees from Akita Biomedical, Alder Biopharmaceuticals, Avanir Pharma, Cipla Ltd, Dr. Reddy's Laboratories, ElectroCore LLC, Novartis, Pfizer Inc, Quest Diagnostics, Scion, Medico- Legal, UptoDate, and Oxford University Press. He receives grants and personal fees from Allergan, Amgen, Eli Lilly and Company, and eNeura Inc. He receives personal fees and other from Trigemina Inc. He reports work, personal fees from Journal Watch, and Massachusetts Medical Society, outside the submitted work. He has a patent on magnetic stimulation for headache licensed to eNeura. OBJECTIVE: To investigate the efficacy of fremanezu- mab in chronic migraine (CM) patients with or without concomitant use of preventive medication. BACKGROUND: Some patients with CM may take more than one preventive medication. Fremanezumab, a fully humanized monoclonal antibody (IgG2a) that se- lectively targets calcitonin gene-related peptide (CGRP), has demonstrated efficacy in migraine prevention. BACKGROUND: Some patients with CM may take more than one preventive medication. Fremanezumab, a fully humanized monoclonal antibody (IgG2a) that se- lectively targets calcitonin gene-related peptide (CGRP), has demonstrated efficacy in migraine prevention. DESIGN/METHODS: In this Phase 3, randomized, double-blind, placebo-controlled, parallel-group study, eli- gible patients with prospectively confirmed CM (≥15 head- ache days and ≥8 migraine days per month) were randomized 1:1:1 to receive subcutaneous injections of fre- manezumab quarterly (675 mg at baseline; placebo at Weeks 4 and 8), fremanezumab monthly (675 mg at baseline; 225 mg at Weeks 4 and 8) or placebo at each time point over a 12-week treatment period. Changes from baseline were assessed in the monthly average num- ber of headache days of at least moderate severity, and in migraine days in patients with or without con- comitant preventive medication. David W. Dodick: Provides consultation to Acorda, Allergan, Amgen, Alder, Dr. Reddy’s, Merck, Promius, eNeura, Eli Lilly & Company, Insys, Autonomic Technolo- gies, Teva, Xenon, Tonix, Trigemina, Boston Scientific, GBS, Colucid, Zosano, Laydenburg Thalmann, Biocentric, Biohaven, Magellan, Charleston Laboratories, Pfizer. Royal- ties: Oxford University Press and Cambridge University Press (Book Royalty). He receives editorial/honoraria from UpToDate. He receives honoraria/publishing or honoraria/ royalties from Chameleon Communications, Medscape, WebMD, Academy for Continued Healthcare Learning, Haymarket Medical Education, Global Scientific Communi- cations, HealthLogix, Academy for Continued Healthcare Learning, MeetingLogiX, Wiley Blackwell, Oxford Univer- sity Press, Cambridge University Press. Stock/options: GBS/ Nocira, Epien, and Mobile Health. He has a consulting use agreement with NAS. Efficacy of fremanezumab in patients with chronic migraine with or without concomitant use of pre- ventive medication DESIGN/METHODS: In this Phase 3, multicenter, randomized, doubleblind, placebo-controlled, parallel- group study, adults with prospectively confirmed CM (≥15 headache days and ≥8 migraine days per month) were randomized 1:1:1 to subcutaneous injections of fre- manezumab quarterly (675 mg at baseline; placebo at Weeks 4 and 8), fremanezumab monthly (675 mg at baseline; 225 mg at Weeks 4 and 8), or matching Peter J. Goadsby1, David W. Dodick2, Stephen D. Silberstein3, Paul P. Yeung4, Tricia Blankenbiller4, Xiaoping Ning4, Ronghua Yang4, Yuju Ma4, Ernesto Aycardi4, Marcelo E. Bigal4 Page 7 of 11 The Journal of Headache and Pain (2018) 19:19 1 NIHR-Wellcome Trust King’s Clinical Research Facility, King’s College, London, UK There were also fewer migraine days with both fremane- zumab regimens. There were also fewer migraine days with both fremane- zumab regimens. y g g 2 Mayo Clinic, Phoenix, Arizona, USA 2 Mayo Clinic, Phoenix, Arizona, USA CONCLUSIONS: Fremanezumab demonstrated effi- cacy in patients with CM, regardless of concomitant pre- ventive medication use. CONCLUSIONS: Fremanezumab demonstrated effi- cacy in patients with CM, regardless of concomitant pre- ventive medication use. 3 Jefferson Headache Center, Thomas Jefferson University, Philadelphia, Pennsylvania, USA 4 Teva Pharmaceuticals, Frazer, Pennsylvania, USA 4 Teva Pharmaceuticals, Frazer, Pennsylvania, USA TRIAL REGISTRATION: ClinicalTrials.gov, NCT026 21931 Correspondence: Peter J. Goadsby (Peter.Goadsby@ ucsf.edu) ETHICS APPROVAL The study was approved by all relevant independent ethics committees or institutional review boards, accord- ing to national or local regulations. The correspondence e-mail address contained an error. The correct e-mail address has now been in- cluded in this correction. Disclosures: He has a board position at King-Devick Inc. p RESULTS: Analyses included 239 patients receiving one concomitant preventive medication (quarterly, N=77; monthly, N=85; placebo, N=77) and 882 patients receiving none (quarterly, N=298; monthly, N=290; pla- cebo, N=294). During the 12-week treatment period, fre- manezumab reduced from baseline the mean number of monthly headache days of at least moderate severity ver- sus placebo in patients receiving concomitant preventive medication (quarterly: –3.8±0.61; monthly: –4.5±0.57; placebo: –2.5±0.61), reaching significance with monthly dosing (P=0.003). Reductions were also significant for fremanezumab quarterly and monthly in those not re- ceiving concomitant preventive medication (quarterly: – 4.6±0.33; monthly: –4.9±0.33; placebo: –2.7±0.33; both, P<0.0001). These reductions were observed as early as 4 weeks after initiation of fremanezumab monthly in pa- tients receiving concomitant preventive medication (P=0.028); similarly early reductions occurred with fre- manezumab monthly and quarterly in patients not re- ceiving concomitant preventive medication (P<0.0001). p RESULTS: Analyses included 239 patients receiving one concomitant preventive medication (quarterly, N=77; monthly, N=85; placebo, N=77) and 882 patients receiving none (quarterly, N=298; monthly, N=290; pla- cebo, N=294). During the 12-week treatment period, fre- manezumab reduced from baseline the mean number of monthly headache days of at least moderate severity ver- sus placebo in patients receiving concomitant preventive medication (quarterly: –3.8±0.61; monthly: –4.5±0.57; placebo: –2.5±0.61), reaching significance with monthly dosing (P=0.003). Reductions were also significant for fremanezumab quarterly and monthly in those not re- ceiving concomitant preventive medication (quarterly: – 4.6±0.33; monthly: –4.9±0.33; placebo: –2.7±0.33; both, P<0.0001). These reductions were observed as early as 4 weeks after initiation of fremanezumab monthly in pa- tients receiving concomitant preventive medication (P=0.028); similarly early reductions occurred with fre- manezumab monthly and quarterly in patients not re- ceiving concomitant preventive medication (P<0.0001). Stephen D. Silberstein: Provides consultation to Alder, Allergan, Amgen, Avanir, Curelater Inc., Depomed, Dr. Reddy’s Laboratories, Ensured Inc., ElectroCore Medical LLC, INSYS Therapeutics, Lilly USA LLC, Supernus Pharmaceuticals Inc., Teva Pharma- ceuticals, Theranica, and Trigemina Inc. Paul P. Yeung: Employee of Teva Pharmaceuticals. Tricia Blankenbiller: Former employee of Teva Pharmaceuticals. Xiaoping Ning: Employee of Teva Pharmaceuticals. Ronghua Yang: Employee of Teva Pharmaceuticals. Y j M E l f T Ph ti l Xiaoping Ning: Employee of Teva Pharmaceuticals. R h Y E l f T Ph i l Yuju Ma: Employee of Teva Pharmaceuticals. TRIAL REGISTRATION: ClinicalTrials.gov, NCT026 21931 TRIAL REGISTRATION: ClinicalTrials.gov, NCT026 21931 2 Teva Pharmaceuticals, Amsterdam, The Netherlands 2 Teva Pharmaceuticals, Amsterdam, The Netherlands 3 Albert Einstein College of Medicine and Montefiore Medical Center, Bronx, New York, USA 3 Albert Einstein College of Medicine and Montefiore Medical Center, Bronx, New York, USA The correspondence e-mail address contained an error. The correct e-mail address has now been in- cluded in this correction. Post hoc analyses evaluated changes in headache and mi- graine frequency and depression in patients with moderate to moderately severe depression (score of 10–19 on the 9-item Patient Health Questionnaire [PHQ-9]) at baseline. Dawn C. Buse: Consultant to Amgen, Allergan, Avanir, Biohaven, Eli Lilly and Promeius. Disclosures: Page 8 of 11 Page 8 of 11 The Journal of Headache and Pain (2018) 19:19 Ernesto Aycardi: Former employee of Teva Pharma ceuticals. at least moderate severity (quarterly: –5.4±0.79; monthly: –5.6±0.75) versus those who received placebo (–2.2±0.84) during the 12-week treatment period (both, P<0.001), with effects observed as early as Week 4 (P<0.0001). Similar treatment differences were observed for change in the mean number of migraine days (P<0.001). Fremanezumab also reduced the mean PHQ-9 score from baseline to Week 12 (quarterly: –10.5±0.68; monthly: –9.5±0.63) ver- sus placebo (–8.7±0.71); the quarterly group reached sig- nificance (P<0.05). Marcelo E. Bigal: Former employee of Teva Pharma ceuticals. The correspondence e-mail address contained an error. The correct e-mail address has now been in- cluded in this correction. Disclosures: Joshua M. Cohen: Employee of Teva Pharmaceuticals. y Paul P. Yeung: Employee of Teva Pharmaceuticals. Ernesto Aycardi: Former employee of Teva Pharmaceuticals. OBJECTIVE: To evaluate the efficacy of fremanezu- mab on migraine symptoms and depression in patients with chronic migraine (CM) and comorbid moderate to moderately severe depression. Marcelo E. Bigal: Former employee of Teva Pharma ceuticals. Ronghua Yang: Employee of Teva Pharmaceuticals. BACKGROUND: Depression is common in CM and contributes to the already substantial burden of disease. Fremanezumab, a fully humanized monoclonal antibody (IgG2a) that selectively targets calcitonin gene-related peptide (CGRP), has demonstrated efficacy in migraine prevention. BACKGROUND: Depression is common in CM and contributes to the already substantial burden of disease. Fremanezumab, a fully humanized monoclonal antibody (IgG2a) that selectively targets calcitonin gene-related peptide (CGRP), has demonstrated efficacy in migraine prevention. Kristen Bibeau: Former employee of Teva Pharma ceuticals. Maja Galic: Employee of Teva Pharmaceuticals. Maja Galic: Employee of Teva Pharmaceuticals. Michael J. Seminerio: Employee of Teva Pharma ceuticals. Richard B. Lipton: Consultant to Teva Pharma ceuticals. DESIGN/METHODS: In this Phase 3, multicenter, randomized, doubleblind, placebo-controlled, parallel- group study, eligible patients aged 18–70, with prospect- ively confirmed CM (≥15 headache days and ≥8 mi- graine days per month) were randomized 1:1:1 to receive subcutaneous injections of fremanezumab quarterly (675 mg at baseline; placebo at Weeks 4 and 8), fremanezumab monthly (675 mg at baseline; 225 mg at Weeks 4 and 8), or matching placebo over a 12-week treatment period. Post hoc analyses evaluated changes in headache and mi- graine frequency and depression in patients with moderate to moderately severe depression (score of 10–19 on the 9-item Patient Health Questionnaire [PHQ-9]) at baseline. RESULTS: Almost 20% (219/1130) of randomized pa- tients had moderate to moderately severe depression at baseline (quarterly, n=74; monthly, n=88; placebo, n=57). As in the overall study population, fremanezumab-treated patients in this subgroup had significant reductions from baseline in the mean number of monthly headache days of DESIGN/METHODS: In this Phase 3, multicenter, randomized, doubleblind, placebo-controlled, parallel- group study, eligible patients aged 18–70, with prospect- ively confirmed CM (≥15 headache days and ≥8 mi- graine days per month) were randomized 1:1:1 to receive subcutaneous injections of fremanezumab quarterly (675 mg at baseline; placebo at Weeks 4 and 8), fremanezumab monthly (675 mg at baseline; 225 mg at Weeks 4 and 8), or matching placebo over a 12-week treatment period. Efficacy of fremanezumab in patients with chronic migraine and comorbid moderate to moderately se- vere depression Joshua M. Cohen1, Paul P. Yeung1, Ernesto Aycardi1, Marcelo E. Bigal1, Ronghua Yang1, Kristen Bibeau1, Maja Galic2, Michael J. Seminerio1, Richard B. Lipton3, Dawn C. Buse3 Joshua M. Cohen1, Paul P. Yeung1, Ernesto Aycardi1, Marcelo E. Bigal1, Ronghua Yang1, Kristen Bibeau1, Maja Galic2, Michael J. Seminerio1, Richard B. Lipton3, Dawn C. Buse3 CONCLUSIONS: Fremanezumab demonstrated effi- cacy in preventive treatment of CM in patients with co- morbid moderate to moderately severe depression, reducing migraine and headache frequency and improv- ing depression. CONCLUSIONS: Fremanezumab demonstrated effi- cacy in preventive treatment of CM in patients with co- morbid moderate to moderately severe depression, reducing migraine and headache frequency and improv- ing depression. 1 Teva Pharmaceuticals, Frazer, Pennsylvania, USA 1 Teva Pharmaceuticals, Frazer, Pennsylvania, USA ETHICS APPROVAL * Correspondence: Joshua M. Cohen (Joshua.Cohen05 @tevapharm.com) * Correspondence: Joshua M. Cohen (Joshua.Cohen05 @tevapharm.com) The study was approved by all relevant independent ethics committees or institutional review boards, accord- ing to national or local regulations. The correspondence e-mail address contained an error. The correct e-mail address has now been in- cluded in this correction. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02 638103 TRIAL REGISTRATION: ClinicalTrials.gov, NCT02 638103 RESULTS: Among patients with medication overuse at baseline (quarterly n=201; monthly n=198; placebo n=188), more fremanezumabtreated patients reported no medication overuse during the 12-week treatment period (quarterly: 111/201 patients [55%], P=0.0389; Achievement of response over time with fremane- zumab in the treatment of chronic and episodic migraine Stephen D. Silberstein1, Richard B. Lipton2, Merle L. Diamond3, Joshua M. Cohen4, Ronghua Yang4, Bo Jiang4 1 Jefferson Headache Center, Thomas Jefferson Uni- versity, Philadelphia, Pennsylvania, USA 2 Albert Einstein College of Medicine, Bronx, New York, USA 3 Diamond Headache Clinic, Chicago, Illinois, USA 4 Teva Pharmaceuticals, Frazer, Pennsylvania, USA Correspondence: Stephen D. Silberstein (Stephen.Silberstein@jefferson.edu) Page 9 of 11 The Journal of Headache and Pain (2018) 19:19 The correspondence e-mail address contained an error. The correct e-mail address has now been in- cluded in this correction. The impact of fremanezumab on medication over- use in patients with chronic migraine Pa- tients with <20% reduction at Month 3 had >40% re- sponse rates of 18% (32/176) at Month 6 and 30% (21/ 69) at Month 9. Patients with <40% reduction at Month 1 had >40% response rates of 36% (99/272) at Month 6 and 51% (55/108) at Month 9. Patients with <40% reduc- tion at Month 3 had >40% response rates of 28% (72/ 253) at Month 6 and 41% (41/101) at Month 9. EM pa- tients with <20% reduction in migraine days at Month 1 had >40% response rates of 53% (53/100) at Month 6 and 62% (26/42) at Month 9. Patients with <20% reduc- tion at Month 3 had >40% response rates of 41% (34/83) at Month 6 and 47% (15/32) at Month 9. Patients with <40% reduction at Month 1 had >40% response rates of 57% (92/162) at Month 6 and 63% (45/72) at Month 9. Patients with <40% reduction at Month 3 had >40% re- sponse rates of 46% (62/135) at Month 6 and 61% (33/ 54) at Month 9. RESULTS: CM patients with <20% reduction in mi- graine days at Month 1 had >40% response rates of 29% (58/197) at Month 6 and 43% (35/81) at Month 9. Pa- tients with <20% reduction at Month 3 had >40% re- sponse rates of 18% (32/176) at Month 6 and 30% (21/ 69) at Month 9. Patients with <40% reduction at Month 1 had >40% response rates of 36% (99/272) at Month 6 and 51% (55/108) at Month 9. Patients with <40% reduc- tion at Month 3 had >40% response rates of 28% (72/ 253) at Month 6 and 41% (41/101) at Month 9. EM pa- tients with <20% reduction in migraine days at Month 1 had >40% response rates of 53% (53/100) at Month 6 and 62% (26/42) at Month 9. Patients with <20% reduc- tion at Month 3 had >40% response rates of 41% (34/83) at Month 6 and 47% (15/32) at Month 9. Patients with <40% reduction at Month 1 had >40% response rates of 57% (92/162) at Month 6 and 63% (45/72) at Month 9. Patients with <40% reduction at Month 3 had >40% re- sponse rates of 46% (62/135) at Month 6 and 61% (33/ 54) at Month 9. The impact of fremanezumab on medication over- use in patients with chronic migraine The impact of fremanezumab on medication over- use in patients with chronic migraine OBJECTIVES: The long-term efficacy of monoclonal antibodies that selectively target calcitonin gene-related peptide (CGRP) in patients with early treatment failure is not well characterized. Based on data from Phase 3 trials in episodic (EM) and chronic migraine (CM) of fremanezumab, a fully humanized monoclonal antibody (IgG2a) that selectively targets CGRP, we assessed long-term treatment response rates in patients with early treatment failure. Stephen D. Silberstein1, Sait Ashina2, Zaza Katsarava3, Kristen Bibeau4, Michael J. Seminerio4, Danielle E. Harlow4, Joshua M. Cohen4 Stephen D. Silberstein1, Sait Ashina2, Zaza Katsarava3, Kristen Bibeau4, Michael J. Seminerio4, Danielle E. Harlow4, Joshua M. Cohen4 1 Jefferson Headache Center, Thomas Jefferson University, Philadelphia, Pennsylvania, USA 2 Beth Israel Deaconess Medical Center Comprehensive Headache Center, Harvard Medical School, Boston, Massachusetts, USA 3 University of Essen, Unna, Germany METHODS: This multicenter, randomized, double- blind, parallelgroup, long-term study, included patients who completed either 12-week Phase 3 study (HALO CM or HALO EM). Patients continued on treatment from the 12-week studies, receiving either subcutaneous fremanezumab quarterly (675 mg every 3 months), fre- manezumab monthly (CM: 675 mg at baseline and 225 mg every month; EM: 225 mg every month) over a 12-month treatment period. The percentage of patients with a reduction in migraine days (response rates) >40% at Months 6 and 9 among patients with low response rates (<40%) at Month 1 was assessed in patients who received active treatment in the 12-week studies. 4 Teva Pharmaceuticals, Frazer, Pennsylvania, USA * Correspondence: Stephen D. Silberstein (Stephen. Silberstein@jefferson.edu) The correspondence e-mail address contained an error. The correct e-mail address has now been in- cluded in this correction. OBJECTIVES: Overuse of acute or symptomatic headache medications (triptans, ergot derivatives, opioids, and combination analgesics) can cause medication over- use headache (MOH), which often accompanies chronic migraine (CM). Fremanezumab, a fully humanized mono- clonal antibody (IgG2a) that selectively targets calcitonin gene-related peptide (CGRP), reduced the frequency and severity of headaches in CM patients. We assessed the ef- fect of fremanezumab on medication overuse and acute headache medication use in CM patients. RESULTS: CM patients with <20% reduction in mi- graine days at Month 1 had >40% response rates of 29% (58/197) at Month 6 and 43% (35/81) at Month 9. The impact of fremanezumab on medication over- use in patients with chronic migraine METHODS: In this multicenter, randomized, double- blind, placebocontrolled, Phase 3 study, CM patients CM were randomized 1:1:1 to receive subcutaneous fre- manezumab quarterly (675 mg at baseline, and placebo at Weeks 4 and 8), fremanezumab monthly (675 mg at baseline, and 225 mg at Weeks 4 and 8), or placebo over a 12-week treatment period. We assessed the proportion of patients who reverted from overusing medications at baseline (use of acute headache medication on ≥15 days, use of migraine-specific acute medication on ≥10 days, or use of combination medications for headache on ≥10 days during the 28-day baseline period) to not overusing medications at Week 12, and the change from baseline in the number of days of acute headache medication use among these patients. Analyses were performed in the full analysis set (all randomized patients who received ≥1 dose of study drug and had ≥10 days of post-baseline efficacy assessments on the primary endpoint). METHODS: In this multicenter, randomized, double- blind, placebocontrolled, Phase 3 study, CM patients CM were randomized 1:1:1 to receive subcutaneous fre- manezumab quarterly (675 mg at baseline, and placebo at Weeks 4 and 8), fremanezumab monthly (675 mg at baseline, and 225 mg at Weeks 4 and 8), or placebo over a 12-week treatment period. We assessed the proportion of patients who reverted from overusing medications at baseline (use of acute headache medication on ≥15 days, use of migraine-specific acute medication on ≥10 days, or use of combination medications for headache on ≥10 days during the 28-day baseline period) to not overusing medications at Week 12, and the change from baseline in the number of days of acute headache medication use among these patients. Analyses were performed in the full analysis set (all randomized patients who received ≥1 dose of study drug and had ≥10 days of post-baseline efficacy assessments on the primary endpoint). CONCLUSIONS: Failure to achieve an early response to fremanezumab does not predict failure at later time points. ETHICS APPROVAL The study was approved by all relevant independent ethics committees or institutional review boards, accord- ing to national or local regulations. Page 10 of 11 Page 10 of 11 The Journal of Headache and Pain (2018) 19:19 monthly: 120/198 patients [61%], P=0.0024) than those who received placebo (87/188 patients [46%]). This re- sponse was seen as early as Week 4 (quarterly: 102/201 patients [51%], P=0.0091; monthly: 107/198 patients [54%], P=0.0014; vs placebo: 73/188 patients [39%]). Among patients who responded (quarterly n=111; monthly n=120; placebo n=87), the baseline number of days with medication overuse was similar across treat- ment groups (quarterly [mean ± standard error]: 16.6 ±0.32 days; monthly: 16.7±0.33 days; placebo: 16.6 ±0.35). Within this population, fremanezumab treatment reduced the days of acute headache medication use over the treatment period (quarterly: –9.0±0.41 days, P=0.0017; monthly: –8.9±0.41 days, P=0.0040) versus those who received placebo (–7.1±0.46 days). framework. Reviews, original studies, and clinical guide- lines in European adults (≥18 years) with EM (<15 head- ache days per month), CM (≥15 headache days with ≥8 migraine days per month), or medication overuse headache (MOH) were included. Searches focused on epidemiology, incidence, prevalence, mortality, morbidity, treatment patterns, clinical guidelines relating to prevent- ive interventions, and unmet need (published 2007–Feb- ruary 1, 2018; geographical limitation: United Kingdom, France, Germany, Spain, Italy, the Netherlands, Poland, Denmark, Finland, Iceland, Norway and Sweden). Searches included: Embase, MEDLINE, and the Cochrane Library databases; specialty medicine associations; and health technology assessment agency websites. gy g y RESULTS: Analysis included 64 publications. The World Health Organization estimated 77 million mi- graine sufferers in Europe, with an incidence of up to 39.2 per 1000 patient-years. Migraine prevalence was higher (2–6-fold) in women than in men in all but one study, with peak prevalence at 25–55 years of age. EM was up to 15 times more prevalent than CM. MOH has an estimated prevalence of 1% in the general adult popu- lation. Preventive therapies are typically betablockers, antidepressants, anticonvulsants (topiramate), or in some countries, onabotulinumtoxinA (for chronic migraine), but most have no proven efficacy in CM. Guidelines vary by country, but the European Headache Federation (EHF) recommends preventive therapy for patients with ≥2 de- bilitating attacks per month; however, ≤13% of patients who qualify for preventive treatment actually receive it. ETHICS APPROVAL Adherence to available preventive therapies is low, and many physicians believe that the disadvantages, including adverse events, drug dependency, and lack of sustained efficacy outweigh the benefits. To manage MOH, with- drawal of treatment is recommended. gy g y RESULTS: Analysis included 64 publications. The World Health Organization estimated 77 million mi- graine sufferers in Europe, with an incidence of up to 39.2 per 1000 patient-years. Migraine prevalence was higher (2–6-fold) in women than in men in all but one study, with peak prevalence at 25–55 years of age. EM was up to 15 times more prevalent than CM. MOH has an estimated prevalence of 1% in the general adult popu- lation. Preventive therapies are typically betablockers, antidepressants, anticonvulsants (topiramate), or in some countries, onabotulinumtoxinA (for chronic migraine), but most have no proven efficacy in CM. Guidelines vary by country, but the European Headache Federation (EHF) recommends preventive therapy for patients with ≥2 de- bilitating attacks per month; however, ≤13% of patients who qualify for preventive treatment actually receive it. Adherence to available preventive therapies is low, and many physicians believe that the disadvantages, including adverse events, drug dependency, and lack of sustained efficacy outweigh the benefits. To manage MOH, with- drawal of treatment is recommended. CONCLUSIONS: Fremanezumab treatment was asso- ciated with reduced overuse of acute medications and fewer days using acute medications. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02 621931 P136 Epidemiology of chronic and episodic migraine in Europe Hicham Benhaddi1, Timothy Fitzgerald2, Sophie McCabe3, Ruth Zeidman3 Hicham Benhaddi1, Timothy Fitzgerald2, Sophie McCabe3, Ruth Zeidman3 1 Teva Pharmaceuticals, Wilrijk, Belgium 1 Teva Pharmaceuticals, Wilrijk, Belgium 1 Teva Pharmaceuticals, Wilrijk, Belgium 2 T Ph i l F P l i USA 2 Teva Pharmaceuticals, Frazer, Pennsylvania, USA 2 Teva Pharmaceuticals, Frazer, Pennsylvania, USA 3 Covance Market Access, London, UK 3 Covance Market Access, London, UK 3 Covance Market Access, London, UK * Correspondence: Hicham Benhaddi (Hicham.Benhaddi @tevaeu.com) * Correspondence: Hicham Benhaddi (Hicham.Benhaddi @tevaeu.com) * Correspondence: Hicham Benhaddi (Hicham.Benhaddi @tevaeu.com) CONCLUSIONS: Migraine is highly prevalent in Eur- ope. Despite improvements in compliance with treat- ment guidelines, the number of patients on preventive therapies remains low, likely due to poor tolerability and efficacy of available therapies and limited access to these medications due to restrictive guidelines. The correspondence e-mail address contained an error. The correct e-mail address has now been in- cluded in this correction. BACKGROUND: Migraine is a debilitating neuro- logical disorder characterized by headaches of varying duration and intensity. Treatment options vary depend- ing on disease severity, frequency, regional practices, and therapy availability. Robert Cowan1, Joshua Cohen2, Erik Rosenman3, Tim Fitzgerald2, Ravi Iyer2* 1 Neurology, Stanford University School of Medicine, Stanford, California, 94305, USA 2 Teva Pharmaceutical Industries, Frazer, Pennsylvania, 19355, USA 2 Teva Pharmaceutical Industries, Frazer, Pennsylvania, 19355, USA 1 Neurology, Stanford University School of Medicine, Stanford, California, 94305, USA Robert Cowan1, Joshua Cohen2, Erik Rosenman3, Tim Fitzgerald2, Ravi Iyer2* ETHICS APPROVAL The study was approved by all relevant independent ethics committees or institutional review boards, accord- ing to national or local regulations. P137 The impact of offering monthly and quarterly dos- ing options for a new class of migraine preventive therapy on likelihood of acceptance and adherence in adults with migraine OBJECTIVE: To conduct a systematic literature review of the epidemiology (incidence, prevalence, mortality, and morbidity), current treatment pathways and patterns, pre- ventive therapy guidelines, and unmet needs of chronic (CM) and episodic migraine (EM) in Europeans. METHODS: Literature searches and evidence screening were structured according to the PICOS (population, intervention, comparators, outcomes, and study types) METHODS: Literature searches and evidence screening were structured according to the PICOS (population, intervention, comparators, outcomes, and study types) METHODS: Literature searches and evidence screening were structured according to the PICOS (population, intervention, comparators, outcomes, and study types) Page 11 of 11 The Journal of Headache and Pain (2018) 19:19 1. http://migraineresearchfoundation.org/about-migraine/ migraine-facts/ Last accessed June 22nd, 2018 1. http://migraineresearchfoundation.org/about-migraine/ migraine-facts/ Last accessed June 22nd, 2018 Background: Migraine affects approximately 39 mil- lion people in the US1. A new class of migraine prevent- ive therapy launching in 2018-2019 will provide physicians and patients with an alternate approach to preventive treatment. This study sought to understand the impact, if any, of having both monthly and quarterly dosing options on acceptance of, and adherence to, the new class of migraine preventive therapy among adults with migraine. Received: 21 September 2018 Accepted: 21 September 2018 Conclusions 3 IQVIA, Inc., Cambridge, Massachusetts, 02139, USA * Correspondence: Ravi Iyer (Ravi.Iyer01@tevapharm. com) Adults with migraine are more likely to fill the new class of preventive therapy and to take it consistently over one year when presented with their preferred dos- ing regimen. The correspondence e-mail address contained an error. The correct e-mail address has now been in- cluded in this correction. Received: 21 September 2018 Accepted: 21 September 2018 Reference h d Benhaddi H et al (2018) 12th European Headache Federation Congress jointly with 32nd National Congress of the Italian Society for the Study of Headaches. J Headache Pain 19(Suppl 1):80 https://doi.org/10.1186/s10194- 018-0900-0 Methods: In this double-blind, observational study, 420 US adults with migraine completed a 20-minute, self- administered online survey. Respondents included 228 moderate-frequency episodic (5-9 headache days / month), 106 high-frequency episodic (10-14 headache days / month), and 86 chronic migraine patients (≥15 headache days / month). Adults with migraine were exposed to three scenarios: 1) only monthly dosing of the new class of migraine preventive therapy is avail- able, 2) only quarterly dosing is available, and 3) both monthly and quarterly dosing are available. In each scenario, and assuming roughly equivalent efficacy re- gardless of dosing schedule, adults with migraine were asked their likelihood to fill the prescription (if pre- scribed) and their likelihood to take it consistently over one year, measured on a 7-point scale where 1 was “not at all likely” and 7 was “extremely likely”. Those that selected a 6 or 7 on the scale were classi- fied as “likely”. At the end of the survey, respondents were then asked if they preferred either monthly or quarterly dosing for this new class of therapy. Data analysis included descriptive statistical analyses and comparison of means through ANOVA testing, with significance set at p<0.05. Results: A similar proportion of adults with migraine preferred monthly (35.7%) and quarterly (39.5%) dosing regimens (24.8% had no preference). Among those who prefer monthly dosing (n=150), a greater proportion in- dicate they are likely to fill the prescription and remain adherent when only monthly is prescribed and available compared to when only quarterly is (77% vs. 56% p<0.001 and 80% vs. 57% p<0.001 respectively). Likewise, among those who prefer quarterly dosing (n=166), a greater proportion indicate they are likely to fill and re- main adherent when only quarterly is prescribed and available compared to when only monthly is (63% vs. 55% p<0.008 and 62% vs. 54% p<0.023 respectively).
https://openalex.org/W3090247382	https://hal.archives-ouvertes.fr/hal-03120867/file/islandora_109673.pdf	English	null	Comparative patterns of modified nucleotides in individual tRNA species from a mesophilic and two thermophilic archaea	RNA	2,020	cc-by	16,380	To cite this version: Philippe Wolff, Claire Villette, Julie Zumsteg, Dimitri Heintz, Laura Antoine, et al.. Comparative patterns of modified nucleotides in individual tRNA species from a mesophilic and two thermophilic archaea. RNA, 2020, 26 (12), pp.1957-1975. 10.1261/rna.077537.120. hal-03120867 Comparative patterns of modified nucleotides in individual tRNA species from a mesophilic and two thermophilic archaea Philippe Wolff, Claire Villette, Julie Zumsteg, Dimitri Heintz, Laura Antoine, Béatrice Chane-Woon-Ming, Louis Droogmans, Henri Grosjean, Eric Westhof Comparative patterns of modified nucleotides in individual tRNA species from a mesophilic and two thermophilic archaea HAL Id: hal-03120867 https://hal.science/hal-03120867v1 Submitted on 25 Jan 2021 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Cold Spring Harbor Laboratory Press on January 21, 2021 - Published by rnajournal.cshlp.org ownloaded from © 2020 Wolff et al. This article, published in RNA, is available under a Creative Commons License (Attribution 4.0 International), as described at http://creativecommons.org/licenses/by/4.0/. ABSTRACT To improve and complete our knowledge of archaeal tRNA modification patterns, we have identified and compared the modification pattern (type and location) in tRNAs of three very different archaeal species, Methanococcus maripaludis (a mesophilic methanogen), Pyrococcus furiosus (a hyperthermophile thermococcale), and Sulfolobus acidocaldarius (an acidophilic thermophilic sulfolobale). Most abundant isoacceptor tRNAs (79 in total) for each of the 20 amino acids were isolated by two-dimensional gel electrophoresis followed by in-gel RNase digestions. The resulting oligonucleotide fragments were separated by nanoLC and their nucleotide content analyzed by mass spectrometry (MS/MS). Analysis of total modified nucleosides obtained from complete digestion of bulk tRNAs was also performed. Distinct base- and/or ri- bose-methylations, cytidine acetylations, and thiolated pyrimidines were identified, some at new positions in tRNAs. Novel, some tentatively identified, modifications were also found. The least diversified modification landscape is observed in the mesophilic Methanococcus maripaludis and the most complex one in Sulfolobus acidocaldarius. Notable observa- tions are the frequent occurrence of ac4C nucleotides in thermophilic archaeal tRNAs, the presence of m7G at positions 1 and 10 in Pyrococcus furiosus tRNAs, and the use of wyosine derivatives at position 37 of tRNAs, especially those decod- ing U1- and C1-starting codons. These results complete those already obtained by others with sets of archaeal tRNAs from Methanocaldococcus jannaschii and Haloferax volcanii. Keywords: mass spectrometry; Archaea; tRNA; modifications; hyperthermophiles PHILIPPE WOLFF,1 CLAIRE VILLETTE,2 JULIE ZUMSTEG,2 DIMITRI HEINTZ,2 LAURA ANTOINE,1 BÉATRICE CHANE-WOON-MING,1 LOUIS DROOGMANS,3 HENRI GROSJEAN,3 and ERIC WESTHOF1 1Architecture et Réactivité de l’ARN, Institut de Biologie Moléculaire et Cellulaire du CNRS, Université de Strasbou F-67084, Strasbourg, France 2Institut de Biologie Moléculaire des Plantes du CNRS, Université de Strasbourg, F-67084, Strasbourg, France 3Laboratoire de Chimie Biologique, Université Libre de Bruxelles, Institut Labiris, B-1070, Belgium 2Institut de Biologie Moléculaire des Plantes du CNRS, Université de Strasbourg, F-67084, Strasbourg, France 3Laboratoire de Chimie Biologique, Université Libre de Bruxelles, Institut Labiris, B-1070, Belgium 2Institut de Biologie Moléculaire des Plantes du CNRS, Université de Strasbourg, F-67084, Strasbourg, France 3Laboratoire de Chimie Biologique, Université Libre de Bruxelles, Institut Labiris, B-1070, Belgium Corresponding authors: e.westhof@ibmc-cnrs.unistra.fr, p.wolff@ibmc-cnrs.unistra.fr Article is online at http://www.rnajournal.org/cgi/doi/10.1261/rna. 077537.120. Freely available online through the RNA Open Access option. INTRODUCTION ribosomal subunits (Selmer et al. 2006). The modifications occur in the whole body of the tRNA but especially in the elbow created by the intricate contacts formed between the D- and T-loops (Machnicka et al. 2014). The second main role played by tRNA modifications is to guarantee fi- delity and efficiency during ribosomal translation at the de- coding site, thereby participating in the regulation of the translational activity and the control of proteostasis (Pollo-Oliveira and de Crécy-Lagard 2019). These modifi- cations occur mainly in the extended anticodon loop of tRNAs (Yarus 1982). Their roles are (i) to maintain a confor- mation of the anticodon loop preorganized for pairing with the mRNA codon in the A site (Vendeix et al. 2012); (ii) to stabilize the weak AU-rich codon/anticodon pairs (Grosjean and Westhof 2016); (iii) to avoid miscoding (e.g., Met/Ile or Trp/Stop) (Cantara et al. 2013); (iv) to allow ribosomal subunits (Selmer et al. 2006). The modifications occur in the whole body of the tRNA but especially in the elbow created by the intricate contacts formed between the D- and T-loops (Machnicka et al. 2014). The second main role played by tRNA modifications is to guarantee fi- delity and efficiency during ribosomal translation at the de- coding site, thereby participating in the regulation of the translational activity and the control of proteostasis (Pollo-Oliveira and de Crécy-Lagard 2019). These modifi- cations occur mainly in the extended anticodon loop of tRNAs (Yarus 1982). Their roles are (i) to maintain a confor- mation of the anticodon loop preorganized for pairing with the mRNA codon in the A site (Vendeix et al. 2012); (ii) to stabilize the weak AU-rich codon/anticodon pairs (Grosjean and Westhof 2016); (iii) to avoid miscoding (e.g., Met/Ile or Trp/Stop) (Cantara et al. 2013); (iv) to allow Transfer RNAs are the most modified RNA molecules in terms of number of modified positions and diversity of chemical modifications. A whole gamut of modification enzymes had to differentially evolve in the three domains of life to mature properly tRNAs (Helm and Alfonso 2014; Boccaletto et al. 2018). In short, tRNA modifications play two central roles. The first one is to guarantee the maintenance of the uniqueness and stability of the tRNA architectural fold (Helm 2006; Motorin and Helm 2010, 2011), a requirement for proper recognition by key factors like aminoacyl-tRNA synthetases (Giégé and Springer 2016), the Elongator complex (Karlsborn et al. tRNA genes and their cellular mature products In the GtRNAdb (Chan and Lowe 2009, 2016), there are 37 genes coding for tRNAs in M. maripaludis and 46 in both P. furiosus and S. acidocaldarius, all predicted with excel- lent scores using tRNAscan-SE 2.0 (Lowe and Eddy 1997). In S. acidocaldarius, 50 tRNA genes are predicted but four have scores below 45.0 and would be considered as pseudogenes (Chan and Lowe 2009, 2016). Based on genomic information, in M. maripaludis, there are only two tRNA genes corresponding to 4-codon boxes and only one gene corresponding to 2-codon boxes, except for Asp, Glu, and Lys where there are two isoacceptors. In P. furiosus and S. acidocaldarius, there are three genes coding for isoacceptors in 4-codon boxes and only one in 2-codon boxes, except again for Glu, Lys, but also Gln, Ile, Arg(AGR), and Leu(UUR) (all codons of 2/3-codon boxes ending with R3 and thus decoded by Y34-contain- ing tRNAs). In the three species, there is one gene coding for tRNA-Meti and another one for tRNA-Met, except in M. maripaludis, where there are two copies of tRNA- Meti. As in all other archaeal genomes sequenced so far, there is no gene coding for A34-containing tRNA. Remark- ably, in M. maripaludis, the C34-containing tRNAs are ab- sent in both 4- and 2-codon boxes except of course in tRNA-Ile(CAU), tRNA-Met and tRNA-Meti(CAU), and tRNA-Trp(CCA). Thus, besides these five particular tRNAs, all the other tRNA anticodon triplets start with either G34 or U34. In contrast, in the two thermophilic archaea, P. fur- iosus and S. acidocaldarius, the C34-containing tRNAs are present, which explains the increase from 37 to 46 naturally occurring tRNA genes. These distributions follow the spar- ing strategies in Archaea described by Grosjean et al. (2010). The percentage of GC-content increases from M. maripaludis (34%) to S. acidocaldarius (37.5%) and to P. furiosus (41.1%). The codon usage is such that U3- and A3-ending codons are highly preferred (Nakamura et al. 2000; Emery and Sharp 2011; Nayak 2013). This is particu- larly striking in the mesophilic M. maripaludis where U3- and A3-ending codons are decoded solely by G34-tRNAs Here, we examined the landscapes of tRNA modifica- tions in three archaeal species with very different evolution- ary history. Two belong to the Kingdom Euryarchaeota: Methanococcus maripaludis, a methane-producing an- aerobic mesophilic archaeon belonging to the same Methanococcales clade as M. INTRODUCTION 2014), and Corresponding authors: e.westhof@ibmc-cnrs.unistra.fr, p.wolff@ibmc-cnrs.unistra.fr Article is online at http://www.rnajournal.org/cgi/doi/10.1261/rna. 077537.120. Freely available online through the RNA Open Access option. 1957 RNA (2020) 26:1957–1975; Published by Cold Spring Harbor Laboratory Press for the RNA Society Cold Spring Harbor Laboratory Press on January 21, 2021 - Published by rnajournal.cshlp.org Downloaded from Cold Spring Harbor Laboratory Press on January 21, 2021 - Published by rnajournal.cshlp.org Downloaded from Wolff et al. the decoding of purine-ending codons in split codon box- es by promoting unusual base-pairings that fit within the decoding ribosomal grip (Rozov et al. 2016). Although the nature and number of the modifications vary consider- ably between the three domains of life, the great majority of the tRNA positions that are modified are highly con- served throughout phylogeny. theoretical corresponding tDNA genes (Chan and Lowe 2009, 2016). In-gel digestion by specific nucleases fol- lowed. The resulting tRNA digests, for which sequences were deduced from their known genomes, were then sep- arated by chromatography and analyzed by mass spec- trometry. The analysis of modified nucleosides of bulk tRNAs was also performed and the results compared with those obtained from the analysis of oligonucleotide sequences. Altogether, our results support the idea that, despite the fact that Archaea share typical modified nucle- otides present in both Bacteria and/or Eukarya, they also display unique and specific modifications. Unfortunately, the complete patterns of tRNA modifica- tions are known for only a limited number of species (e.g., E. coli in Bacteria or S. cerevisiae in Eukarya). In Archaea, al- though studied for a long time, the landscape of tRNA modifications remained disperse, and known only for a few particular tRNA isoacceptors or bulk cellular tRNAs (see for examples: Kuchino et al. 1982; Edmonds et al. 1991; Tomikawa et al. 2013). Only in the cases of Haloferax volcanii (Gupta 1984, 1986; Grosjean et al. 2008a) and very recently in the case of Methanocaldococcus jannaschii (Yu et al. 2019), the modificationlandscape of acomplete set of cellular tRNAs has been elucidated. These data reveal that a few modifications are unique to certain archaea, while others are present in most, if not all archaeal species stud- ied so far. Among the archetypal ones are a N1-methylated pseudouridine at position 54 instead of a thymine (Pang et al. 1982; Gupta 1984), the presence of archaeosine, or 7-formamidino-7-deazaguanosine, at position 15 (Wata- nabe et al. INTRODUCTION 1997), certain wyosine derivatives like imG, imG2 and mimG at position 37 (de Crécy-Lagard et al. 2010) and the presence at position 34 of tRNA-Ile of agma- tidine, a modified C where the carbonyl group is replaced by decarboxy-arginine (Ikeuchi et al. 2010; Mandal et al. 2010). Modified nucleotides in tRNAs from three archaea and U34-tRNAs, respectively. In M. maripaludis, the per- centage of GC-content at the third position is 25.6%, while it is 28.8% in P. furiosus and as high as 39.3% in S. acido- caldarius. Such differences in decoding strategies have consequences on the modification identity found at posi- tion 34 and in the extended anticodon stem–loop of indi- vidual isoacceptor tRNA (see below and Grosjean and Westhof 2016). The knowledge of tRNA genes and copy numbers does not allow yet to predict which identified genes correspond to lowly or highly expressed cellular tRNAs (minor/major species). The low abundance of cer- tain cellular tRNAs can be below the detection mapping method, which explains why some tRNA isoacceptors are missing in our analyses of bulk tRNAs. However, for each archaeon, representative tRNAs corresponding to each of the 20 amino acids could be analyzed. They probably correspond to the most abundant naturally occurring spe- cies. Lastly, in Archaea, especially the hyperthermophilic ones, tRNA genes often contain introns (Sugahara et al. 2008). Among the three archaeal species studied, the S. acidocaldarius contains the highest number of tRNAs with introns (21 out of 46) (Chan and Lowe 2009, 2016). Such intron-containing tRNAs are often the targets of site-specific 2′-O-ribose methylations or uridine isomeriza- tion into pseudouridine via the sRNA-guided FlpA C/D box or the Cbf5 H/ACA enzymatic machinery, respectively (see below). tRNA sequences especially for the highly conserved D- and TΨC-loops. To be sure to assign unambiguously an MS/MS sequencing spectrum, each tRNA isoacceptor was analyzed separately (see Materials and Methods). All CID (Collision Induced Decay) MS/MS spectra were manu- ally examined and sequenced. Although LC MS/MS spec- tra of digestion products allow the localization of a modified nucleotide within the sequence, some of the modifications unfortunately share the same (m/z) mass. For example, methylation can be detected but, solely on the basis of the mass spectra, it is not possible to localize the methyl group (either on the ribose or on the base). The same situation occurs between uridine and pseudour- idine. However, knowing the presence of a given modified nucleotide at the same position in a homogous tRNA of a closely related archaeon, together with the existence of corresponding modification enzymes (and its ORF in the genome), sometimes allows to assign the most probable chemical modification after verification that a homolog gene exists in the genome of the archaea studied (Supplemental Table S2). Purification and sequencing of isolated tRNA species Each tRNA isoacceptor was isolated using two-dimension- al PAGE. The process leads to a series of tRNA spots. Each spot contains generally one isoacceptor, while some con- tain two or rarely three isoacceptors (Supplemental Fig. S1). For each spot, all CID spectra were manually inspected and sequenced. The tRNAs were identified by unique se- quences in RNase digestion products (see Materials and Methods). From such 2D-gel electrophoresis, we could pu- rify and analyze 27 post-transcriptionally matured tRNAs corresponding to obviously major naturally occurring spe- cies from M. maripaludis and 30 tRNAs from P. furiosus (over 37 and 46, respectively) but only 22 (over 46) from S. acidocaldarius. In this latter case, of the 21 intron-con- taining primary transcripts predicted from the genome se- quence only six were identified as matured species in the purified bulk matured tRNAs, while 16 over 25 of the pre- dicted intron-less tRNAs were detected. In P. furiosus and M. maripaludis, only two tRNAs (specific for Met [CAU] and Trp[CCA]) are transcribed as intron-containing species and each of them was obtained and analyzed as the matured species. Modified nucleotides in tRNAs from three archaea The ambiguous cases that are left are discussed in the text or legends. Analyses of total modified nucleosides of bulk tRNAs were also performed allowing to identify the presence of modified nucleotides that escape the above analysis of sufficiently long oligonu- cleotides (Supplemental Figs. S2, S3). The code for modi- fied nucleosides used throughout this paper, except when specifically mentioned, follows either the one used in MODOMICS (Boccaletto et al. 2018) or the chemically based nomenclature (Motorin and Helm 2011; Helm and Alfonso 2014). tRNA genes and their cellular mature products jannaschii (but the latter is thermophilic) and Pyrococcus furiosus, an anaerobic hyper- thermophilic archaeon belonging to the Thermococcales clade (Forterre 2015). The third species studied here is Sul- folobus acidocaldarius (an obligate aerobic, acidophilic, sulfur-oxidizing thermophile that belongs to the Sulfolo- bales clade of the Crenarchaeota Kingdom). The last two archaea therefore belong to families not yet systematically analyzed and S. acidocaldarius isthe firstcrenoarchaeonfor which the modification landscape is reported. Further, all euryarchaeal tRNAs analyzed so far are from anaerobic mi- croorganisms (with thermophilic or hyperthermophilic character), while S. acidocaldarius is an aerobic moderate thermophile. As performed earlier in the case of H. volcanii (Gupta 1984, 1986), two-dimensional gel electrophoresis was used to separate individual tRNA isoacceptors from puri- fied bulk cellular tRNAs of each of the three archaea, allow- ing the analysis of 79 cellular tRNAs out of a total of 116 1958 RNA (2020) Vol. 26, No. 12 Cold Spring Harbor Laboratory Press on January 21, 2021 - Published by rnajournal.cshlp.org Downloaded from Cold Spring Harbor Laboratory Press on January 21, 2021 - Published by rnajournal.cshlp.org Downloaded from Cold Spring Harbor Laboratory Press on January 21, 2021 - Published by rnajournal.cshlp.org Downloaded from Modified nucleotides in tRNAs from three archaea Structural tRNA alignments Alignments of the sequenced archaeal tRNAs studied in this work, including the modified nucleotides we have been able to detect atthe oligonucleotide digest products, are shown in Figure 1 (see also Table 1; Supplemental Fig. S10). These alignments follow the usual nomenclature of tRNA structure, and they are structural in the sense that equivalent positions in the three-dimensional structure (as- sumed by homology with known crystal structures) are ver- tically aligned. They show that the tRNA sequences follow the expected patterns of covariations and conservations as observed in the majority of cellular tRNAs of all three do- mains of life, demonstrating that they do conform to the known three-dimensional structure of tRNAs. The numbers of Watson–Crick base pairs (often GC-rich and with a small number of GU pairs, especially in the thermophiles) in the stems are as usual: seven in the acceptor stem (AA-stem), four in the dihydrouridine stem (D-stem), five in the antico- don stem (AC-stem) and the thymine stem (TΨC-stem). Also, a long variable region is always present in the long- arm tRNAs specific for leucine and serine. The tRNA-Leu (anticodons YAG, Y = U/C) in M. maripaludis and P. furiosus A critical point in the present experimental strategy is to assign a modified position to the tRNA it belongs to. Indeed, because archaeal tRNAs are highly GC-rich (espe- cially in stems), there is a high level of similarities between 1959 www.rnajournal.org Cold Spring Harbor Laboratory Press on January 21, 2021 - Published by rnajournal.cshlp.org Downloaded from l Cold Spring Harbor Laboratory Press on January 21, 2021 - Published by rnajournal.cshlp.org Downloaded from Cold Spring Harbor Laboratory Press on January 21, 2021 - Published by rnajournal.cshlp.org Downloaded from Wolff et al. FIGURE 1. Compilation of modified tRNA sequences from S. acidocaldarius, M. maripaludis, and P. furiosus. Red nucleotides indicate fragments obtained by RNase T1, and/or RNase A, and/or RNase U2 digestion, while black nucleotides represent regions that could not be analyzed. Nucleotides in gray are modified nucleotides with a mass corresponding to several possible modifications. FIGURE 1 Compilation of modified tRNA sequences from S acidocaldarius M maripaludis and P furiosus Red nucleotides indicate fragments FIGURE 1. Compilation of modified tRNA sequences from S. acidocaldarius, M. maripaludis, and P. furiosus. Red nucleotides indicate fragments obtained by RNase T1, and/or RNase A, and/or RNase U2 digestion, while black nucleotides represent regions that could not be analyzed. Structural tRNA alignments Nucleotides in gray are modified nucleotides with a mass corresponding to several possible modifications. both contain, after the conserved U8, U9 instead of the very common R9 (R = A/G). This is also the case in Thermo- plasma acidophilum, a thermo-acidic Euryarchaeon where s4U8 and s4U9 have been detected (Tomikawa et al. 2013). This occurrence of two consecutive Us at positions 8 and 9 is surprisingly accompanied by an unusual G38 opposite to U32 in the anticodon loop (especially with a CAG antico- don). Although we could not detect s4U at either position 8 or 9, the nucleoside analysis (Supplemental Figs. S2, S3) confirms the presence of s4U in both M. maripaludis and P. furiosus. An analysis of the GtRNAdb shows that these correlations stand out in Euryarchaeota and Thau- marchaeota (Chan and Lowe 2009, 2016). The D-stem presents also some particularities like a preference for G10-Y25 and Y13-G22 with the central two base pairs maintained as Watson–Crick. It is likely that some, if not all, of the uridines at positions 13, 22, and 25 of the D- stem and position 39 of the anticodon stem (AC-stem) are pseudouridines (Ψ) which cannot be differentiated from U by mass spectrometry. Moreover, the AC-stem has a pro- nounced preference for a G30–C40 pair, while in other tRNAs, especially of mesophilic organisms, a A30–U40 pair is present (Marck and Grosjean 2002). There are seven residues in the AC- and T-loops with the conserved residues U33 and the favored C32/A38 opposition in the anticodon loop. In the Sulfolobales clade of the Crenarch- aeota, the tRNA-Cys(GCA) has an unusual C33 (together with G27oU43), but unfortunately we could not isolate 1960 RNA (2020) Vol. 26, No. 12 Cold Spring Harbor Laboratory Press on January 21, 2021 - Published by rnajournal.cshlp.org ownloaded from Modified nucleotides in tRNAs from three archaea ABLE 1. Structural tRNA alignments List of modified positions in different Archaea Halophile Methano Coccales Pyro Coccales Sulfolobales osition Haloferax volcanii Methanococcus maripaludis Methanocaldococcus jannaschii Thermococcus kodakarensis Pyrococcus furiosus Sulfolobus acidocaldarius 2H-m7G ac4C ac4C m2G Cm m2G/m2 2G ac6A (s4U)∗ s4U s4U Um/(s4U)∗ (m1A/m1G)∗ m1A/m1G m1A 0>> m2G/m2 2G m2G/m22G m2G/m2 2G m2 2G m7G/2H-m2,7Gm/(m2 2G∗) m2G/m2 2G 5>> G+ G+ G+ G+ G+ G+ 7 s2C s2C s2C Cm/Am 8 Gm Gm 0 Um Am Um 2 Gm Gm 3 Gm 5 Cm/Um 6>> m2G/m2 2G m2 2G m2 2G/m2 2Gm (m2 2G)∗ m2G/m2 2G/m2 2Gm m2G/m2 2G/m2 2Gm 7 m5C m5C 9 Gm 2 Cm Cm/(s2C)∗ Cm/Um/s2C m5Cm/(s2C∗ s2C Cm 3 s2U 4>37 Anticodon and its 5′ flanking purine-37 (see Table 2) 8 Am 9 Um/m5C s2U Cm m2 2G/Cm Cm 0 m5C ac4C Cm ac4C 2 Gm Cm/ac4C/Gm 4 Um 7 xU Am/m2 2G/xU 8>> m5C (m5C)∗ m5C m5C m5C m5C 9>> m5C (m5C)∗ m5C m5C m5C m5C 0 Gm Um/Cm Gm 4 m1Y m1Y m1Y m5s2U m5U/m5s2U s2U/sxmxU Continued www.rnajournal.org 1961 Cold Spring Harbor Laboratory Press on January 21, 2021 - Published by rnajournal.cshlp.org Downloaded from Cold Spring Harbor Laboratory Press on January 21, 2021 - Published by rnajournal.cshlp.org Downloaded from Cold Spring Harbor Laboratory Press on January 21, 2021 - Published by rnajournal.cshlp.org Downloaded from Wolff et al. Co t ued Halophile Methano Coccales Pyro Coccales Sulfolobales Position Haloferax volcanii Methanococcus maripaludis Methanocaldococcus jannaschii Thermococcus kodakarensis Pyrococcus furiosus Sulfolobus acidocaldarius 55 56>> Cm Cm Cm Cm Cm Cm 57>> m1I m1I m1I m1I m1I m1I 58 m1A m1A m1A m1A m1A 62 Cm 63 ac4C 64 ac4C Um 67 m2G m2G 68 Gm 69 Cm 72 (m5C)∗ (m5C)∗ Um/m5C See Table 2 for positions 34–37 in the anticodon loop. Note that, in the case of T. kodakarensis, the information corresponds to the single tRNA-Trp (Hirata et al. 2019), with the supplemental information in- dicated by an asterisk that should exist in other tRNAs of that archaeon (Krishnamohan et al. 2019 and Supplemental Table S2). New modified positions found in the three Archaea of this study are in bold. Pseudouridines are not indicated, except at position 54 (Y). In the case of methylation, our technique does not always allow to place unambiguously the methyl group (either on the base or on the ribose). New modified positions Table 1, Figure 1, Supplemental Figure S10, and the corre- sponding Supplemental Table S2 list all the modified nu- cleotides we have detected, among them several are present at newly identified positions (mostly methylations and acetylations in the two thermophilic archaea). In eu- karyotic tRNAs, the m2 2G modification is exclusively found at position 26 (exceptionally at position 27) at the interface between the D- and AC-stems (Machnicka et al. 2014), while in archaeal tRNAs, it is present at many other loca- tions. The m2 2G10 is observed when residue 25 is a U, in which case the m2 2G modification stabilizes the G10oU25 wobble pair (discussed in Urbonavicius et al. 2006). Similarly, the 26-44 pair displays a frequent G26 modifica- tion (m2G, m2 2G but also m2 2Gm), forming either G26oU44 or G26–A44 pairs (Supplemental Fig. S11D), rarely G26– C44 (with a slight bias toward m2 2G at position 26 when pairing with U44, especially for the long-arm tRNAs Leu and Ser). Surprisingly, in tRNA-Ser(GGA) of P. furiosus, MS/MS data and T1 cleavage analysis (Supplemental Table S1) indicate the presence of m2 2G at positions 39, which would induce a pronounced propeller-twist of the C31 = m2 2G39 pair (as seen in Supplemental Fig. S11D). A similar situation exists for m2 2G6–C67 in the amino acid stem. Only thermophilic archaea present the triple methyl- ation (m2 2Gm) on both the base and the ribose. Such “doubly” modified nucleosides (xNm) are hallmarks of hy- perthermophilic tRNAs (McCloskey et al. 2001; Grosjean et al. 2008b; Hori et al. 2018). Very recently, Sas-Chen et al. (2020) published a thorough analysis of ac4C modifications in rRNAs and tRNAs across phylogeny including several archaeal spe- cies. They found high concentrations of ac4C in the hyper- thermophiles Thermococcus kodakarensis, P. furiosus, Thermococcus sp. AM4, and S. solfataricus, with a prefer- ence for CCG sites, the modified C being the middle C. Residues ac4C most certainly contribute to the stabiliza- tion of base pairs (Kawai et al. 1992). In this work we iden- tified 13 ac4C modifications in the isolated tRNAs (Table 1; Fig. 1) and all, except the one at the wobble position 34 of tRNA-Gln (ac4CmUG—see below), are found in the middle position of a CCG motif within a stem. For example, in S. acidocaldarius, ac4C is found at position 41 of the AC-stem of tRNA-Leu(UAG) and at position 40 of tRNA- Ala(UGC). Structural tRNA alignments Altogether, these pe- culiarities correspond to tRNAs well stabilized, even in the mesophilic M. maripaludis. at position 49 of T. acidophilum tRNA-Leu (UAG) (Tomi- kawa et al. 2013). Here, we report the presence of m7G at position 10 of tRNA-Meti (CAU) of P. furiosus. We sug- gest also the presence of a reduced neutral form of 2H-m7G at positions 1 and 10 of tRNA-Glu (CUC) (for posi- tion 10 we suggest an unexpected and unique case of tri-methylated 2H-m2 7Gm). These suggestions are based onthefollowing observations.The monomethylatednucle- otide at position 10 of tRNA-Meti has a mass of 359 Da with a neutral loss of 165 Da that is characteristic of a normal m7G, while at position 1 of tRNA-Glu, the methylated nu- cleotide has a mass of 361 Da(Supplemental Fig. S4),which could correspond to the reduced form of m7G (2H-m7G) (Supplemental Fig. S4D; Wintermeyer and Zachau 1975). The same reduced form exists for 2H-m2 7Gm at position 10 of tRNA-Glu. The accuracy is less than 0.05 Da; however, in the absence of standard substance to compare against, these suggestions are still tentative. The presence of an m7G derivative at position 1 of an archaeal tRNA would in- deed be remarkable. Usually, such guanosine derivatives are found in the form of a positively charged cap-like struc- ture protecting the tRNA against 5′-exonucleolytic degra- dation (see for example Ohira and Suzuki 2016). In m7G cap, the 5′ extremity is a 5′OH, while our MS/MS data show a classical 5′P extremity on the P. furiosus tRNA-Glu (see Supplemental Fig. S4B, which corresponds to the MS/MS spectrum of the 5′-end of tRNA-Glu where, in the ion series c, c1 corresponds to p2H-m7G). A putative protecting role of such terminal m7Gp against specific exo- nucleolytic degradation processes remains to be demon- strated in Archaea. Structural tRNA alignments However, knowing the type of chemical modification at equivalent positions in tRNAs of other closely related archaea, it is often possible to guess for putative types of modification. For example, in both S. sulfolobus and T. kodakarensis, A9 was demonstrated to be catalyzed by N1-methylating enzyme Trm10 (Kempenaers et al. 2010; Krishnamohan et al. 2019), an enzyme for which the corresponding gene (PF0678) also exists in P. furiosus (Supplemental Table S2). Likewise, the detected monomethylation of C56 in M. maripaludis, P. furiosus and S. acidocaldarius was interpreted as Cm56, again based on the presence in their genomes of the gene coding for a well characterized almost ubiquitous Trm56 enzyme that is present in all archaea sequenced so far (Supplemental Table S2). All these putative modifica- tions are indicated as (XXX)∗. When uncertain, the methylation is noted as a 2′O-methyl and underlined. Likewise, the modifications not completely identified are underlined. Data for H. volcanii are from Gupta (1984, 1986) and Grosjean et al. (2008a), data for M. jannaschii are from Yu et al. (2019). RNA (2020) Vol. 26, No. 12 1962 Cold Spring Harbor Laboratory Press on January 21, 2021 - Published by rnajournal.cshlp.org Downloaded from Cold Spring Harbor Laboratory Press on January 21, 2021 - Published by rnajournal.cshlp.org Downloaded from Cold Spring Harbor Laboratory Press on January 21, 2021 - Published by rnajournal.cshlp.org Downloaded from Cold Spring Harbor Laboratory Press on January 21, 2021 - Published by rnajournal.cshlp.org Downloaded from Modified nucleotides in tRNAs from three archaea those tRNAs. The T-loop is closed by an invariant G53–C61 pair with always the possibility to form a U54/A58 trans Watson–Crick/Hoogsteen stacking against it. The trans Watson–Crick/Hoogsteen between U8 and A14 is always present and triple formation with A21 is possible in the large majority of sequences. Interestingly, 15–48 is always a G15–C48 pair (Supplemental Fig. S11A) that stacks with the invariant A60 (from the T-loop). In three tRNA sequenc- es (Ile, elongator Met, and Thr) of P. furiosus, residue 60 is G instead of the usual U, and is preceded by A59, which forms a rare combination. C48, as expected, is often methylated on C5 (m5C); in which case, the methyl group would be in the tRNA core and in the neighborhood of the charged for- mamidino group of G15 (G+). The positive charge on G15 points in a cavity surrounded by three negative phosphate groups from residues 7, 14, and 15. Modifications in D-loops In mesophilic bacterial and eukaryotic tRNAs, U17 as well as a few other Us at positions 16, 20, and 20a of the D-loop, are usually modified to dihydrouridine (D). In thermophilic bacteria and hyperthermophilic archaea, this thermolabile dihydrouridine D, is rare or even totally absent (Edmonds et al. 1991), while in psychrophilic bacteria D is abundant (Dalluge et al. 1997). From these observations, Dalluge and coworkers (1977) suggested an interesting functional role for D in the maintenance of a certain degree of confor- mational flexibility in tRNAs, especially important to organ- isms growing at low temperatures where the dynamics of thermal motions of tRNAs are severely compromised. Unexpectedly, in tRNAs of M. maripaludis and of P. furio- sus we found 2-thiocytidine at position 17 (Table 1; Fig. 1), a situation found also in the hyperthermophilic tRNA- Trp of T. kodakarensis (Hirata et al. 2019). However, in S. acidocaldarius, position 17 contains instead a methyl group on either C or A, most probably a 2′-O-methyl (dis- cussed above) (Table 1). Usually, s2C and Cm are found at position 32 of anticodon loops (Jühling et al. 2009; Boccaletto et al. 2018). Thiolation of pyrimidines and methylation of 2′-O-ribose of nucleotides are known to fa- vor stacking, thus limiting local flexibility of the RNA (Plesiewicz et al. 1976; Larsen et al. 2015), a property that is obviously important in organisms thriving at high temperatures. In most crystal structures, residue 17 bulges out of the tRNA core structure and is exposed to solvent. In addition, residues 16 and 17 are in the vicinity of two other bulging residues from the T-loop, 59 and 60, and modifications in the D-loop may limit the tendency of residues 16 or 17 to bulge out of the loop. The locations of residues 16 or 17, either within or outside the tRNA core, may influence inter- actions between other residues in the T-D environment. When residue 16 is a pyrimidine, it is often observed that it forms a pair with residue 59, especially when 59 is also a pyrimidine. However, in the present archaeal tRNAs, res- idue 59 is always A and residue 60 is mostly U. In that case, a couple of crystal structures show A59 stacked on G15– C48 and U60 forming an H-bond between N3(U60) and the phosphate group between the two invariant G18– G19 of the D-loop (see for example PDB entry 2DU3, Supplemental Fig. New modified positions m5C40 is found in tRNA-Ile of H. volcanii (Grosjean et al. 2008a). In position 40, the modification cannot be a ribose methylation (Cm40) because the O2′ (C40) is functionally engaged in H-bonds with the con- served A1339 of the 16S rRNA during the P state of trans- lation (Selmer et al. 2006; Watson et al. 2020). Indeed, the base pairs of the AC-stem 30–40 and 29–41 are both in- volved in contacts with, respectively, G1338 and A1339 of the 16S rRNA in the P state (Supplemental Fig. S12A, In tRNAs of both Bacteria and Eukarya, the positively charged m7G is often found at position 46 in the variable region (Machnicka et al. 2014). m7G has been identified in unfractionated bulk tRNAs of two thermophilic archaea, Thermoproteus neutrophilium and Thermoplasma acido- philium (Edmonds et al. 1991). Recently m7G was located 1963 www.rnajournal.org Cold Spring Harbor Laboratory Press on January 21, 2021 - Published by rnajournal.cshlp.org Downloaded from Cold Spring Harbor Laboratory Press on January 21, 2021 - Published by rnajournal.cshlp.org Downloaded from Cold Spring Harbor Laboratory Press on January 21, 2021 - Published by rnajournal.cshlp.org Downloaded from Wolff et al. B). In tRNA-Meti(CAU) of S. acidocaldarius, residue G29 pairing with C41 is methylated but in this case the 2′-hy- droxyl group is far enough from G1338 and therefore can accommodate a methyl group, leading to a final assign- ment of 2′O-methyl G29 (Gm). In S. acidocaldarius, we found a m5C residue at position 72 in the acceptor stem of tRNA-Asp(GUC), while at the same position in tRNA- Gln(UUG), an undetermined monomethylated U72 (indi- cated as Um) was found. Residue m5C72 has been already reported in S. solfataricus tRNA-Glu/-Gly/-Met (Wagner et al. 2004), as well as in tRNA-Cys and tRNA-Thr of humans (Haag et al. 2015). Residue m5C is quite common at posi- tions 40 (see above), 48, 49 of tRNAs in both Eukarya and Archaea (Machnicka et al. 2014). Worth to note is that, in tRNAs of S. acidocaldarius (and to a lower extent of P. furiosus), many residues are monomethylated all over the molecules. Some are catalyzed by site-specific pro- tein-only methyltransferases while others are catalyzed by Fibrillarin-C/D box sRNP guide machinery acting on specif- ic intron-containing pre-tRNAs (Gaspin et al. 2000; Omer et al. 2000; Clouet d’Orval et al. 2001; Dennis et al. 2001). New modified positions From the database maintained by Todd Lowe (pro- vided at http://lowelab.ucsc.edu/snoRNAdb/), this enzy- matic machinery in S. acidocaldarius could target site- specific methylations to tRNA-Gly(CCC) at C50 in the D- arm (Tang et al. 2005; Zago et al. 2005) and to tRNA-Gln (UUG) at U34 and G18 of the AC- and D-loops, respectively (see also Ziesche et al. 2004), a situation indeed observed in the present study. However, in S. acidocaldarius, a few other observed monomethylated nucleotides remain or- phan and await corresponding snoRNAs or stand-alone specific methyltransferases to be identified (Table 1; Supplemental Table S2, they are indicated as Xm). Likewise, in tRNAs of P. furiosus, several positions were pre- dicted to be methylated via the Fibrillarin-C/D box machin- ery: tRNA-Trp(CCA) at C32, C34, C39, tRNA-Leu(UAA) at G47, tRNA-Leu(CAA) at C34, tRNA-Gln at C16, tRNA-Val and tRNA-Gly at G26 and tRNA-Asp(GUC) at U35 (http ://lowelab.ucsc.edu/snoRNAdb/). However, although we do observe Cm34, Am38, and m2 2Gm26 in some tRNAs, the other putative methylated positions listed above were presently not detected in the set of tRNAs we analyzed. In M maripaludis most modified nucleotides were ex conserved residue, is thiolated (s2U) (Supplemental Fig. S6). An s2U33 has already been found in trypanosomid mi- tochondrial tRNA-Trp(CCA) (Crain et al. 2002), where the stop codon is translated as Trp (Alfonso et al. 1999). The probability to find site-specific 2′O-methylations guided by snoRNA in M. maripaludis, is meager as the frequency of occurrence of potential intron-containing tRNA targets in mesophilic archaea is much lower than in the hyperther- mophilic archaea (Sugahara et al. 2008). Modifications in TΨC-loops Nucleotide U54 is nearly always modified to thymine or 5-methyluridine (m5U) in Eukaryotes and Bacteria (Machnicka et al. 2014). However, in most Archaea (mainly Euryarchaeota), a 1-methylpseudouridine (m1Ψ) is general- ly found instead (Pang et al. 1982; Gupta 1984; McCloskey et al. 2001; Chatterjee et al. 2012; Yu et al. 2019). Such a modification adds a methyl group to the pseudouridine (Ψ) at the free N1 atom, a position structurally equivalent to the C5 atom of T54. In the case of Ignococcus hospitalis, a crenarchaeon thriving at temperatures up to 100°C, m1Ψ54 was shown to be further hypermodified into s4m1Ψ (Rose et al. 2020). The sulfur atom at position 4 in Ψ54 is structurally equivalent to the sulfur atom at position 2 of U54 (see Fig. 6 of Rose et al. 2020). In a few other ar- chaea belonging to the thermococcales clade among the Euryarchaeota, as P. abyssi, P. furiosus, and T. kodakaren- sis, the bacterial-like m5s2U has been identified instead (Kowalak et al. 1994; Urbonavicius et al. 2008; Hirata et al. 2019; for review, see Hori et al. 2018). Residue 56 is a conserved C, usually 2′-O-methylated on its ribose in almost all archaeal tRNAs analyzed so far (Clouet-d’Orval et al. 2005; Renalier et al. 2005). The ri- bose of C56 is highly accessible in the turn of the TΨC- loop and methylation allows protection against hydrolysis, especially at high temperatures. Cm56 forms a Watson– Crick pair with the conserved G19 of the D-loop (G19– C56) and therefore cross-bridges the two parts of the tRNA core. Residue 57 is either G or A where A57 is often doubly modified first into m1A, positively charged, and then into m1I neutral (Grosjean et al. 1995). Residue 57 in- tercalates between the invariant G18 and the conserved G19–C56 pair. With G57, there is an H-bond between N2(G57) or N1(G57) and one anionic oxygen atom of the phosphodiester bond between 18 and 19 and with m1I57 the methyl group will be on the solvent exposed sur- face of the T-loop. It is noteworthy that nucleotide 17 is probably 2′-O-methylated on the ribose in S. acidocaldar- ius and P. furiosus, and 2-thiolated in P. furiosus and M. maripaludis (Fig. 3). The 2′-O-methyl group of Cm56 could protect the bent backbone from cleavage. Modifications in TΨC-loops Both Cm56 and m1I57 are unique and frequent in archaeal tRNAs (Table 1), while m1A58 is also present in many bacterial and eukary- otic tRNAs (Jühling et al. 2009). In this work, we confirm the presence of m1Ψ54 in the mesophilic M. maripaludis, and in the hyperthermophilic P. furiosus a mix of m5U54 and m5s2U54 (Supplemental Figs. S2 and S9). For P. furiosus, the result is consistent with the fact that the 2-thiolation process occurs after 5- methylation of U54 and 1-methylation of A58 (Shigi et al. 2006). For S. acidocaldarius, the MS/MS sequencing spec- tra show the presence of thiolated U/Ψ (Supplemental Fig. S9A) and, only in tRNA-Val, the presence of a methylthio- lated U/Ψ (Supplemental Fig. S9B). In the latter case a neu- tral loss of 142 was observed, which corresponds to a modified U with a methyl group and a sulfur atom, a situa- tion that was not observed in the case of m5s2U54-contain- ing tRNAs of P. furiosus. The total absence of m5s2U is also evident in the chromatogram profile of nucleoside digests from S. acidocaldarius, in comparison to P. furiosus (Supplemental Fig. S2). Altogether, these facts strongly suggest the presence of a methylthiolated derivative like s4m1Ψ as in I. hospitalis (Rose et al. 2020). This situation is however perplexing. Indeed, examination of the S. acid- ocaldarius genome reveals the lack of genes coding for both Pus10 (catalyzing formation of Ψ55 and Ψ54) and TrmY (catalyzing m1Ψ), while genes coding for TtuA and TtuB responsible for thiolation of U/Ψ (into s2U or s4Ψ) are present (Supplemental Table S2). It might be that in S. acidocaldarius a new type of s2U54/or s4Ψ54-methylat- ing enzyme exists. Therefore, we prefer to indicate that po- sition as sxmxU/Ψ54. Modified nucleotides in tRNAs from three archaea 2007). As stated above the modified G bearing a positively charged formamidino group (G+, archaeosine), a hallmark of archaeal tRNAs, also contributes to the global stability of the 3D-core of tRNA. In Bacteria and Eukarya, nucleo- tide 15 is never modified. In all three archaea analyzed, A58 is methylated at posi- tion N1 (m1A58), adding a positive charge on the base while still allowing for the formation of the usual trans Watson–Crick/Hoogsteen 54–58 pair (Supplemental Fig. S11B). In sum, thiolation of m5U, m1Ψ and U/Um at posi- tion 54 of tRNA appears as a hallmark of (hyper)thermo- philic archaea. In thermophilic bacteria, such as Thermus thermophilus, the thiolation process was demonstrated to be thermo-inducible, as the level of 2-thiolation of m5U54 increases with the cultivation temperature (Shigi et al. 2006). The van der Waals radius of the sulfur atom is 0.3 Å larger than that of the oxygen atom and its pres- ence may fill the cavity present around that position in the overall compact T-loop, thereby excluding solvent molecules and promoting stacking. Modifications in D-loops S12C; Fukunaga and Yokoyama In M. maripaludis, most modified nucleotides were ex- pected, except a methylated G22 (Gm) in tRNA-Ala (UGC) and an unidentified adenine derivative in the accep- tor stem of tRNA-Val(GAC). The corresponding nucleoside A∗7 displays a mass of 309.1 Da corresponding to either hypermodified m6 2Am or monomodified N6-acetylA. We tend to favor the latter (ac6A), since such acetylated aden- osine has been discovered in another methanogen (Sauerwald et al. 2005) with its location, however, tenta- tively assigned to residue 37. In M. maripaludis, a mass cor- responding to f6A37 was found instead in tRNA-Asp (GUC) (see below). Intriguingly, in tRNA-Leu(UAG), the C32 is un- modified but U33 of the AC-loop, a very rarely modified RNA (2020) Vol. 26, No. 12 1964 Cold Spring Harbor Laboratory Press on January 21, 2021 - Published by rnajournal.cshlp.org Downloaded from Modified nucleotides in tRNAs from three archaea Modifications at position 34 (Table 2) The majority of the modifications observed at position 34 are the same as those identified in several tRNAs, mostly from Bacteria, such as cmnm5s2U, mnm5U, cnm5U, mchm5U, Cm, Um, ac4C, except that, in the present ther- mophilic archaeal tRNAs, some of them are doubly modi- fied with an extra 2′-O-methylribose, mchm5Um, ac4Cm, s2Um (Grosjean et al. 2008a,b, 2010; Jühling et al. 2009). In tRNA-Meti of M. maripaludis and P. furiosus (recogniz- able by the invariant last three G = C pairs of the AC- stem [Kuchino et al. 1982]) and tRNA-Trp of S. acidocaldar- ius, there is a modified Cm34 (Table 2). The methyl group in that position occupies a tight space in the decoding site locking C34 for pairing only with G3 (Met codon AUG or 1965 www.rnajournal.org Cold Spring Harbor Laboratory Press on January 21, 2021 - Published by rnajournal.cshlp.org Downloaded from Cold Spring Harbor Laboratory Press on January 21, 2021 - Published by rnajournal.cshlp.org Downloaded from Wolff et al. TABLE 2. Modifications at position 34 (Table 2) Interestingly, ac4C residues were also found in the acceptor stems of P. furiosus and S. acidocaldarius, as well as in the AC-stem of S. acidocaldarius (see above and Table 1). Modification of U34 is necessary for decod- ing G3 ending codons. The modification in U34 (U34∗) changes the chemical structure of the U34 so that a pair U34∗–G3, with the U displaced into the minor groove, and not into the major groove, can be stabilized (Rozov et al. 2016; Westhof et al. 2019). Several U34-containing tRNAs were observed modified either at position C5 and/or at position C2. The case of xU34 in tRNA-Leu of M. maripaludis is explained below. ognizing codons CCN (Pro), CGN (Arg), CUG (Leu), that is, in tRNAs decoding in the codon quadrant starting with C1 (Fig. 2; Supplemental Fig. S10). All the other tRNAs, belonging to the three remaining decoding quad- rants, harbor either an unmodified A37 or a modified A37 (m2A, t6A, m6t6A, ms2i6A) with large modifications in the codon quadrants starting with U1 or A1 (Fig. 2; Supplemental Fig. S10). In the halophilic mesophilic H. volcanii, m1G37 occurs in tRNAs decoding codons starting with C1 (G-ending codons) and U1 (A-ending codons, with the exceptions of tRNA-Ser(CGA), tRNA-Ser(GGA) where A38 is found and tRNA-Glu(CUU) with m1G; Gupta 1986). In all the other tRNAs of H. volcanii, A37 or a mod- ified A37 is used (Grosjean et al. 2008a). j In M. jannaschii, P. furiosus, M. maripaludis, and S. acid- ocaldarius, the landscape is striking. Indeed, the tRNAs for the codon quadrants starting with C1 and U1 contain m1G but also imG-14/imG2, a wyosine derivative of m1G37 that is reminiscent of the yW37 found exclusively in tRNA-Phe of Eukaryotes (de Crécy-Lagard et al. 2010). One finds in- deed imG-14 or mimG at position 37 of tRNA-Phe(GAA) in M. maripaludis and P. furiosus with a wyosine-like (xG) (as discussed below), in tRNA-Tyr(GUA) of M. maripaludis and mimG37 in tRNA-Arg(GCG) of P. furiosus (Fig. 2; Supplemental Fig. S10). In S. acidocaldarius, wyosine de- rivatives are present in tRNA-Asp(GUC), tRNA-Ser(UGA), tRNA-Phe(GAA), tRNA-Trp(CCA), and tRNA-Tyr(GUA). This surprising result was noted in the case of M. jannaschii (Yu et al. 2019) where besides tRNA-Phe, the tRNA-Arg (UCG), tRNA-Cys(GCA), tRNA-Leu(UAA), tRNA-Ser(GGA), and tRNA-Tyr(GUA) contain wyosine derivatives (Fig. 2). In T. kodakarensis (Hirata et al. 2019), mimG was found at position 37 of tRNA-Trp. Modifications at position 34 (Table 2) Continued Amino acid Anticodon AC-loop Val GAC [Cm]UGAC[m1G]C Ser UGA CU[mchm5Um]GA[mimG]A This list is not as complete as those in the other figures or tables. Red nucleotides indicate fragments obtained by RNase T1 and/or RNase A digestion, while black nucleotides represent regions that could not be analyzed. Nucleotides in gray are modified nucleotides with a mass corresponding to several chemistries for modifications. Positions 34, 35, and 36 are underlined. Modified nucleotides in tRNAs from three archaea Cold Spring Harbor Laboratory Press on January 21, 2021 - Published by rnajournal.cshlp.org Downloaded from Modified nucleotides in tRNAs from three archaea TABLE 2. Continued Amino acid Anticodon AC-loop Val GAC [Cm]UGAC[m1G]C Ser UGA CU[mchm5Um]GA[mimG]A This list is not as complete as those in the other figures or tables. Red nucleotides indicate fragments obtained by RNase T1 and/or RNase A digestion, while black nucleotides represent regions that could not be analyzed. Nucleotides in gray are modified nucleotides with a mass corresponding to several chemistries for modifications. Positions 34, 35, and 36 are underlined. This list is not as complete as those in the other figures or tables. Red nucleotides indicate fragments obtained by RNase T1 and/or RNase A digestion, while black nucleotides represent regions that could not be analyzed. Nucleotides in gray are modified nucleotides with a mass corresponding to several chemistries for modifications. Positions 34, 35, and 36 are underlined. Trp codon UGG) and preventing mispairing of the C34 res- idue with A3 (Ile codon AUA or stop codon UGA). The Cm34 modified nucleotide found in tRNA-Trp in S. acido- caldarius was previously observed in H. volcanii (Gupta 1984) and in T. kodakarensis (Hirata et al. 2019). In tRNA- Ile, the anticodon CAU has to read exclusively the Ile co- don AUA and the C34-tRNA is modified into agmatidine (C+) (Ikeuchi et al. 2010; Mandal et al. 2010). The corre- sponding mass spectrum for tRNA-Ile from M. maripaludis is shown on Supplemental Figure S8. In tRNA-Gln(CUG) of P. furiosus, the modified residue ac4Cm34, the same as in the homolog tRNA-Gln of H. volcanii, was found (Gupta 1984). NMR studies have shown that ac4Cm is exceptional- ly rigid in conformation owing to the additive nature of the acetylation and methylation modifications which stabilize the 3′-endo sugar conformation (Kawai et al. 1992). The same remark probably applies to Um34 and s2Um34 that occur in tRNA-Gln(UUG) and tRNA-Leu(UAG) of S. acido- caldarius. Modifications at position 34 (Table 2) Obviously, the presence of wyosine derivatives is more prevalent in Archaea than in Eukarya, especially in the U1-quadrant. Modifications at position 34 (Table 2) List of the archaeal tRNAs for which we could fully characterize the tRNA anticodon regions Amino acid Anticodon AC-loop Methanococcus maripaludis 32–38 Asn GUU CUGUU[hn6A]A Asp GUC CUGUC[f6A]C Glu UUC CU[mnm5U]UC[m1G]A Gly UUC CU[cnm5U]CCAA Ile GAU CUGAU[hn6A]A Ile CAU CU[C+]AU[hn6A]A Lni CAU CUCAUAA Leu UAG C[s2U][xU]AGAA Lys UUU CU[cmnm5s2U]UU[t6A]A Met CAU CU[Cm]AU[hn6A]A Phe GAA [Cm]UGAA[imG-14]A Tyr GUA [Cm]UGUA[xG]A Pyrococcus furiosus 32–38 Ala GGC UUGGCAA Ala UGC CU[cnm5U]GCAA Arg CCU [s2C]UCCU[t6A])A Arg UCU CU[cnm5U]CUAA Gln UUG CU[cnm5U]UGGA Gln CUG CU[ac4Cm]UGGA Glu CUC CUCUC[m1G]A Gly CCC CUCCCAA Gly GCC CUGCCAA Gly UCC CU[cnm5U]CC[m1G]A Ile GAU CUGAU[hn6A]A Lni CAU CUCAUAA Leu CAG UUCAGGG Leu UAG UU[cnm5U]AGGG Lys CUU CUCUU[ms2t6A]A Met CAU CU[Cm]AU[hn6A]A Phe GAA CUGAA[mimG]A/[Am] Pro UGG UU[cnm5U]GG[m1G]GA Thr GGU UUGGU[t6A]/[hn6A]A Thr UGU CU[cnm5U]GU[hn6A]A Ser GCU CUGCU[t6A]A Val GAC CUGACAA Sulfolobus acidocaldarius 32–38 Ala UGC [Cm]U[Um]GCAA Asp GUC CUGUC[m1G]/[imG2]/[mimG]A Gln UUG CU[Um]/[s2Um]UG[m1G]C Glu CUC [Cm]UCUCAA Gly GCC CUGCCAC Leu CAG [Cm]U[Cm]AG[m1G]C L UAG [C ]U[ 2U ]AG[ 1G]C Methanococcus maripaludis Asn GUU Asp GUC Glu UUC Gly UUC Ile GAU Ile CAU Lni CAU Leu UAG Lys UUU Met CAU Phe GAA Tyr GUA Pyrococcus furiosus Ala GGC Ala UGC Arg CCU Arg UCU Gln UUG Gln CUG Glu CUC Gly CCC Gly GCC Gly UCC Ile GAU Lni CAU Leu CAG Leu UAG Lys CUU Met CAU Phe GAA Pro UGG Thr GGU Thr UGU Ser GCU Val GAC Sulfolobus acidocaldarius Ala UGC Asp GUC Gln UUG Glu CUC Gly GCC Leu CAG Leu UAG Lni CAU Trp CCA Phe GAA Tyr GUA Continued RNA (2020) Vol. 26, No. 12 1966 TABLE 2. Continued Amino acid Anticodon AC-loop Val GAC [Cm]UGAC[m1G]C Ser UGA CU[mchm5Um]GA[mimG]A This list is not as complete as those in the other figures or tables. Red nucleotides indicate fragments obtained by RNase T1 and/or RNase A digestion, while black nucleotides represent regions that could not be analyzed. Nucleotides in gray are modified nucleotides with a mass corresponding to several chemistries for modifications. Positions 34, 35, and 36 are underlined. Modified nucleotides in tRNAs from three archaea Cold Spring Harbor Laboratory Press on January 21, 2021 - Published by rnajournal.cshlp.org Downloaded from Cold Spring Harbor Laboratory Press on January 21, 2021 - Published by rnajournal.cshlp.org Downloaded from Cold Spring Harbor Laboratory Press on January 21, 2021 - Published by rnajournal.cshlp.org ownloaded from TABLE 2. Wyosine and modifications at position 37 The modifications observed frequently have a large font and are bold, while those observed only once or in a single tRNA species have a smaller font and are not bold. in M. maripaludis, a mass corresponding to ac6A, a modified nucleotide in the same biochemical pathway as f6A, was ob- served for residue 7 of the acceptor stem. The homologous tRNA in M. jannaschii harbors an unmodified A. There is also one report of the presence of m6 2A in Mycobacterium bovis, butwithoutidentification ofeither tRNA speciesortRNApo- sition (Chan et al. 2011). Also, in E. coli, tRNA-Val(UAC) con- tains m6A37 (Golovina et al. 2009). decoding on the ribosome, while for R1-Y36 another type of stacked hypermodified A37 is favored. In addition to residue 37, other elements of the extended anticodon stem–loop, including generally simpler chemical modifica- tions on the base and/or the ribose also contribute to the global efficiency and accuracy of the translation process, whatever the temperature at which the archaeon is grow- ing. These rules are more restrictive than those observed in other organisms like E. coli, S. cerevisiae or H. volcanii (Fig. 2; see Grosjean and Westhof 2016). Thus, as a rule, in Archaea, tRNAs decoding the codon quadrant starting with C1 and U1 harbor either unmodified A37 or mostly modified G37. The G1-quadrant has a pref- erence for unmodified A or slightly modified A with some occurrences of modified G37. While tRNAs decoding the codon quadrant starting with A1 seem to harbor mostly hypermodified A37. In short, the stacking power of a G37 derivative (modified or not) on the first codon–antico- don base pair (Y1-R36) is obviously preferred for efficient Wyosine and modifications at position 37 Residue 37 is commonly a purine, often (hyper)modified, that stacks on the first 1–36 bp formed between the codon and the anticodon during translation on the ribosome. Residue 37 should not be 2′-O-methylated because it forms an H-bond with N6(A1913) of helix H69 from the large subunit in the A state in known crystal structures of ribosomes (Supplemental Fig. S12D; Selmer et al. 2006). The type of modification at base 37 usually correlates with the rest of the so-called extended anticodon stem– loop, especially with the adjacent nucleotide 36 of the an- ticodon (Yarus 1982; Grosjean and Westhof 2016). In E. coli, for example, m1G exclusively occurs in tRNAs rec- The tRNAs corresponding to the codon quadrants start- ing with A1 still prefer large modifications on A37 (t6A, ms2t6A, hn6A), while the G1-quadrant prefers A and in a few isolated cases also m1G (Fig. 2; Supplemental Fig. S10). In the case of tRNA-Asp of M. maripaludis, a modified A37, which hasthesame nucleosidemass (295.1) than either di-methyl-A (m6 2A) or N6-formyl-A (f6A) was found. The lat- ter f6A derivative is the most probable modification. The modified f6A derivative has been identified in mammalian mRNAs (Fu et al. 2013). Interestingly, as discussed below, 1967 www.rnajournal.org Cold Spring Harbor Laboratory Press on January 21, 2021 - Published by rnajournal.cshlp.org Downloaded from Wolff et al. FIGURE 2. Patterns of distribution of modified nucleotides at position 37 in E. coli, S. cerevisiae and five archaeal species on the wheels of the genetic code (Grosjean and Westhof 2016). In that representation, GC-rich codons are at the top and AU-rich at the bottom of the wheel. The codon positions are numbered 1, 2, 3 so that G1A2C3 gives an Asp codon. The four quadrants are characterized by the first codon position. Data for E. coli and S. cerevisiae are from MODOMICS (Boccaletto et al. 2018). Data for H. volcanii are from Gupta (1984, 1986) and Grosjean et al. (2008a), data for M. jannaschii are from Yu et al. (2019). The wheels for E. coli, S. cerevisiae, and H. volcanii are adapted from Grosjean and Westhof (2016). In each quadrant, the observed modified nucleotides at position 37 are shown (red for G derivatives and black for A deriv- atives; the very unusual U37 of tRNA-Ala(GGC) of M. jannaschii is also shown in black). Wyosine and modifications at position 37 The modifications observed frequently have a large font and are bold, while those observed only once or in a single tRNA species have a smaller font and are not bold. FIGURE 2. Patterns of distribution of modified nucleotides at position 37 in E. coli, S. cerevisiae and five archaeal species on the wheels of the genetic code (Grosjean and Westhof 2016). In that representation, GC-rich codons are at the top and AU-rich at the bottom of the wheel. The codon positions are numbered 1, 2, 3 so that G1A2C3 gives an Asp codon. The four quadrants are characterized by the first codon position. Data for E. coli and S. cerevisiae are from MODOMICS (Boccaletto et al. 2018). Data for H. volcanii are from Gupta (1984, 1986) and Grosjean et al. (2008a), data for M. jannaschii are from Yu et al. (2019). The wheels for E. coli, S. cerevisiae, and H. volcanii are adapted from Grosjean and Westhof (2016). In each quadrant, the observed modified nucleotides at position 37 are shown (red for G derivatives and black for A deriv- atives; the very unusual U37 of tRNA-Ala(GGC) of M. jannaschii is also shown in black). The modifications observed frequently have a large font and are bold, while those observed only once or in a single tRNA species have a smaller font and are not bold. FIGURE 2. Patterns of distribution of modified nucleotides at position 37 in E. coli, S. cerevisiae and five archaeal species on the wheels of the genetic code (Grosjean and Westhof 2016). In that representation, GC-rich codons are at the top and AU-rich at the bottom of the wheel. The codon positions are numbered 1, 2, 3 so that G1A2C3 gives an Asp codon. The four quadrants are characterized by the first codon position. Data for E. coli and S. cerevisiae are from MODOMICS (Boccaletto et al. 2018). Data for H. volcanii are from Gupta (1984, 1986) and Grosjean et al. (2008a), data for M. jannaschii are from Yu et al. (2019). The wheels for E. coli, S. cerevisiae, and H. volcanii are adapted from Grosjean and Westhof (2016). In each quadrant, the observed modified nucleotides at position 37 are shown (red for G derivatives and black for A deriv- atives; the very unusual U37 of tRNA-Ala(GGC) of M. jannaschii is also shown in black). Tentative identification of three novel modified nucleosides maripaludis tRNA-Tyr, on the basis of previous work, one would therefore expect to find either imG, yW-86 or yW-72 (see G37 pathway #5 in Fig. 4 of de Crécy-Lagard et al. 2010). Taking into account that CID fragmentation of xG37 occurs between guanine and the modification, the MS/MS spectrum shows the complete mass of guano- sine (345 Da) and a neutral loss of 109 Da representing the mass of the adduct (Supplemental Fig. S5). The nucleoside mass of xG (392 Da) could therefore correspond to yW-72 (436.17 Da) with the loss (natural or accidental) of the car- boxyl group (44.17 Da). other archaeal tRNAs (Mandal et al. 2014). We propose that xU34 in M. maripaludis is the simpler 5-cyano-U (cn5U), with the cyano group directly linked to the C5 atom of uracil. Such a derivative is known from organic chemistry (Mao et al. 2018) but was never identified in tRNA so far. A third unidentified modification, with a nucleoside mass of 338 Da, was found at position U47 of two tRNAs of S. acidocaldarius, tRNA-Val(GAC) (Supplemental Fig. S7A) and tRNA-Gly(GCC) (Supplemental Fig. S7B), two tRNAs of S. acidocaldarius, tRNA-Met elongator(CAU) (Supplemental Fig. S7C) and tRNA-Thr(UGU) of P. furiosus. In Bacteria and Eukarya, 3-(3-amino-3-carboxypropyl)-uri- dine (acp3U, nucleoside mass 345.1 Da) is widely con- served in the D- and variable loops (Takakura et al. 2019). It is likely that in hyperthermophilic archaea U47 is modified differently. The second novel modification is xU at position 34 of tRNA-Leu(xUAG) of M. maripaludis with a nucleoside mass of 269 Da. Again, in several tRNAs of M. jannaschii (Yu et al. 2019), either 5-cyanomethyl-U (cnm5U) or 2-thio- lated-5-cyanomethyl-U (cnm5s2U) have been found, as in Tentative identification of three novel modified nucleosides The sequence analysis of each tRNA allowed the detection of three possibly novel chemical modifications (designated xG and xU). The first one was found at position G37 of 1968 RNA (2020) Vol. 26, No. 12 Cold Spring Harbor Laboratory Press on January 21, 2021 - Published by rnajournal.cshlp.org Downloaded from Modified nucleotides in tRNAs from three archaea FIGURE 2. Continued. FIGURE 2. Continued. tRNA-Tyr(GUA) of M. maripaludis (Supplemental Fig. S5) with a nucleoside mass of 392 Da, much higher than for the expected m1G37. In the homolog tRNA-Tyr of M. jan- naschii, G37 is unexpectedly imG-14 of the wyosine me- tabolism (Yu et al. 2019). If this is also the case for M. maripaludis tRNA-Tyr, on the basis of previous work, one would therefore expect to find either imG, yW-86 or yW-72 (see G37 pathway #5 in Fig. 4 of de Crécy-Lagard et al. 2010). Taking into account that CID fragmentation of xG37 occurs between guanine and the modification, the MS/MS spectrum shows the complete mass of guano- sine (345 Da) and a neutral loss of 109 Da representing the mass of the adduct (Supplemental Fig. S5). The nucleoside mass of xG (392 Da) could therefore correspond to yW-72 (436.17 Da) with the loss (natural or accidental) of the car- boxyl group (44.17 Da). other archaeal tRNAs (Mandal et al. 2014). We propose that xU34 in M. maripaludis is the simpler 5-cyano-U (cn5U), with the cyano group directly linked to the C5 atom of uracil. Such a derivative is known from organic chemistry (Mao et al. 2018) but was never identified in tRNA so far. A third unidentified modification, with a nucleoside mass of 338 Da, was found at position U47 of two tRNAs of S. acidocaldarius, tRNA-Val(GAC) (Supplemental Fig. S7A) and tRNA-Gly(GCC) (Supplemental Fig. S7B), two tRNAs of S. acidocaldarius, tRNA-Met elongator(CAU) (Supplemental Fig. S7C) and tRNA-Thr(UGU) of P. furiosus. In Bacteria and Eukarya, 3-(3-amino-3-carboxypropyl)-uri- dine (acp3U, nucleoside mass 345.1 Da) is widely con- served in the D- and variable loops (Takakura et al. 2019). It is likely that in hyperthermophilic archaea U47 is modified differently. tRNA-Tyr(GUA) of M. maripaludis (Supplemental Fig. S5) with a nucleoside mass of 392 Da, much higher than for the expected m1G37. In the homolog tRNA-Tyr of M. jan- naschii, G37 is unexpectedly imG-14 of the wyosine me- tabolism (Yu et al. 2019). If this is also the case for M. DISCUSSION Supplemental Table S2 lists all the archaeal tRNA modification enzymes (and their corresponding cod- ing genes) that have been experimentally validated so far in independent works or deduced from their close similar- ities with genuine modification enzymes in each of the Methanococcales, Pyrococcales and Sulfolobales groups of Archaea. Altogether, 79 naturally occurring fully ma- tured isoacceptor tRNAs coding for 20 amino acids have been analyzed out of a theoretical total set of 116 species. Thus, the possibility still exists that a few modifications that are specific to the missing tRNAs (especially in the antico- don loop including the wobble position 34) still escaped our analysis. Fibrillarin-C/D box sRNP guide machinery; (iv) depending on the tRNA species, nucleotides at positions 32, 38, and 39 of the extended anticodon loop are frequently but di- versely modified; (v) beyond those found at positions 34 and 37 in the anticodon loop, a few characteristic modifica- tions are found in the body of most if not all isoacceptor species of the three archaea (G+15 in the D-loop, m2G/ m2 2G at positions 10 and 26 at the beginning and the end of the D-arm, m5C at positions 48/49 of the variable loop, Cm56, m1I57 and m1A58 in the TΨC-loop), some of them are hallmarks of archaeal tRNAs; (vi) except for ac4C at the wobble position 34, this modified residue is present in the amino-acceptor and anticodon stems of only thermophilic archaeal tRNAs; (vii) at variance with the situation in bacterial and eukaryal tRNAs, m2G/m2 2G are found at positions other than 10 and 26; (viii) remark- ably, the chemical adducts on the conserved U54 of the T-loop depends on archaeon analyzed (m1Ψ, m5U/ m5s2U, or s4m1Ψ); (ix) the suggested presence of m7G modifications at the nucleotides 1 and 10 in P. furiosus would also be remarkable. Nevertheless, the major general conclusions that came out from this comparative analysis of modified nucleotide patterns in three very different archaeal species are the fol- lowing: (i) the least diversified chemical modification pat- tern is observed in the mesophilic M. maripaludis; (ii) a larger diversity of modifications is found in the two hyper- thermophiles; (iii) the largest amount of 2′-hydroxyl ribose methylations occurs in the acidophilic hyperthermophile S. DISCUSSION The different modifications observed are reported on a typical cloverleaf two-dimensional structure. Underlined nucleotides are modified nucleotides with a mass corre- sponding to several possible chemistries for modifications, most of them corresponding to simple monomethylation products (see legend to Table 1). A “/” between two notations means a mixture of modifications. An “x” before a nucleotide, for example, xG, or after a modification symbol, for example, mx, means that the modification has not been formally identified (see text). Modified nucleotides indicated in brackets fol- lowed by an asterisk correspond to modifications deduced from the convergence of information from both the analysis of total tRNA and the presence of the gene coding for the corresponding modification enzyme in the genome of the particular archaeon. The modifications, uncertain or not completely identified, are underlined. Pseudouridines are not represented, except in the case of N1-methylpseudouridine (m1Y) because of the methyl group. FIGURE 3. Nucleoside modification patterns in archaeal tRNAs determined by experimental MS/MS sequencing. The different modifications observed are reported on a typical cloverleaf two-dimensional structure. Underlined nucleotides are modified nucleotides with a mass corre- sponding to several possible chemistries for modifications, most of them corresponding to simple monomethylation products (see legend to Table 1). A “/” between two notations means a mixture of modifications. An “x” before a nucleotide, for example, xG, or after a modification symbol, for example, mx, means that the modification has not been formally identified (see text). Modified nucleotides indicated in brackets fol- lowed by an asterisk correspond to modifications deduced from the convergence of information from both the analysis of total tRNA and the presence of the gene coding for the corresponding modification enzyme in the genome of the particular archaeon. The modifications, uncertain or not completely identified, are underlined. Pseudouridines are not represented, except in the case of N1-methylpseudouridine (m1Y) because of the methyl group. Supplemental Figure S10 align the intron-less tRNA sequences as deduced from the genomes on which the modified nucleotides are indicated; Figure 3 summarizes the data in cloverleaf representations; Supplemental Figure S3 displays the relative amounts of modified nucle- osides identified in bulk tRNAs; Supplemental Tables S2 and S3 list the oligonucleotide fragments obtained after RNase digests. DISCUSSION All the tRNA modifications identified in this work are com- piled in several figures and tables. Figure 1 and 1969 www.rnajournal.org www.rnajournal.org Cold Spring Harbor Laboratory Press on January 21, 2021 - Published by rnajournal.cshlp.org Downloaded from Cold Spring Harbor Laboratory Press on January 21, 2021 - Published by rnajournal.cshlp.org Downloaded from Wolff et al. FIGURE 3. Nucleoside modification patterns in archaeal tRNAs determined by experimental MS/MS sequencing. The different modifications observed are reported on a typical cloverleaf two-dimensional structure. Underlined nucleotides are modified nucleotides with a mass corre- sponding to several possible chemistries for modifications, most of them corresponding to simple monomethylation products (see legend to Table 1). A “/” between two notations means a mixture of modifications. An “x” before a nucleotide, for example, xG, or after a modification symbol, for example, mx, means that the modification has not been formally identified (see text). Modified nucleotides indicated in brackets fol- lowed by an asterisk correspond to modifications deduced from the convergence of information from both the analysis of total tRNA and the presence of the gene coding for the corresponding modification enzyme in the genome of the particular archaeon. The modifications, uncertain or not completely identified, are underlined. Pseudouridines are not represented, except in the case of N1-methylpseudouridine (m1Y) because of the methyl group. FIGURE 3. Nucleoside modification patterns in archaeal tRNAs determined by experimental MS/MS sequencing. The different modifications observed are reported on a typical cloverleaf two-dimensional structure. Underlined nucleotides are modified nucleotides with a mass corre- sponding to several possible chemistries for modifications, most of them corresponding to simple monomethylation products (see legend to Table 1). A “/” between two notations means a mixture of modifications. An “x” before a nucleotide, for example, xG, or after a modification symbol, for example, mx, means that the modification has not been formally identified (see text). Modified nucleotides indicated in brackets fol- lowed by an asterisk correspond to modifications deduced from the convergence of information from both the analysis of total tRNA and the presence of the gene coding for the corresponding modification enzyme in the genome of the particular archaeon. The modifications, uncertain or not completely identified, are underlined. Pseudouridines are not represented, except in the case of N1-methylpseudouridine (m1Y) because of the methyl group. FIGURE 3. Nucleoside modification patterns in archaeal tRNAs determined by experimental MS/MS sequencing. DISCUSSION acidocaldarius, most of them appear being catalyzed by All these observations complete and reinforce similar conclusions made by others about the importance of certain post-transcriptional modifications for correct tRNA folding and on final cellular stability of the already G/C-rich tRNAs in thermophiles (Edmonds et al. 1991; Kowalak et al. 1994; McCloskey et al. 2001; Noon et al. 2003; for reviews, see Machnicka et al. 2014; Lorenz et al. 2017; Hori et al. 2018). As a rule, methylations RNA (2020) Vol. 26, No. 12 1970 Cold Spring Harbor Laboratory Press on January 21, 2021 - Published by rnajournal.cshlp.org Downloaded from Cold Spring Harbor Laboratory Press on January 21, 2021 - Published by rnajournal.cshlp.org Downloaded from Cold Spring Harbor Laboratory Press on January 21, 2021 - Published by rnajournal.cshlp.org Downloaded from Cold Spring Harbor Laboratory Press on January 21, 2021 - Published by rnajournal.cshlp.org Downloaded from Modified nucleotides in tRNAs from three archaea promote precise H-bonded pairs (e.g., m1A favors Hoogsteen pairs or m2 2G favors GoU or GoΨ pairs) and electrostatic charges introduced by the chemical adducts are localized in shielded pockets of the tRNA fold (m1A, m7G, archaeosine G+). Modifications in the tRNA core, al- though not close in sequence, tend to form clusters of modifications filling empty space on the surface of the compact tRNA core. In addition, methylations on the base and/or the ribose affect the hydration shells in com- plex ways (Auffinger and Westhof 2001). Thiolation of U or C, acetylation of C, and isomerization of U to Ψ, can stabilize the 3′-endo sugar conformation, fill in space and enhance stacking power or base-pairing (Kawai et al. 1992; Davis 1995; Larsen et al. 2015; Sas-Chen et al. 2020). High-resolution crystallographic structures would be necessary to apprehend the effects of such complex modification scaffolds. column (130 Å, 1.7 µm, 75 µm × 200 mm) using a nanoAcquity sys- tem (Waters). The column was equilibrated in buffer A containing 7.5 mM TEAA (Triethylammonium acetate), 7.0 mM TEA (Triethylammonium) and 200 mM HFIP (Hexafluoroisopropanol) at a flow rate of 300 nL/min. Oligonucleotides were eluted using a gradient from 15% to 35% of buffer B (100% methanol) for 2 min followed by elution with an increase of buffer B to 50% in 20 min. MS and MS/MS analyses were performed using SYNAPT G2-S (quadrupole time-of-flight mass spectrometer) from Waters Corporation. Culture and tRNA isolation Total tRNA of M. maripaludis and P. furiosus were prepared as de- scribed in de Crécy-Lagard et al. (2010). Total tRNA of S. acidocal- darius was obtained from a 12 L culture using the procedure described in Buck et al. (1983). Data analysis by MassSpec-Toolkit for RNAs tRNA isoacceptors were isolated using two-dimensional gel elec- trophoresis as previously described (Antoine et al. 2019; Antoine and Wolff 2020). Briefly, the total tRNA of each organism was sep- arated in a first dimension gel under denaturing conditions using 12.5% polyacrylamide gel and 8 M urea, followed by a second di- mension under semi-denaturing conditions using 20% polyacryl- amide gel and 4 M urea at room temperature (Supplemental Fig. S1). Gel are staining with an ethidium bromide solution (10 µg.l–1) for 10 min. Spots containing tRNAs are visualized and excised un- der UV light (302 nm). The identification and characterization of modified tRNAs by LC- MS/MS spectrum analysis is difficult and time-consuming. To help in this process, we implemented MassSpec-Toolkit for RNAs, a Python web application (http://labex-ibmc.u-strasbg.fr/ MassSpec-Toolkit/, accessible upon request) linked to a local MongoDB database that stores user-provided RNA genomic se- quences and their theoretical digestion products obtained by specific ribonucleases. For each studied species, mature tRNA se- quences retrieved from GtRNAdb (Chan and Lowe 2009, 2016) were submitted to the application and digested in silico with RNases T1, A and/or U2, in the “RNA Digestion” module. Genomic tRNA sequences longer than 100 nt were discarded and for the remaining ones with a nonambiguous anticodon po- sition, fragments with common U34 or G34 and/or A37 or G37 modifications were generated when appropriate. In addition, fragment variants containing up to five additional methylations were also computed for each digestion product. Experimental data such as parent ion masses or manually reconstructed subse- quences could then be compared to the theoretical ones in the “RNA Search” module. Additional criteria, like the species of in- terest, the ribonuclease used, as well as the presence of expected methylations or modifications at specific positions, can be speci- fied to reduce the search space in the database. Candidate tRNAs are given a score comprised between 0 and 1 depending on the number of matching masses or subsequences they present with the list provided by the user. Besides these two main modules, the application gathers a set of “Additional Tools” under a third module that includes some of the tools present in the Mongo Data analysis All CID were deconvoluted using MassLynx software from Waters and manually sequenced by following the y and/or c series (w ions were also useful when sequencing was difficult or in order to con- firm the sequence). Experimental masses of parents and frag- ments were compared to the theoretical masses obtained by the Mongo Oligo Mass Calculator (https://mods.rna.albany.edu/ masspec/Mongo-Oligo; Cantara et al. 2011). tRNA identification was done by comparisons with the genomic sequences obtained from GtRNAdb (http://gtrnadb.ucsc.edu/; Chan and Lowe 2009, 2016). Data about nucleoside modification were obtained from Modomics (Boccaletto et al. 2018). DISCUSSION All experiments were performed in negative mode with a capillary voltage set at 2.6 kV and a sample cone voltage set at 30 V. Source was heated to 130°C. The samples were ana- lyzed overan m/z range from 500 to 1500 for the full scan, followed by fast data direct acquisition scan (Fast DDA). In-gel RNase digestion Gel spots containing tRNAs were dried and rehydrated by 20 µL of 0.1 U/µL of RNase T1 (ThermoFisher Scientific) or by 20 µL of 0.01 U/µL of RNase A (Thermo Fisher Scientific) in 100 mM ammo- nium acetate (pH is not adjusted). For a few selected samples, spots were digested by RNase U2, by using 50 µL of RNase U2 (homemade) at 0.3 ng.µL−1 in 100 mM ammonium acetate (pH is not adjusted). The spots were incubated 4 h at 50°C. Using ZipTip C18 (Milllipore) samples were desalted by several washes with 200 mM ammonium acetate and eluted with 50% acetonitrile in milliQ water and dried under vacuum. LC-MS/MS of nucleosides Antoine L, Wolff P, Westhof E, Romby P, Marzi S. 2019. Mapping post- transcriptional modifications in Staphylococcus aureus tRNAs by nanoLC/MSMS. Biochimie 164: 60–69. doi:10.1016/j.biochi .2019.07.003 Antoine L, Wolff P, Westhof E, Romby P, Marzi S. 2019. Mapping post- transcriptional modifications in Staphylococcus aureus tRNAs by Total tRNA was desalted by ethanolic precipitation with 200 mM ammonium acetate (Supplemental Figs. S2, S3). For nucleoside analysis, tRNAs are diluted to a concentration of 5 µg/µL in H2O. Digestion was carried out in the following order: 14 µL H2O; 2 µL buffer P1 10× (2 mM ZnCl2, 250 mM NH4OAc, pH 5.0); 21 µL of tRNA and 2 µL of P1 (0.5 U/µL). The mixture is incu- bated at 37°C for 2 h followed by addition of 2 µL of snake venom phosphodiesterase (0.1 U/µL) for 4 h at 37°C. After digestion, 20 µL of BAP (1.5 U/µL in 100 mM NH4OAc) were added to the mix- ture. The latter was then incubated at 37°C for 2 h, dried under vacuum SpeedVac and resuspended with 100 µl of methanol. Nucleosides were analyzed by liquid chromatography coupled to mass spectrometry using an Ultimate 3000 (Thermo) chroma- tography coupled to an EvoQ triple quadrupole (Bruker). Separation was performed on an Acquity UPLC HSST3 column (1.8 µm, 2.1 × 100 mm, Waters) equipped with an Acquity UPLC HSST3 precolumn (1.8 µm, 2.1 × 5 mm, Waters). A gradient of sol- vent A (H2O, 0.1% formic acid [Sigma Aldrich]) and solvent B (methanol [Fisher Chemicals], 0.1% formic acid [Sigma Aldrich]) was used as follows: 2% B during 2 min, 7% B at 4 min, 100% B at 12 min, hold during 1.5 min and back to 2% of B at 13.5 min, hold during 1.5 min for a total run time of 15 min. The column was operated at 35°C with a flow rate of 0.32 mL/min; 10 µL of samples were injected for each run. The triple quadrupole was used in positive ion mode, the spray voltage was set at 3500 V and cone temperature at 350°C. Nucleosides were identified us- ing multiple reaction monitoring (MRM) with one to three transi- nanoLC/MSMS. Biochimie 164: 60–69. doi:10.1016/j.biochi .2019.07.003 Auffinger P, Westhof E. 2001. Hydrophobic groups stabilize the hydra- tion shell of 2′-O-methylated RNA duplexes. Angew Chem Int Ed Engl 40: 4648–4650. NanoLC-MS/MS of RNA oligonucleotides Pellet containing RNase digestion products is resuspended in 3 µL of milliQ water and separated on an Acquity peptide BEH C18 1971 www.rnajournal.org Cold Spring Harbor Laboratory Press on January 21, 2021 - Published by rnajournal.cshlp.org Downloaded from Cold Spring Harbor Laboratory Press on January 21, 2021 - Published by rnajournal.cshlp.org Downloaded from Cold Spring Harbor Laboratory Press on January 21, 2021 - Published by rnajournal.cshlp.org Downloaded from Cold Spring Harbor Laboratory Press on January 21, 2021 - Published by rnajournal.cshlp.org Downloaded from Wolff et al. Oligo Mass Calculator (Cantara et al. 2011) and “Total Mass Decipherer.” The latter program can be very useful to identify modifications in case of incomplete MS/MS series, since it com- putes all combinations of a chosen set of (modified) nucleotides matching the mass of an RNA fragment obtained after cleavage by RNase T1. Oligo Mass Calculator (Cantara et al. 2011) and “Total Mass Decipherer.” The latter program can be very useful to identify modifications in case of incomplete MS/MS series, since it com- putes all combinations of a chosen set of (modified) nucleotides matching the mass of an RNA fragment obtained after cleavage by RNase T1. tions per nucleotide. The identifications were based on the retention time, m/z of the parent ion and m/z of the daughter ions in MS Data Review software (Bruker), with a signal-to-noise (S/N) ratio set at 10 and a search window of ±0.2 min. REFERENCES Alfonzo JD, Blanc V, Estevez AM, Rubio MA, Simpson L. 1999. 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https://openalex.org/W2336048935	https://link.springer.com/content/pdf/10.1007%2Fs10877-016-9876-y.pdf	English	null	Factors related to monitoring during admission of acute patients	Journal of clinical monitoring and computing	2,016	cc-by	6,107	Thomas Schmidt1 • Camilla N. Bech2 • Mikkel Brabrand2,3 • Uffe Kock Wiil1 • Annmarie Lassen2 Received: 17 December 2015 / Accepted: 5 April 2016 / Published online: 12 April 2016 The Author(s) 2016. This article is published with open access at Springerlink.com Abstract Understanding the use of patient monitoring systems in emergency and acute facilities may help to identify reasons for failure to identify risk patients in these settings. Hence, we investigate factors related to the uti- lization of automated monitoring for patients admitted to an acute admission unit by introducing monitor load as the proportion between monitored time and length of stay. A cohort study of patients admitted and registered to patient monitors in the period from 10/10/2013 to 1/10/2014 at the acute admission unit of Odense University Hospital in Denmark. Admissions with at least one measurement were analyzed using quantile regression by looking at the impact of distance from nursing ofﬁce, number of concurrent patients, wing type (medical/surgical), age, sex, comorbidities, and severity conditioned on how much patients were monitored during their admissions. We reg- istered 11,848 admissions, of which we were able to link patient monitor readings to 3149 (26.6 %) with 50 % being monitored \1.4 % of total admission time. Distance from nursing ofﬁce had little inﬂuence on patients monitored \10 % of their admission time. But for other patients, being positioned further away from the ofﬁce reduced the level of monitoring. Higher levels of severity were related to higher degrees of monitoring, but being admitted to the surgical wing reduce how much patients were monitored, and periods with many concurrent patients lead to a small increase in monitoring. We found a signiﬁcant variation concerning how much patients were monitored during admission to an acute admission unit. Our results point to potential patient safety improvements in clinical proce- dures, and advocate an awareness of how patient moni- toring systems are utilized. Electronic supplementary material The online version of this article (doi:10.1007/s10877-016-9876-y) contains supplementary material, which is available to authorized users. & Thomas Schmidt schmidt@mmmi.sdu.dk Camilla N. Bech Camilla.noergaard.bech@rsyd.dk Mikkel Brabrand mikkel.brabrand@rsyd.dk Uffe Kock Wiil ukwiil@mmmi.sdu.dk Annmarie Lassen annmarie.lassen@rsyd.dk 1 The Maersk Mc-Kinney Moeller Institute, University of Southern Denmark, Campusvej 55, 5230 Odense, Denmark 2 Department of Emergency Medicine, Odense University Hospital, Odense, Denmark 3 Department of Emergency Medicine, Hospital of South West Jutland, Esbjerg, Denmark Keywords Emergency departments Computerized decision support Patient monitoring J Clin Monit Comput (2017) 31:641–649 DOI 10.1007/s10877-016-9876-y ORIGINAL RESEARCH Factors related to monitoring during admission of acute patients Thomas Schmidt1 • Camilla N. Bech2 • Mikkel Brabrand2,3 • Uffe Kock Wiil1 • Annmarie Lassen2 1 Background Patients of all sorts and with a wide range of diagnoses are treated in emergency departments (ED) around the world every single day. Keeping track of such a diverse group of patients challenges both clinicians and systems. To cope with this, several health information systems have been developed speciﬁcally for managing the ﬂow and treatment of patients. Still, a substantial number of acutely admitted patients deteriorate during their admission with an increased risk of adverse outcomes [1]. There is wide- spread consensus that the risk of such deterioration can be 1 The Maersk Mc-Kinney Moeller Institute, University of Southern Denmark, Campusvej 55, 5230 Odense, Denmark 2 Department of Emergency Medicine, Odense University Hospital, Odense, Denmark 3 Department of Emergency Medicine, Hospital of South West Jutland, Esbjerg, Denmark 12 3 J Clin Monit Comput (2017) 31:641–649 642 reduced by a more frequent and rigorous approach to monitoring of patient vital signs [2]. However, the decision to continuously monitor a patient’s vital signs can still be a result of multiple causes; e.g., raised patient concern, or to optimize working procedures by not having to attach sen- sors repeatedly on patients requiring frequent registrations. Or perhaps also as a mean for boosting situational aware- ness in high load periods [3]. As such, monitoring can be viewed as an important part of the afferent limb as it provides feedback needed to initiate interventions [4]. The notion that an increased rate of vital sign registrations reduce the risk of adverse events has spurred a surge in quality assurance programs worldwide, despite concerns about the effectiveness of routinely measured vital signs have been raised [5]. Partly because the process of vital sign registrations is associated with both human and machine related errors [6]. Evidently, there exists a gap between the clinical reality and the vital sign registration procedures deﬁned by guidelines [7, 8], and as most research on automated monitoring has been conducted in the settings of intensive care units (ICU) [9] we in this work focus instead on monitoring in acute settings. deemed necessary, an intensive care consultant is contacted and need and relevancy for transfer has to be acknowl- edged by both parties. The ward is structured into three wings, one wing for surgical patients (12 beds), and two wings for medical patients (18 and 16 beds). 2.1 Data description We expect very sick patients to be monitored more than the less sick; and, it has been documented that clinicians are prone to skip vital sign registrations of less severe patients [8]. This can potentially lead to dire consequences for these patients as the risk of deterioration is present across all severity levels [10]. Understanding the utilization of patient monitoring systems in the dispersed and shared working environments of EDs and acute wards may help to identify some of the reasons for failure to rescue patients [11]. All vital signs from all monitors at the ward in the period from 10th of October 2013 to 1st of October 2014 are captured in a research database using a customized appli- cation written in Java. The department relies on Philips IntelliVue MP30/50 monitors in a networked setup as monitoring information from beds are aggregated on Phi- lips IntelliVue Information Centers in each nursing ofﬁce. When a patient is attached to a monitor our system receives a packet containing vital sign information at different intervals. Every minute we register heart rate and respira- tion rate from 3-lead ECG, pulse rate and peripheral oxy- gen saturation (spO2) measured via pulse oximetry. Depending on the clinical assessment of the patient, sys- tolic and diastolic blood pressures are registered in inter- vals from 5 to 60 min using cuffs. In this project, nurses are asked to enter patient identiﬁcation into the Philips moni- tors by personal identiﬁcation number (PIN) and name, thus enabling us to link the vital signs from a given bed location to a speciﬁc patient. Apart from this, the data collection instills no further change to existing clinical practice. We include all patients registered on the monitor with at least one measurement. However, not all patients get their information entered into the monitors, and con- sequently our system holds an amount of vital values which we cannot associate with speciﬁc patients. The character- istics of the not-identiﬁed patients are included in our analysis to enable between-group comparisons. Although increased levels of automated monitoring may improve the detection of patient deterioration, several factors may inﬂuence the extent to which a patient is being monitored. 2.1 Data description The purpose of this paper is to investigate the use of automated monitoring of patients admitted to an acute admission unit by analyzing how much the effects of distance from the nursing ofﬁce, number of concurrently admitted patients, wing type (medical/surgical), age, sex, comorbidities, and severity change conditioned on how much patients are monitored during admission. 1 Background The processing and management of patients in this ED has been documented in an earlier ﬁeld study conducted by the ﬁrst author [12]. In relevance to this paper, the most important aspects are the department’s reliance on a 5-level triage and observation regimen system which deﬁnes a baseline level of clinical alertness for each level (Blue, Green, Yellow, Orange, Red), and that the bedside ward is structured into three distinct wings, with a nurse ofﬁce in the center of each wing. Each bed on every wing is equipped with its own vital signs monitoring unit. The degree of monitoring for each patient is deﬁned by the attending physician based on the observation regimen, and in some cases adjusted by nurses afterwards. The assigned observation regimen is registered in the patient’s electronic medical record. 2.2 Analysis We aim to describe patient and department related factors and their relationship to how much patients are monitored. During their admission, patients will be intermittently attached to bedside vital sign monitors. We use the extent to which a patient is monitored as our point of interest by deﬁning monitor load as the proportion between time attached to monitor and length of stay. A monitor load percentage of 100 % means that the patient is being con- tinuously monitored throughout their entire admission; which in the study settings translates to 1 automatic reading per minute. Between group comparison for distribution of triage categories as severity, and comorbidities between patients registered on the monitors, and not-registered patients are evaluated using Chi squared tests. The preprocessing and regression analysis is conducted in R (version 3.1.1) using the quantreg package [21]. The data is preprocessed by calculating the all the aggregated exposure variables such as distance, comorbidity and concurrent load. Access to the registry of patient data was approved by the Danish Data Protection Agency (Datatilsynet—J.nr. 2013-41-2238), and the Danish Health and Medicines Authority (Sundhedsstyrelsen—J.nr. 3-3013-518/1). The study has been presented to the Research Ethics Committee of Southern Denmark, but as this is a non-interventional study an approval was not needed according to Danish law. p Table 1 provides an overview of the exposure variables used in the model. Categorical variables are automatically converted to dummy variables. Concurrent patient load is calculated based on the number of active beds in the wing during each patient’s admission period. The analysis includes exposures relating speciﬁcally to each individual; age, observation regimen, Charlson comorbidity index [15], and sex. And external factors; distance from nursing ofﬁce, concurrent load, and wing type. The relationship between monitor load and each of the exposure variables are investigated via scatter or box-plots. We focus specif- ically on the relationship between distance from nursing ofﬁce and monitor load using univariate linear regression analysis, and investigate how the relationship between these variables change conditioned on what quantile of monitor load we look at. All variables are combined in a multivariate model to examine the partial effects of each variable when controlling for all others [17–19]. We apply QR for the quantiles s = (0.10, 0.25, 0.50, 0.75, 0.90) and linear multiple Ordinary Least Squares regression. 2 Methods Our work is based on a cohort study conducted at the acute admission unit at Odense University Hospital, a 1000 bed teaching hospital serving as a primary hospital for a local population of 280,000 citizens. After initial assessment in the ED, admitted patients projected for short-term stays of up to 48 h are transferred to the admission unit. Patients can be transferred to intensive care on clinical indication. If Using the PIN, we link the collected data with supple- mental information from population based national patient registries. Arrival, admission, and discharge information 123 643 J Clin Monit Comput (2017) 31:641–649 are retrieved from the Danish National Patient Registry [13, 14]. We correct for multiplicity using the Holm–Bonferroni method, and investigate issues with multicollinarity between exposure variables using the variance inﬂation factor (VIF) [20]. Finally, we test for differences in regression coefﬁcients between the quantiles using the ANOVA method. 3 Results During the data collection period there were 11,848 admissions to the acute ward representing 35,727 days. Of these we are able to link monitor use to 3149 admissions (26.6 %) for 10,844 days (30.4 %), representing 1031 fully monitored days. Patient monitor utilization was also reg- istered for patients who we could not identify on their monitors, equating to 1271 fully monitored days. Patients in our dataset are on average admitted to the ward for 3.3 days, compared to 2.9 days for not-included patients. 115 of the patients admitted to monitors in the dataset Table 1 Overview of exposure variables Independent variable Type Values Description Comorbidity Index (CI) [15] Ordinal A, B, C, D A: CI = 0, B: CI = 1; C: CI = 2; D: CI [ 2 Severity Ordinal Regimen levels (1–5) See [16] Age Ordinal 15 - x Sex Nominal Female/male Distance Ordinal 0 - x Distance in meters from ofﬁce on each wing Wings Nominal MAU1, Surgial, MAU2 MAU1-2: (Medical Admission Unit) wings Concurrent load Ordinal 1 - x Average number of patients admitted to the wing per day during the admission period of the patient Table 1 Overview of exposure variables 123 644 J Clin Monit Comput (2017) 31:641–649 Figure 2 exempliﬁes this by showing the linear regres- sion line of the relationship between distance from nursing ofﬁce and monitor load in Fig. 2a, and quantile regression lines based on the 0.20, 0.50 (the median) and 0.80 quan- tiles in Fig. 2b. From the regression coefﬁcients, we observe that the association between monitor load and distance from nursing ofﬁce grows stronger for the upper quartiles of monitor load. Online Supplement 1 (Figure 4) shows the individual relationships between each exposure variable (age, sex, comorbidity group, triage, wing type, and the number of other patients treated during admission) and monitor load. experienced respiratory distress, seven patients suffered strokes, and one patient had both respiratory and heart related deterioration during admission. Table 2 summa- rizes data for patients identiﬁable from the monitors, and from patients not registered to monitors. While the differences in proportions for both comor- bidities and triage between patients identiﬁable on the monitors, and other patients, are statistically signiﬁcant, there are no substantial clinical differences between these factors. We do however observe that a lower percentage of surgical patients are identiﬁable on the monitors. In Fig. 3 Results 1, we observe the highly skewed distribution of how much patients are monitored. 50 percent of all the admissions have a monitor load of less than 0.014; meaning that half of all the cases are monitored less than 1.4 percent of their admission. Moving upwards, 70 percent of all admissions are monitored less than 28 percent of their total admission length. Thus, as the distribution of monitor load is heavily right skewed, standard Ordinary Least Squares regression cannot provide plausible insight. However, applying a QR approach enables us to analyze the rela- tionship between the different exposure variables and monitor load conditioned on monitor load. The results of the multivariate QR results are shown for all exposures in Fig. 3 as quantile process plots from the 0.10th up to the 0.90th quantile. The solid horizontal line for each variable indicates the Ordinary Least Squares regression coefﬁcient, and the dotted horizontal lines show the conﬁdence interval. Similarly the QR regression results at each quartile are marked with the regression coefﬁcient of the exposure variable, and the conﬁdence interval as the grey band. E.g., we ﬁnd that distance from nursing ofﬁce has the strongest inﬂuence for patients who are monitored a lot (i.e., admissions in the upper quantiles of Fig. 1). For Table 2 Exposure characteristics Admitted to monitor Not admitted to monitor Number of admissions 3149 8699 Number of patients 2815 4104 Male [n (%)] 1526 (48.4) 4314 (49.6) Mean age Male 63.8 years, SD = 18.5 years 60.9 years, SD = 21.1 years Female 66.8 years, SD = 20.9 years 63.8 years, SD = 22.6 years Comorbidity (Charlson Score (CS)) [n (%)] (A) CS = 0 1124 (35.7) 3481 (40.0) (B) CS = 1 641 (20.3) 1643 (18.9) (C) CS = 2 498 (15.8) 1297 (14.9) (D) CS [ 2 886 (28.2) 2278 (26.2) Triage [n (%)] Missing 514 (16.4) 1554 (17.9) Blue 7 (0.2) 27 (0.3) Green 431 (13.7) 1341 (15.4) Yellow 1301 (41.3) 3333 (38.3) Orange 842 (26.7) 2315 (26.6) Red 54 (1.7) 129 (1.5) Average number of registered vital signs/admission 408 registrations, SD = 633 – Wing [n (%)] Surgical 809 (25.7) 3948 (45.4) MAU1 1015 (32,2) 4751 (54.6) [both medical wings] MAU2 1325 (42.1) 12 645 J Clin Monit Comput (2017) 31:641–649 bservation regimens we ﬁnd that Orange classes have a several exposure variables but the ANOVA ﬁnds that all ig. 3 Results 1 Quantile plot for the esponse variable—illustrating he distribution of monitor load y its quantile distribution ig. 2 Univariate regression plot of Distance from nursing ofﬁce and registered Monitor load. a Ordinary Least Squares (mean ased) linear egression. b Mean linear regression, Median (Q50), 20th Quantile and 80th Quantile linear regression Clin Monit Comput (2017) 31:641–649 645 Fig. 1 Quantile plot for the response variable—illustrating the distribution of monitor load by its quantile distribution Fig. 2 Univariate regression plot of Distance from nursing ofﬁce and registered Monitor load. a Ordinary Le regression. b Mean linear regression, Median (Q50), 20th Quantile and 80th Quantile linear regression Fig. 2 Univariate regression plot of Distance from nursing ofﬁce and registered Monitor load. a Ordinary Least Squares (mean ased) linear regression. b Mean linear regression, Median (Q50), 20th Quantile and 80th Quantile linear regression several exposure variables, but the ANOVA ﬁnds that all QR coefﬁcients are signiﬁcantly different from one another. observation regimens, we ﬁnd that Orange classes have a stronger inﬂuence across the quantiles of monitoring load, but also that its impact decreases for highly monitored patients. An example of how to interpret the results from Table 3 in Online Supplement 2 and Fig. 3 is provided in Online Supplement 3. Table 3 in Online Supplement 2 conveys the results of both regression approaches. Our multiple linear regression model has an adjusted R2 of 0.1719, and are thus compa- rable to those of [22], and the model is overall statistically signiﬁcant. The VIF is below 1.62 for all exposure vari- ables, and we thus dismiss issues of multicollinarity. The Holm–Bonferroni adjustment changes the signiﬁcance of 3.1 Sensitivity analysis To address and investigate the potential impact of missing values in the dataset, we reran the analysis with missing 12 3 J Clin Monit Comput (2017) 31:641–649 646 Fig. 3 Quantile regression process plots for exposures—showing the regression coefﬁcients for the quantiles of exposure variables and the intercept when controlling for all factors Fig. 3 Quantile regression process plots for exposures—showing the regression coefﬁcients for the quantiles of exposure variables and the intercept when controlling for all factors values removed. This had little impact on the distribution of the remaining triage coefﬁcients, and did not substan- tially alter the exposure coefﬁcients or their signiﬁcance. decreeing more monitoring of patients, does not necessarily reduce the proportion of patients with adverse events [29]. Vital sign readings are often used to support clinical intu- itive hunches, and less as objective points of Ref. [30]. Even so, little research on what determine frequency of vital sign registrations have been published [22]. Since most assessment systems rely on intermittent or spot-driven observations, continuous monitoring in its current state may simply yield excessive amounts of data which can only be utilized fully through integration into clinical decision support systems. Also, the risk of more monitoring leading to alarm fatigue and habituation has to be factored in by careful consideration of calibrating the alarm thresholds [31, 32]. 123 4 Discussion We ﬁnd that distance from nursing ofﬁce has little inﬂu- ence on patients monitored less than 10 % of their admission time. But for other patients who are monitored more than this, distance from nursing ofﬁce becomes has more impact in reducing the degree of monitoring. We also note that higher levels of observation regimens have a signiﬁcant impact on monitoring load. Being admitted to the surgical wing greatly reduces how much patients are monitored, and periods with a high amount of concurrent patients have little effect on the degree of monitoring. Recent studies have rectiﬁed the assumption that deviance from protocol is solely due to clinical misjudg- ments, and instead taken a more holistic approach to the problem by investigating several factors such as day of week, time of day, and characteristics of both patients and clinicians [33, 34]. In this study, we ﬁnd evidence for adherence to observation regimen protocols through insight into how much patients are actually monitored during admission. Along these lines it is problematic that patients on the surgical wing are monitored much less than medical wing patients given that adverse events are also associated The increased focus on identiﬁcation of deteriorating patients can be seen in the body of published work on Early Warning Scores [23], Track & Trigger systems [24, 25], and Rapid Response Teams [26]. Although few of the existing deterioration detection systems in use have been rigidly validated [27, 28], the need to identify efﬁcient means for keeping an eye on multiple patients is evident as the pressure on EDs is ever increasing. However, simply 123 J Clin Monit Comput (2017) 31:641–649 647 nurses. The percentage of patients who were identiﬁable by the monitors was highest in the early phases the data acquisition stage, and then gradually decreased. The monitor registration identiﬁcation eventually plateaued, indicating that a dedicated subset of nurses persisted in registering patients to the vital sign monitors for us. This naturally induces a permutation of selection bias that is difﬁcult to overcome in this kind of project. This selection bias is also evident as identiﬁable patients are slightly older, have longer hospital stays, are sicker, and are deemed in need of more frequent observations (Table 2). 6 Conclusion As expected, there is signiﬁcant variation concerning the how much patients are monitored during their admission to an acute admission unit, but the effect of the investigated factors varies depending on how much patients are moni- tored. We conﬁrm that patients assigned to more severe observation regimen categories, are monitored more, but also show that both distance from the wing’s nursing ofﬁce inﬂuence monitoring for most patients. Number of simul- taneously admitted patients has a small effect across all levels of monitoring. Finally, we ﬁnd a big difference between the extent to which monitoring is utilized at medical and surgical wings. 4 Discussion p g y g Cabled monitoring as found in the settings of this study has several downsides; immobilization of patients, patient induced stress due to perceived severity, and loss of data during out of bed activities [39]. Consequently, much research effort has been put into the potential of wireless monitoring, but several practical obstacles such as battery life and poor communication networks still persist [40–42]. However, given that wireless monitoring could support temporary storage of vital signs on the device, would enable a smoother transition between hospital departments and reduce loss of information in out-of-bed periods. In this scenario, all patients could achieve a monitoring load of 100 %, thereby enabling more complete representation of their states and trajectories. Another limitation is missing data, and inaccurate date and time values in the coupled registries. Issues with timestamps in data retrieved from Patient Administration Systems are well known in the scientiﬁc community. Also, the observation regimen classes originate from the triage classes assigned at arrival time, generally there is a direct mapping between triage and observation regimen for patients admitted to the acute admission unit, but not necessarily for all admissions. Finally, external validity of our ﬁndings may be challenged by the single site nature of our study. Yet, assessing the monitor load of patients may be of value to similar studies, and the design of future patient monitoring systems. Interestingly, the decreasing impact of the most inﬂu- ential coefﬁcients in our statistical analyses for patients who are continuously monitored, indicate that factors not included in our model prompt higher degrees of monitor- ing. Seeking to capture the complexity of patient moni- toring in just seven exposure variables yields a very simpliﬁed model at best, and shows that patient monitoring is a complex and subjective endeavor. In this perspective, it would be interesting to include staff speciﬁc features such as clinical experience, department seniority, team compo- sition, and clinical concern in future work. An important aspect we intentionally left out of the analysis is temporal inﬂuences. As both clinical work, and the vital signs of patients follow a circadian rhythm, these aspects may reveal valuable insight for the evaluation of existing clin- ical protocols. 4 Discussion Although, the identiﬁable admissions in our analysis only account for 27 % of the total admissions in the entire period, the linked vital signs account for 45 % of all vital signs registered in the same period. This may either be a sign of issues with linking the vital signs accurately to admissions, but is also likely a seasonal indicator as the ﬁrst 6 months had the highest inclusion rate, and took place during Q4-2013 till Q1-2014. with post-surgical situations [35, 36]. This is probably a combined effect of differences in working procedures, culture between specialties as mobilization of post-surgical patients is considered important by surgical nurses, and the fact that many pre-surgical patients are unaffected until surgery, and that many orthopedic patients are admitted with minor surgical problems. Quantifying the extent to which a patient is being monitored, may be an aid to bridge the current gap between usage of automated and manual monitoring as clinical work will continue to depend on tacit knowledge and intuition [37, 38]. Since the use of monitoring is increasing in all types of hospital departments, and as technology becomes more pervasive, the insight from this paper may provide guidance for system designers and clinicians a like. Cabled monitoring as found in the settings of this study has several downsides; immobilization of patients, patient induced stress due to perceived severity, and loss of data during out of bed activities [39]. Consequently, much research effort has been put into the potential of wireless monitoring, but several practical obstacles such as battery life and poor communication networks still persist [40–42]. However, given that wireless monitoring could support temporary storage of vital signs on the device, would enable a smoother transition between hospital departments and reduce loss of information in out-of-bed periods. In this scenario, all patients could achieve a monitoring load of 100 %, thereby enabling more complete representation of their states and trajectories. Quantifying the extent to which a patient is being monitored, may be an aid to bridge the current gap between usage of automated and manual monitoring as clinical work will continue to depend on tacit knowledge and intuition [37, 38]. 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https://openalex.org/W4319065876	https://www.researchsquare.com/article/rs-2234473/latest.pdf	English	null	Effect of increased mechanical strength on the machinability of graphite cast irons in face milling	The international journal of advanced manufacturing technology/International journal, advanced manufacturing technology	2,023	cc-by	9,186	Effect of Increased Mechanical Strength on the Machinability of Graphite Cast Irons in Face Milling Alcione dos Reis Leonardo R Ribeiro da Silva (  leonardo.rrs@gmail.com ) Universidade Federal de Uberlandia https://orcid.org/0000-0003-2777-4500 Aline Elias da Silva Lucas Melo Queiroz Barbosa Álisson Rocha Machado Cássio Luiz Francisco de Andrade Wilson Luiz Guesser Research Article Keywords: Cast iron machinability, Microstructural characteristics, Face milling, Tool wear, Surface roughness Posted Date: November 17th, 2022 DOI: https://doi.org/10.21203/rs.3.rs-2234473/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at The International Journal of Advanced Manufacturing Technology on February 4th, 2023. See the published version at https://doi.org/10.1007/s00170-023-11012-0. Effect of Increased Mechanical Strength on the Machinability of Graphite Cast Irons in Face Milling Alcione dos Reis Leonardo R Ribeiro da Silva (  leonardo.rrs@gmail.com ) Universidade Federal de Uberlandia https://orcid.org/0000-0003-2777-4500 Aline Elias da Silva Lucas Melo Queiroz Barbosa Álisson Rocha Machado Cássio Luiz Francisco de Andrade Wilson Luiz Guesser Research Article Keywords: Cast iron machinability, Microstructural characteristics, Face milling, Tool wear, Surface roughness Posted Date: November 17th, 2022 DOI: https://doi.org/10.21203/rs.3.rs-2234473/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at The International Journal of Advanced Manufacturing Technology on February 4th, 2023. See the published version at https://doi.org/10.1007/s00170-023-11012-0. Effect of Increased Mechanical Strength on the Machinability of Graphite Cast Irons in Face Milling Alcione dos Reis Leonardo R Ribeiro da Silva (  leonardo.rrs@gmail.com ) Universidade Federal de Uberlandia https://orcid.org/0000-0003-2777-4500 Aline Elias da Silva Lucas Melo Queiroz Barbosa Álisson Rocha Machado Cássio Luiz Francisco de Andrade Wilson Luiz Guesser Aline Elias da Silva Lucas Melo Queiroz Barbosa Álisson Rocha Machado Cássio Luiz Francisco de Andrade Wilson Luiz Guesser Abstract Due to the low cost and high vibration damping capacity, gray cast irons are commonly used in machine tool bases, in addition to applications with noise restrictions, such as engine blocks, housings, and brakes. The matrix's graphite, sulfides, and ferrite/pearlite ratio are some of the most important parameters governing the machinability of the gray cast irons. This work aims to evaluate the machinability of high-resistance gray cast irons of the FC 300 grade, in two versions, with the addition of molybdenum (FC 300 (Mo)) and with refined graphite and addition of molybdenum (FC 300 (Mo+RG)), for use in cylinder heads and engines blocks, compared to materials that have been used for this purpose, gray cast iron FC 250 and the compacted graphite cast iron FV450. The face milling process was chosen for the tests, as it is widely used in manufacturing cylinder heads and engine blocks. Uncoated cemented carbide tools with tangential rhomboid geometry were used in the experiments. Analysis of tool life and wear mechanisms and machined surfaces' quality (Ra roughness parameter) where the output variables are considered. The materials were characterized according to the cementite interlayer spacing and microhardness of the perlite matrix, the number of eutectic cells, and the distribution of manganese sulfide inclusions, with those characterizations being correlated with the machinability results. The cutting speed and feed were varied, and the dry condition was used. Among the gray cast irons investigated, the FC 300(Mo+RG) presented worse machinability rates because of its greater mechanical resistance and hardness. Regarding the surface finish, at the beginning of the tool life tests (without considering tool wear) and employing the highest cutting speed, the FC 300(Mo+RG) showed the best results, but at the lowest cutting speed, the worst. Research Article Research Article Keywords: Cast iron machinability, Microstructural characteristics, Face milling, Tool wear, Surface roughness Posted Date: November 17th, 2022 DOI: https://doi.org/10.21203/rs.3.rs-2234473/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at The International Journal of Advanced Manufacturing Technology on February 4th, 2023. See the published version at https://doi.org/10.1007/s00170-023-11012-0. Page 1/30 Page 1/30 1. Introduction Stricter regulations on gas emissions have led car manufacturers to explore different vehicle engine options. Currently, to improve performance in the manufacture of cylinder blocks and cylinder heads of high-powered diesel engines, as well as other castings, a certain number of alloying elements are generally added to gray cast iron. Cast iron grade FC300 materials are widely used to manufacture various industrial products. The reasons are good castability, machinability, and abrasion resistance, and its vibration-dampening capacity is much higher than that of light steels [1, 2, 3]. Given this, the mechanical resistance of cast iron, that is, their ability to withstand external stresses without causing them to cause plastic deformations, and consequently their grades, are conditioned to their final structure obtained [4, 5]. Therefore, this property depends on the shape and amount of graphite and the amount of ferrite and/or perlite in the metallic matrix, in which the resistance is increased with higher levels of perlite and the reduction of the interlayer spacing of the perlite. [5, 6, 7]. The usual mechanism for increasing the tensile strength and hardness of gray cast irons is the addition of alloying elements, such as chromium (Cr), molybdenum (Mo), tin (Sn), and copper (Cu). Additional amounts of copper and tin (pearlized elements) promote the refining of the pearlite or reduction of interlayer spacing, which results in increased strength. On the other hand, Chromium and molybdenum act in the formation of carbides, and, like Sn and Cu, Mo can also be used as a perlite refiner [8, 9]. Page 2/30 Page 2/30 To achieve a minimum tensile strength of 300 MPa, the carbon content is also reduced from 3.2 to 3.0%, which results in smaller graphite lamellae, thus reducing the risk of starting and crack propagation. In this way, a 10 to 20% increase in mechanical properties is achieved from this [10, 11]. The addition of alloying elements depends on the elemental composition and manufacturing method to provide the desired mechanical properties. Therefore, reducing or eliminating Cu use by technical measures without reducing the mechanical properties and processability in mass production will bring some economic and technological benefits [12, 13]. The study on machinability, cutting strategies, and application technology of gray cast iron is today a priority in large companies and research institutions worldwide, becoming an effective guarantee for high-quality products [14]. 1. Introduction Gray cast iron has become a popular material among other cast metals, being widely applied in modern industrial production, mainly for its low cost (20 to 40% less than steel), in addition to having a wide variety of mechanical properties achievable, as good castability, machining property convenient and good wear resistance [15, 16]. The microstructure of gray cast iron is characterized by graphite lamellas dispersed in the ferrous matrix. The casting practice can influence the nucleation and growth of graphite flakes so that the size and type improve the desired properties. The quantity and size of graphite, morphology, and distribution of these lamellae are critical in determining the mechanical behavior of gray cast iron [15, 17]. According to Guesser [9], the machinability of gray cast irons increases as you move towards higher strength grades due to the increased abrasiveness with the increase in the amount of perlite in the matrix and due to the decrease in lubricating action and consequent reduction in ease chip breaking with a decrease in the amount of graphite. Another way to evaluate the machinability of cast iron is by combining the hardness test with an evaluation of microstructure due to some microconstituents that adversely affect machinability (ASM). Thus, it is emphasized that the microstructure plays a crucial role in altering the mechanical properties of any material [18]. Controlling the microstructure, optimizing the process parameters, and adding alloy elements are highly necessary [18, 19]. The main constituent elements of cast iron are mainly carbon, phosphorus,s and silicon, among others. The presence of silicon and phosphorus determines the solubility of carbon in the molten metal [20] Another way to evaluate the machinability of cast iron is by combining the hardness test with an evaluation of microstructure due to some microconstituents that adversely affect machinability (ASM). Thus, it is emphasized that the microstructure plays a crucial role in altering the mechanical properties of any material [18]. Controlling the microstructure, optimizing the process parameters, and adding alloy elements are highly necessary [18, 19]. The main constituent elements of cast iron are mainly carbon, phosphorus,s and silicon, among others. The presence of silicon and phosphorus determines the solubility of carbon in the molten metal [20] Silicon is one of the essential elements in producing gray cast iron because it is a stabilizing element of graphite. Thus, it promotes graphite development at the site of iron carbides. 1. Introduction It is verified from the experience that the Si content of around 3% restricts the formation of iron carbide because no carbon is left in the chemical form [21]. Silicon is one of the essential elements in producing gray cast iron because it is a stabilizing element of graphite. Thus, it promotes graphite development at the site of iron carbides. It is verified from the experience that the Si content of around 3% restricts the formation of iron carbide because no carbon is left in the chemical form [21]. Adding nickel refines the pearlite structure and graphite gray cast iron, improving strength and hardness to balance the differences in thick sections. High hardness, like that of white cast iron bars, produced by adding sodium chloride salt to gray cast iron, is recommended for wear resistance [22, 23]. Adding nickel refines the pearlite structure and graphite gray cast iron, improving strength and hardness to balance the differences in thick sections. High hardness, like that of white cast iron bars, produced by adding sodium chloride salt to gray cast iron, is recommended for wear resistance [22, 23]. Page 3/30 As for molybdenum, this is a carbide-forming element used to strengthen and harden iron because of the transformation of austenite into fine perlite and bainite, generally added in gray cast iron to refine perlite [24, Page 3/30 25, 26]. Molybdenum is not a promoter of perlite; however, it is usually added as a ferromolybdenum containing 60 to 70% (Mo) [27, 28]. Copper and molybdenum in gray cast iron guarantee greater hardness and tensile strength than the common pearlitic types of gray cast iron [11]. 25, 26]. Molybdenum is not a promoter of perlite; however, it is usually added as a ferromolybdenum containing 60 to 70% (Mo) [27, 28]. Copper and molybdenum in gray cast iron guarantee greater hardness and tensile strength than the common pearlitic types of gray cast iron [11]. The gray cast irons of FC300 class possess good mechanical properties, where combinations of multiple structures with low silicon content, fine graphite, and perlite make them stronger and increase their resistance [29, 30]. The combination of microstructure with graphite reinforcement and excellent mechanical strength has led the cast iron alloys of the FC300 class to be used in a wide range of industrial applications, such as automotive parts, internal combustion engines, piping systems, and construction parts [31]. 1. Introduction However, compared with conventional FC250 and FC300, the class is considered a lower-cut material. The problems encountered during the machining of the FC300 class are caused by changes in the microstructure formation due to the addition of alloying elements [29, 31]. This is an issue to be considered in industrial environments because it becomes difficult to predict the actual tool life and to specify the most suitable cutting conditions for a given cast alloy. The integrity of the surface is directly related to the quality achieved in the final machining, which strongly affects the product's performance. Among the factors that can influence the quality of the product's surface are the cutting speed, the feed, the depth of cut, the geometry of the tool, the wear of the tool, and the properties of the part [29, 32]. In practice, machining operations can only fully develop the potential of machine tools and cutting tools after optimizing the cutting parameters to obtain the best work efficiency and tool life [33, 34]. The present study aims to evaluate the influence of the microstructure on the machinability of gray high- strength cast iron, of the FC300 class, for use in diesel engine blocks and heads. For this purpose, two versions of FC300 gray cast iron were produced, one with graphite refining (FC 300 (RG)) and the other, which in addition to graphite refining, has molybdenum addition (FC 300 (Mo + RG)). The FC250 gray cast iron and vermicular iron FV450 were also investigated for comparison purposes. These materials were machined in the front milling, with carbide tools, with comparisons of the tool life and the surface roughness of the part, considering in the analysis and discussions of the results the microstructural characteristics of the materials. 2.1. Materials characterization Material Matrix Graphite Form % Nodularity FC 250 100% Pearlitic I - FC 300 (Mo) 100% Pearlitic I - FC 300 (Mo+RG) 100% Pearlitic I - FV 450 99% Pearlitic III - VI 9 The metallographic analysis allowed us to observe the appearance of graphite in the three gray irons as thin and uniform flakes with random orientation, typical of form I and type A [8]. In the compacted graphite cast iron, the shapes observed as graphite III and VI correspond, respectively, to the form of worms and nodules, as shown in Fig. 1d. For all the materials, this information about the metallography of gray cast iron is crucial for analyzing the machining results since the number of eutectic cells reflects directly on the volume and size of the graphites. A larger number of cells tend to have a thinner structure with a smaller graphite size, which red ces the crack propagation condition making machining diffic lt Table 3 sho s the Brinell hardness of the Table 2 Characteristics of graphite and matrix of the materials [7]. Material Matrix Graphite Form % Nodularity FC 250 100% Pearlitic I - FC 300 (Mo) 100% Pearlitic I - FC 300 (Mo+RG) 100% Pearlitic I - FV 450 99% Pearlitic III - VI 9 Table 2 Characteristics of graphite and matrix of the materials [7]. Material Matrix Graphite Form % Nodularity FC 250 100% Pearlitic I - FC 300 (Mo) 100% Pearlitic I - FC 300 (Mo+RG) 100% Pearlitic I - FV 450 99% Pearlitic III - VI 9 The metallographic analysis allowed us to observe the appearance of graphite in the three gray irons as thin and uniform flakes with random orientation, typical of form I and type A [8]. In the compacted graphite cast iron, the shapes observed as graphite III and VI correspond, respectively, to the form of worms and nodules, as shown in Fig. 1d. For all the materials, this information about the metallography of gray cast iron is crucial for analyzing the machining results since the number of eutectic cells reflects directly on the volume and size of the graphites. A larger number of cells tend to have a thinner structure with a smaller graphite size, which reduces the crack propagation condition, making machining difficult. 2.1. Materials characterization The materials tested through the face milling process were the high-strength gray cast iron FC 300 (Mo), which has molybdenum as an alloying element, the FC 300 (Mo + RG), which also has molybdenum as an alloying element, as well as graphite refinement, the gray cast iron FC 250 and the compressed graphite cast iron FV 450. The last two will be investigated to serve as references in the comparisons. The micrographs of the materials were obtained using a scanning electron microscope (SEM - Hitachi TM 3000) as well as the micrographs showing 100% pearlitic matrix in both, obtained by optical microscopy (Olympus microscope), this being a technique used to view a sample close up with the magnification of a lens Page 4/30 with visible light. Figure 1 shows the distribution of the graphites of the investigated materials. The graphite morphology was evaluated using scanning electron microscopy (SEM), and the pearlitic matrix using optical microscopy after etching with Nital (3%). The chemical composition, mechanical properties, and characteristics of the matrix and graphite of the materials were made available by the manufacturer [7] and detailed in Table 1 and Table 2. Table 1 Chemical composition of the evaluated materials [7]. Material Si [%] Mn [%] P [%] S [%] Cr [%] Ti [%] Sn [%] Cu [%] Mo [%] Ni [%] Cu eq. FC 250 1.900 - 2.000 0.500 - 0.600 0.020 - 0.030 0.100 0.270 0.01- 0.130 0.280 0.020 - 2.38 FC 300 (Mo) 2.11 0.50 0.036 0.10 0.23 0.01 0.06 0.67 0.23 - 4.07 FC 300 (Mo+RG) 2.12 0.66 0.035 0.10 0.23 0.01 0.06 0.67 0.22 - 4.12 FV 450 2.210 0.320 0.019 0.003 0.031 0.007 0.070 0.990 - 0.016 - Table 1 Chemical composition of the evaluated materials [7]. Material Si [%] Mn [%] P [%] S [%] Cr [%] Ti [%] Sn [%] Cu [%] Mo [%] Ni [%] Cu eq. FC 250 1.900 - 2.000 0.500 - 0.600 0.020 - 0.030 0.100 0.270 0.01- 0.130 0.280 0.020 - 2.38 FC 300 (Mo) 2.11 0.50 0.036 0.10 0.23 0.01 0.06 0.67 0.23 - 4.07 FC 300 (Mo+RG) 2.12 0.66 0.035 0.10 0.23 0.01 0.06 0.67 0.22 - 4.12 FV 450 2.210 0.320 0.019 0.003 0.031 0.007 0.070 0.990 - 0.016 - Table 2 Characteristics of graphite and matrix of the materials [7]. 2.1. Materials characterization Table 3 shows the Brinell hardness of the materials, the Vickers microhardness of the pearlite, and the mechanical strength of all the tested materials. Page 5/30 Page 5/30 Table 3 Hardness and tensile strength of the evaluated materials [7]. Properties FC 250 FC 300 (Mo) FC 300 (Mo+RG) FV 450 Hardness [HB] 187 207 217 229 Pearlite microhardness [HV] 278 291 313 364 Ultimate tensile strength [MPa] 259 278 283 524 To determine the amount of manganese sulfide inclusions (MnS/mm2), for each cast iron sample, ten images at a magnification of (500x) were used, and for area evaluation, 150 particles of manganese sulfide were randomly selected from the images. Figure 2 (a) shows examples of the measured particle areas. The cementite interlayer spacing of the perlite was determined by etching the samples with 3% Nital reagent. Twenty-five images were taken at a magnification of 20000x by scanning electron microscopy of different regions, with 25 points being selected at random to measure the intersections of the cementite lamellae according to the methodology established by Vander Voort and Roosz [9]. 2.2. Material samples, cutting toolS, and milling TESTS The work materials were supplied by TUPY S.A [7] in the form of rectangular bars whose dimensions were approximately 400 mm long, 240 mm wide, and 40 mm thick. Before the face milling tests, the specimens had their surfaces pre-machined. This process was employed to remove residues from the sand-casting process and the chill zone, as both can increase the tool wear, masking the influence of the input parameters. The cutting tools used in this research were uncoated cemented carbide inserts manufactured by Walter Tools, ISO code - LNHU130608R-L55T Tiger-tec® Silver, grade WKP 25S. These inserts have a double-sided tangential rhomboid geometry suitable for finishing. These tools were mounted on a 90 ° angle cutter head, BLAXX model, code F5141.B27.080.Z10.12, with a diameter of 80 mm and capacity for ten inserts, also manufactured by Walter Tools. Figure 3 and the table shows the details of the tools used in the experiments. Only one insert was mounted in the milling cutters for the trials, which according to Richetti, et al. [10], does not compromise the comparative results. Page 6/30 Page 6/30 Page 6/30 Table 4 The geometry of the tool used in the tests. Description Symbol Value Indexable insert tolerance class H Number of cutting edges 4 Indexable insert width l2 12 mm Cutting edge length l 13 mm Insert thickness s 6.8 mm Corner radius r 0.8 mm Wiper cutting-edge length b 2.2 mm Table 4 The tool life tests followed a factorial design of experiments (DOE) of 23, with two quantitative variables (cutting speed, vc; feed rate, f) and a qualitative variable (workpiece material). The high (+) and low (-) levels of the variables are shown in Table 5. In a predominantly concordant milling cutting, the axial depth of cut (ap) and the radial depth of cut (ae) remained constant at 1 mm and 60 mm, respectively. To guarantee statistical reliability, all tests were repeated twice, totaling three tests in each condition (test and two replicates). Table 5 Levels of the design of the experiments used in the tests. 2.2. Material samples, cutting toolS, and milling TESTS Level vc [m/min] f [mm/rev] Work material (+) 350 0,2 FC 250 230 0,1 FC 300 (Mo); FC 300 (Mo+RG); FV 450 (-) The levels of the input variables were defined to provide a tool life suitable for the carbide inserts so that they were not too long (at low levels), consuming much time and material, nor too short (at high levels), offering little information about the test. As the qualitative variable (material of the workpiece) has 4 levels, instead of 8 tests, 16 were necessary, totaling 48 tests with repetitions. The tests were carried out under dry conditions, in a vertical CNC machining center Discovery model 760, with main spindle power of 11 kW and a maximum rotation of 10 000 RPM, from the manufacturer ROMI - Bridgeport. For the tool life tests, maximum flank wear (VBBmax) equal to 0.40 mm was used as the end-of-tool life criterion. At the end of each run, the tool wear and surface roughness of the workpiece was measured. After reaching the end of their lives, the tools had their wear mechanisms evaluated by scanning electron microscopy. Page 7/30 Page 7/30 To measure the surface roughness parameters (Ra) was used a portable roughness meter, model SJ201 P / M from MITUTOYO. This instrument has a needle probe with a 2 µm diamond tip radius and 0.1 µm resolution. A 0.8 mm cut-off filter was used, following the recommendation of the /ISO 4288 standard [11]. 3. Results And Discussions 3.1. Distribution of MnS particles and pearlite interlayer spacing The results of the average amount of MnS inclusions/mm² and the average area of these inclusions in the gray cast iron are shown in Fig. 4. The vermicular cast iron, in turn, does not present MnS inclusions in its microstructure since sulfur is reduced in these materials. Figure 4a shows slight differences in the averages of these particles. The cast iron with graphite refining FC 300 (Mo + RG) has, on average, 418 MnS/mm² particles, in which 27% of the inclusions measured in the sample have areas between 11 and 20 µm². On the other hand, 21% of the particles have areas between 6 and 10 µm², and less than 6 µm² represents about 13% of total inclusions, as shown in Fig. 4b. The FC 250 presented 391 MnS/ mm2 particles, with 33% of inclusions having areas between 11 and 20 µm², 20% of the particles having areas between 6 and 10 µm², and 7% of inclusions having areas between 1 and 5 µm². The FC 300 (Mo) has, on average, 351 MnS/mm² particles that are present in the material as follows: 25% of the analyzed particles have areas between 11 and 20 µm², 24% of the inclusions have areas between 6 and 10 µm² and only 5% of the MnS particles have areas smaller than 6 µm². To check if there are statistically significant differences in the number of inclusions per square millimeter among the three materials, an analysis of variance (ANOVA) was carried out regarding the average amount of MnS/mm2 particles. Table 6 shows the results of the analysis of variance for the average amount of MnS particles / mm2, while Table 7 shows the results of the analysis of the distribution of the areas of the MnS particles. Table 6 Result of the analysis of variance for the average amount of MnS particles / mm2. Analysis of variance for quantity average of MnS / mm2 particles Source of Variation SS dl MS F P-value Between Groups 7406,888889 2 3703,444444 122,540441 0,000013 Within Group 181,333333 6 30,222222 Total 7588,222222 8 Page 8/30 Page 8/30 Page 8/30 Table 7 Result of the MnS particle area distribution analysis. Distribution analysis of MnS particle areas Source of Variation SS dl MS F P-value Between Groups 14083 3 4694,33333 18,499835 0,008281 Within Group 1015 4 253,75 Total 15098 7 Table 7 Table 7 Result of the MnS particle area distribution analysis. 3. Results And Discussions 3.1. Distribution of MnS particles and pearlite interlayer spacing The data were obtained using a 95% confidence interval and a 5% significance level using Matlab® software. The analysis of variance (ANOVA) was also used for the cementite interlayer spacing (µm) of the perlite and the size of the MnS particle areas of the materials used in the research. The results of the pearlite interlayer spacing of the tested materials are shown in Fig. 5. Note that, in terms of average values, the FC 300 (Mo + RG) has the smallest spacing between layers, an average of 0.29 µm. Thus, this factor may have influenced the hardness of this material, which was greater than the FC 300 (Mo) and FC 250 (Table 3). The variance (ANOVA) analysis was used to confirm a significant difference between the material's interlayer spacing (µm). Table 8 shows the results of the analysis of variance for interlayer spacing (µm). It can indicate that, for gray cast iron with molybdenum and graphite refining, there will be an increase in tensile strength and abrasiveness, thus lowering the machinability. Table 8 Result of the analysis of variance for interlayer spacing (µm). Source of Variation SS dl MS F P-value Between Groups 0,065664 3 0,021888 17,315181 0,009353 Within Group 0,005056 4 0,001264 Total 0,070721 7 3.2 Tool life and wear mechanisms Figure 6 illustrates the average results for the tool life of the evaluated materials for the cutting conditions shown in Table 5. The results were interpolated to a VBBmax = 0.4 mm, using the methodology proposed by da Silva, et al. [12]. Based on the results, it is observed that the FC 250 presented the best machinability among the analyzed materials, the explanation being that its mechanical properties are inferior compared to the other analyzed materials, as shown in Table 3. To show statistical differences between all materials, an analysis of variance (ANOVA) with 95% reliability was performed according to the values illustrated in Fig. 6, with the result in Table 9. Based on these results, it is observed that all input variables significantly influenced the process, with the cutting speed the variable with the most significant influence and the type of material the variable with the lowest. Page 9/30 Table 9 Analysis of variance for the tool life. Effect SS DoF MS F p Intercept 10223.74 1 10223.74 553.4243 0.000000 Material 563.62 3 187.87 10.1698 0.002211 vc 870.99 1 870.99 47.1477 0.000044 f 825.27 1 825.27 44.6729 0.000055 Error 184.74 10 18.47 Figure 7 illustrates the individual effects of each input variable indicated in Table 9 about the machining time. Figure 7a shows a clear relationship between the increase in mechanical properties and the reduction of machinability, similar to that observed by da Silva, et al. [3] in the drilling process of similar materials. Figure 7b illustrates that the increase in cutting speed resulted in a decrease in machinability, which, according to da Silva, et al. [13], is explained for graphitic cast irons by the increase in forces and temperatures at the cutting interface due to the increase in cutting speed. Figure 7c indicates that the increase in feed resulted in a decrease in machinability, which can be explained by the greater volume of material removed by tool interaction resulting in greater forces on the cutting edge [14]. Figure 8 illustrates the combined effects of the material, cutting speed, and feed-on material machinability. Figure 8 illustrates the combined effects of the material, cutting speed, and feed-on material machinability. 3.2 Tool life and wear mechanisms This analysis allows us to infer that the most significant differences in machinability between the materials are observed for the lowest speed (vc = 230 m/min), with the tool life of the materials being similar for the most severe cutting conditions (vc = 350 m/min, f = 0.2 mm/rev), corroborating the fact that the cutting parameters have a much more significant effect on machinability than the materials evaluated as indicated by the analysis of variance in Table 9. This graph also shows that the FC 250 has machinability considerably superior to the other materials for the lower cutting conditions, confirming the challenge of machining high- strength graphitic cast irons, particularly at higher cutting speeds. Through an approach similar to that of da Silva, et al. [3], the average machinability (based on the tool lives) values for each material about the parameters indicated in Table 5 were linearly correlated with Table 3 and Fig. 5. Among the variables analyzed, only the macrohardness of the materials was found to have a significant correlation with the machinability of the process, despite the microhardness of the matrix showing a linear correlation of almost 70%. The more significant correlation of hardness between UTS and interlayer spacing is because hardness is the property that most correlates with the dynamic shear strength of the material for the high shear rates observed in machining [14]. The more significant correlation of Brinell macrohardness about the Vickers microhardness of the matrix can be explained by the greater measurement area of the macrohardness, which also takes into account the influence of graphite on the stress/strain behavior of the material, which according to Collini, et al. [15] is a defining characteristic of the mechanical behavior of graphitic cast irons. Page 10/30 Page 10/30 At the end of the tool life tests, the cutting tools were taken to a scanning electron microscope (SEM) to analyze the wear mechanisms of the tools. The predominance of adhesion mechanisms (attrition), microabrasion, and chipping was evident in the machining of the four cast iron alloys, regardless of the cutting conditions tested. Analysis by dispersive energy spectrometry (EDS) detected significant amounts of workpiece material adhered to the worn region of the tool in all tests. Figure 10 and Fig. 3.2 Tool life and wear mechanisms 11 illustrate the images obtained by SEM of the worn regions of the tools used in machining the four tested materials, respectively, in the lowest cutting conditions (vc = 230 m/min and f = 0.1 mm/rev) and the most severe (vc = 350 m/min and f = 0.2 mm/rev). It is observed that the flank wear is more pronounced in the most severe conditions, but the wear mechanisms are practically unchanged, concluding that they are only accelerated with higher cutting and feed speeds. When machining compacted graphite cast iron (FV 450) and gray cast iron of a greater resistance (FC 300 (Mo+RG) and FC 300 (Mo)), the presence of micro/macro chipping is always observed, while in machining the less resistant gray cast iron (FC 250) chipping does not appear. When machining the FC 250, the photos in Fig. 10d and Fig. 11d show the predominance of the micro-abrasive mechanism. However, considering that in this material, there is no evidence of the presence of precipitates of very high hardness, the micro abrasion must have originated in the particles of the tool itself, lost by attrition (adhesion), leading to the conclusion of the occurrence of this wear mechanism as well [16]. It is observed that the lowest flank wear among all the tests occurred in the cast iron alloy FC 250. According to da Silva, et al. [3], this is due to the high carbon content associated with the lower number of eutectic cells, a higher percentage of broad graphite lamellae, and the lower hardness of this material. Another relevant aspect is the presence of oxygen in the worn surfaces of the tools. This presence indicates that in this region, there was the penetration of atmospheric air and the possible formation of oxides. These oxides adhere to the tool's surface and, subsequently, are pulled out by the flow of workpiece material, interacting in the tribological system to promote adhesive wear during the milling process [17]. 3.3. Analysis of roughness of the machined surfaces 3.3. Analysis of roughness of the machined surfaces Figure 12 illustrates the average roughness (Ra) of the machined surfaces of each material for each cutting condition. Due to the difficulty in finding correlations between machining parameters and the resulting surface roughness, an analysis of variance (ANOVA) was performed for the results illustrated in Fig. 12, with this analysis being illustrated in Table 10. The analysis of variance indicated that for a 95% confidence interval, the variation of the input parameters has no statistical effect on Ra. Page 11/30 Table 10 Analysis of variance for the Ra. Effect SS DoF MS F p Intercept 2.133133 1 2.133133 134.8657 0.000000 Material 0.058617 3 0.019539 1.2353 0.347674 vc 0.045422 1 0.045422 2.8718 0.121007 f 0.033079 1 0.033079 2.0914 0.178738 Error 0.158167 10 0.015817 Table 10 Analysis of variance for the Ra. According to Guesser, et al. [18], the graphite shape significantly impacts the surface finish when machining graphite cast irons. This is due to its stress-concentrating effect dictating the shear mode of the material in the cutting zone and its ejection from the die through the phenomenon known as open grain, directly impacting the roughness of the machined surface. Based on these facts, a new analysis of variance was carried out and illustrated in Table 11, excluding the compacted graphite cast iron, since the presence of vermicular and nodular graphites in it makes the tribosystem at the cutting interface completely different [3]. Table 11 Analysis of variance for the Ra considering only the gray cast irons Effect SS DoF MS F p Intercept 1.742568 1 1.742568 106.5811 0.000017 Material 0.046424 2 0.023212 1.4197 0.303701 vc 0.083339 1 0.083339 5.0973 0.048528 f 0.026756 1 0.026756 1.6365 0.241577 Error 0.114448 7 0.016350 The analysis of variance illustrated in Table 11 indicated that among the input variables analyzed, only the cutting speed presented, for a 95% confidence interval, statistical significance about the roughness of the machined surface. This effect can best be seen in Fig. 13, which illustrates that higher cutting speeds resulted in lower roughness values. The possible explanation for this phenomenon is the fact that higher shear rates due to the higher cutting speed decrease the shear of the material in the primary and secondary shear planes [19], in addition to making graphite ejection difficult in the open-grain phenomenon by decreasing the time that the material remains undergoing plastic deformation with the increase in the shearing rate [20]. In Fig. 4. Conclusions This paper studied the tool life, wear mechanisms, and surface roughness in the face milling process of four different graphitic cast irons, for two levels of cutting speed and feed rate. The output variables were compared with the material's mechanical properties, allowing a better understanding of the results. The investigation allowed the following conclusions to be drawn: • The FC 250 gray cast iron outperformed the other materials in the tool life. This performance, however, was more pronounced for the lower cutting speed and feed tested. • The FC 250 gray cast iron outperformed the other materials in the tool life. This performance, however, was more pronounced for the lower cutting speed and feed tested. • The compacted graphite cast iron FV 450 presented the poorest machinability about the tool life, however, with results close to the high strength gray cast irons (FC 300 (Mo) and FC 300 (Mo+RG)) at the more aggressive cutting conditions. • The Brinell hardness was the material property that better correlated with the tool life, as it evaluates both the matrix and the graphitic phase of the materials. • Adhesion (attrition), microabrasion, and microchipping were the prevailing wear mechanisms for all machining conditions tested. The flank wear was more pronounced at the more aggressive cutting conditions. • The input variables did not show statistical significance on the surface roughness of the evaluated materials. However, when the FV 450 was taken out of consideration, the cutting speed presented statistical significance about the surface Ra. • Both the UTS and the Brinnel macrohardness are correlated with the surface roughness of the machined surface, as those parameters represent the properties of the matrix and the graphitic phase, which are linked with the shear behavior of the materials. • Substituting a more resistant material in block engines, for example, is advantageous for the performance of the motor, but this has a price. These more resistant materials (FC 300 (Mo) and FC 300 (Mo+RG)) have lower machinability in terms of tool life as compared to the commonly used FC250 gray cast iron but higher than the compact graphite cast iron FV450. The difference tends to reduce when using the more severe cutting conditions (high cutting speed and feed rate). In terms of surface roughness, the same cannot be said; it depends on the cutting conditions. 3.3. Analysis of roughness of the machined surfaces 14, correlations were drawn between the mechanical properties of the material and the average roughness for all the cut variables evaluated, in a similar way done for the tool life, Fig. 9, but now, only for the gray cast irons. Linear correlations above 70% were observed only for the macroscopic mechanical properties that consider the matrix and graphite, i.e., the tensile strength and Brinell macro-hardness. Since these properties consider the stress concentration of the graphitic phase and its interactions with the material matrix, the overlap of the effects of the phenomena caused by the presence of this second phase on the surface Page 12/30 Page 12/30 integrity of the material is evident, i.e., the ejection of the graphite (open grain) and the shear in the cutting zone. integrity of the material is evident, i.e., the ejection of the graphite (open grain) and the shear in the cutting zone. 4. Conclusions In the more severe cutting conditions tested (vc = 350 m/min), the more resistant gray cast irons showed better surface roughness than the FC250. Funding The authors declare that no funds, grants, or other support were received during the preparation of this manuscript. The authors declare that no funds, grants, or other support were received during the preparation of this manuscript. Author Contributions All authors contributed to the study conception, design, writing, material preparation, data collection, and analysis. All authors read and approved the final manuscript. All authors contributed to the study conception, design, writing, material preparation, data collection, and analysis. All authors read and approved the final manuscript. All authors contributed to the study conception, design, writing, material preparation, data collection, and analysis. All authors read and approved the final manuscript. Consent for publication All the authors declare that all items in this work present consent for publication. Conflicts of interest/Competing interests The authors have no relevant financial or non-financial interests to disclose The authors have no relevant financial or non-financial interests to disclose. Availability of data and material All the authors declare that this paper has no available data or material. All the authors declare that this paper didn't need consent to participate. The authors are grateful to the Brazilian research agencies CNPq, FAPEMIG, and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001 for financial support to The authors are grateful to the Brazilian research agencies CNPq, FAPEMIG, and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001 for financial support to Page 13/30 Page 13/30 Page 13/30 Tupy S.A. for providing the work material and technical support and to Walter for the donation of the tooling. Funding References 1. J. Li, P. Wang, X. Cui, K. Li, R. Yi, Gray cast iron cylinder head thermal mechanical fatigue analysis, Proceedings of the FISITA 2012 World Automotive Congress, Springer, 2013, pp. 243-257. 1. J. Li, P. Wang, X. Cui, K. Li, R. Yi, Gray cast iron cylinder head thermal mechanical fatigue analysis, Proceedings of the FISITA 2012 World Automotive Congress, Springer, 2013, pp. 243-257. 2. L. Da Silva, H. 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Molybdenum Steels Irons Alloys, Climax Molybdenum Company, 2014, pp 15-16, 42, 52, 288-304. 30. A.B. Mohd Hadzley1, M.R. Nurul Fatin1, R.A. Raja Izamshah1, N.I.S. Hussein1, A. Siti sarah1, e and S. Sivarao1, “Surface Integrity of a High-Speed Milling FC300 Gray Cast Iron” Faculty of Manufacturing Engineering, Universiti Teknikal Malaysia Melaka (UTeM), Hang Tuah Jaya, 76100 Durian Tunggal, Melaka, Malaysia, 2014. 31. K. Hato, S. Kazuhiro and S. Hitoshi, "Cutting performance of a binder-less sintered cubic boron nitride tool in the high-speed milling of grey cast iron", Journal of Material Processing Technology. 127 217-221 - 2002. 32. R. Elliott, "Cast Iron Technology" Butterworths & co. (publishers) Ltd. United Kingdom 1988. 32. R. Elliott, "Cast Iron Technology" Butterworths & co. (publishers) Ltd. United K 33. Y. Sahin and A.R. 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References Machado, Cutting Temperatures in End Milling of Compacted Graphite Irons, Procedia Manufacturing, 26, 2018, 474-484. 15 VP Astakhov Tribology of metal cutting Elsevier 2006 15. V.P. Astakhov, Tribology of metal cutting, Elsevier, 2006. 16. L. Collini, G. Nicoletto, R. Konečná, Microstructure and mechanical properties of pearlitic gray cast iron, Materials Science and Engineering: A, 488, 2008- 529-539. 17. L. da Silva, F. Souza, W. Guesser, M. Jackson, A. Machado, Critical assessment of compacted graphite cast iron machinability in the milling process, Journal of Manufacturing Processes, 56,2020, 63-74. 18. S. Dawson, I. Hollinger, M. Robbins, J. Daeth, U. Reuter, H. Schulz, The effect of metallurgical variables on the machinability of compacted graphite iron, SAE transactions, 2001 334-352. 19. W.L. Guesser, F.S. Pereira, L. Boehs, Surface changes during turning of grey cast iron, International Journal of Machining and Machinability of Materials, 18 ,2016 - 313-324. 19. W.L. Guesser, F.S. Pereira, L. Boehs, Surface changes during turning of grey cast iron, International Journal of Machining and Machinability of Materials, 18 ,2016 - 313-324. 20. S. Thamizhmanii, S. Hasan, Analyses of roughness, forces and wear in turning gray cast iron, Journal of achievement in Materials and Manufacturing Engineering, 17, 2006. 20. S. Thamizhmanii, S. Hasan, Analyses of roughness, forces and wear in turning gray cast iron, Journal of achievement in Materials and Manufacturing Engineering, 17, 2006. 21. P.W. "Materials, Effects of austempering process on mechanical behavior properties of compacted graphite iron", 2019. 21. P.W. "Materials, Effects of austempering process on mechanical behavior properties of compacted graphite iron", 2019. 22. AN Overview PrachI Singhal, Kuldeep K. Saxena, "Effect of silicon addition on microstructure and mechanical properties of grey cast Iron", Department of Mechanical Engineering, GLA University, Mathura, UP, 281406, India, 2020. 23. G. Goodrich, Case histories of cast iron machinability problems, Modern casting, 87-1997. 30-33 23. G. Goodrich, Case histories of cast iron machinability problems, Modern casting, 87-1997. 30-33 24. Johnson O. Agunsoye1, Talabi S. Isaac2, Olumuyiwa I. Awe3, Afemefuna T. Onwuegbuzie1 "Effect of Silicon Additions on the Wear Properties of Grey Cast Iron" 1Department of Metallurgical and Materials Engineering, University of Lagos, Lagos, Nigeria, 2Department of Materials and Metallurgical Engineering, University of Ilorin, Ilorin, Nigeria,3Department of Metallurgical Engineering, Yaba College of Technology, Lagos, Nigeria, 2013. Page 15/30 Page 15/30 25. Janowak and Gundlach, "A modern approach to alloying gray Iron", AFS, Vol.90, 1982, pp 847-862. 26. Rosenthal, "Theory of metal casting", TMH Co. References “Caracterização da Integridade de Superfícies Usinadas Para Produção de Moldes e Matrizes”- Pró-reitora de Pós-graduação e Pesquisa Coordenadoria de Pós-graduação Programa de Pós- graduação em Materiais - Universidade de Caxias do Sul-Caxias do Sul. RS 2006. 47. Oliveira, J. M. “Caracterização da Integridade de Superfícies Usinadas Para Produção de Moldes e Matrizes”- Pró-reitora de Pós-graduação e Pesquisa Coordenadoria de Pós-graduação Programa de Pós- graduação em Materiais - Universidade de Caxias do Sul-Caxias do Sul. RS 2006. 48. ASM. "Speciality Handbook: Cast Irons",Estados Unidos: ASM International, 1996, p. 33-267. 48. ASM. "Speciality Handbook: Cast Irons",Estados Unidos: ASM International, 1996, p. 33-267. 45. Bagetti, J. H. “Análise da Usinabilidade, Deformação e Temperatura no Fresamento dos Ferros Fundidos Vermicular e Cinzento”. Dissertação (Mestrado em Engenharia Mecânica) – Departamento de Engenharia Mecânica, Universidade Federal de Santa Catarina, 2009. References Bonney, Influence of the number of inserts for tool life evaluation in face milling of steels, International Journal of Machine Tools and Manufacture, 44 695-700, 2004. 39. ABNT NBR ISO 4288 – “Especificações geométricas de produto (GPS) - Rugosidade: Método do perfil - Regras e procedimentos para avaliação de rugosidade”, 2008. 40. Doré, C. “Influência da Variação da Nodularidade na Usinabilidade do Ferro Fundido Vermicular”. Dissertação (Mestrado em Engenharia Mecânica) – Departamento de Engenharia Mecânica, Universidade Federal de Santa Catarina, Florianópolis, 2007. 41. Xavier, F. A. “Aspectos Tecnológicos do Torneamento do Ferro Fundido Vermicular com Ferramentas de Metal-duro, Cerâmica e CBN”. Dissertação (Mestrado em Engenharia Mecânica) – Departamento de Engenharia Mecânica, Universidade Federal de Santa Catarina, 2003. 42. König, W.; Klocke, F. Fertigungsverfahren: Drehen, Frasen und Bohren. Berlim: Heidelberg, 2008. p. 562. 42. König, W.; Klocke, F. Fertigungsverfahren: Drehen, Frasen und Bohren. Berlim: Heidelberg, 2008. p. 562. 43. Machado, Á. R.; Abrão, A. M.; Coelho, R. T.; Da Silva, M. B. “Teoria da Usinagem dos Materiais”. 3. ed. São Paulo – SP: Edgard Blücher, 2015. p. 407. 43. Machado, Á. R.; Abrão, A. M.; Coelho, R. T.; Da Silva, M. B. “Teoria da Usinagem dos Materiais”. 3. ed. São Paulo – SP: Edgard Blücher, 2015. p. 407. Page 16/30 44. Reuter, U., Schulz, H., Dawson, S., Hollinger, I., Robbins, M., Daeth, J. "The Effect of Metallurgical Variables on the Machinability of Compacted Graphite Iron". Society of automotive engineers, Inc, Alemanhã, 2001, Page 16/30 p 1-18. 45. Bagetti, J. H. “Análise da Usinabilidade, Deformação e Temperatura no Fresamento dos Ferros Fundidos Vermicular e Cinzento”. Dissertação (Mestrado em Engenharia Mecânica) – Departamento de Engenharia Mecânica, Universidade Federal de Santa Catarina, 2009. 46. Guenza, J.E. “Análise do Desempenho do Fresamento em Altas Velocidades de Corte do Ferro Fundido GG25 em Aplicação Industrial” Dissertação (Mestrado em Engenharia) - Programa de Pós-graduação em Engenharia Mecânica e de Materiais, Universidade Tecnológica Federal do Paraná, Curitiba, 134 p. 2008. 46. Guenza, J.E. “Análise do Desempenho do Fresamento em Altas Velocidades de Corte do Ferro Fundido GG25 em Aplicação Industrial” Dissertação (Mestrado em Engenharia) - Programa de Pós-graduação em Engenharia Mecânica e de Materiais, Universidade Tecnológica Federal do Paraná, Curitiba, 134 p. 2008. 47. Oliveira, J. M. 46. Guenza, J.E. “Análise do Desempenho do Fresamento em Altas Velocidades de Corte do Ferro Fundido GG25 em Aplicação Industrial” Dissertação (Mestrado em Engenharia) - Programa de Pós-graduação em Engenharia Mecânica e de Materiais, Universidade Tecnológica Federal do Paraná, Curitiba, 134 p. 2008. Figures Page 17/30 Figure 1 Micrographs of material surfaces with and without 3% Nital attack, obt (SEM) and optical microscope: a) FC 250; b) FC 300 (Mo); c) FC 300 (Mo Figure 1 Micrographs of material surfaces with and without 3% Nital attack, obtained by scanning electron microscope (SEM) and optical microscope: a) FC 250; b) FC 300 (Mo); c) FC 300 (Mo+RG); d) FV450. Figure 1 Micrographs of material surfaces with and without 3% Nital attack, obtained by scanning electron microscope (SEM) and optical microscope: a) FC 250; b) FC 300 (Mo); c) FC 300 (Mo+RG); d) FV450. Micrographs of material surfaces with and without 3% Nital attack, obtained by scanning electron microscope (SEM) and optical microscope: a) FC 250; b) FC 300 (Mo); c) FC 300 (Mo+RG); d) FV450. Page 18/30 Figure 2 a) Identification of MnS inclusions; b) Determination of the cementite interlayer spacing of the perlite. Figure 2 a) Identification of MnS inclusions; b) Determination of the cementite interlayer spacing of the perlite. Figure 2 a) Identification of MnS inclusions; b) Determination of the cementite interlayer spacing of the perlite. Page 19/30 Figure 3 Figure 3 Figure 3 Page 19/30 Page 19/30 The geometry of the tool used in the tests. Figure 4 (a) Average amount of MnS/mm² in gray cast iron. (b) MnS particle size distribution in gray Figure 4 (a) Average amount of MnS/mm² in gray cast iron. (b) MnS particle size distribution in gray (a) Average amount of MnS/mm² in gray cast iron. (b) MnS particle size distribution in gray cast iron. Page 20/30 Figure 5 Pearlite interlayer spacing. Page 21/30 Figure 6 Maximum flank wear (VBBmax) about Tool life, interpolated for a VBBmax = 0.4 mm. Fi 6 Figure 6 Maximum flank wear (VBBmax) about Tool life, interpolated for a VBBmax = 0.4 mm. Maximum flank wear (VBBmax) about Tool life, interpolated for a VBBmax = 0.4 mm. Page 22/30 Figure 7 Effects of the test variables in the tool life. (a) material; (b) cutting speed; (c) feed rate. Figure 7 Effects of the test variables in the tool life. (a) material; (b) cutting speed; (c) feed rate. Page 23/30 Figure 8 Combine the effects of the machined material and cutting speed in relation to the tool life for (a) f = 0. / f 0 2 / Figure 8 Combine the effects of the machined material and cutting speed in relation to the tool life for (a) f = 0. Figure 8 Combine the effects of the machined material and cutting speed in relation to the tool life for (a) f = 0.1 mm/rev; f = 0.2 mm/rev. Page 24/30 Page 24/30 Figure 9 Correlations between mechanical properties and the overall machinability of the tool life, interpolated for a VBBmax = 0.4 mm. (a) Pearlite interlayer spacing; (b) Ultimate tensile strength; (c) Microhardness of the pearlite; (d) Brinell hardness. Correlations between mechanical properties and the overall machinability of the tool life, interpolated for a VBBmax = 0.4 mm. (a) Pearlite interlayer spacing; (b) Ultimate tensile strength; (c) Microhardness of the pearlite; (d) Brinell hardness. (d) Brinell hardness. Page 25/30 Page 25/30 Page 25/30 Figure 10 View of the rake surfaces of the tools used at cutting speed vc = 230 (m/min) and feed per rev f = 0.1 (mm/rev): a) FV 450; b) FC 300 (Mo+RG); c) FC 300 (Mo); d) FC 250. Figure 10 Figure 10 View of the rake surfaces of the tools used at cutting speed vc = 230 (m/min) and feed per rev f = 0.1 (mm/rev): a) FV 450; b) FC 300 (Mo+RG); c) FC 300 (Mo); d) FC 250. Page 26/30 Figure 11 View of the rake surfaces of the tools used at cutting speed vc = 350 (m/min) and feed per rev f = 0.1 (mm/rev): a) FV 450; b) FC 300 (Mo+RG); c) FC 300 (Mo); d) FC 250. Figure 11 Figure 11 Effect of the material's parameters on the roughness of the machined surface (a) Pearlite interlayer spacing; (b) Ultimate tensile strength; (c) Microhardness of the pearlite; (d) Brinell hardness. Figure 11 View of the rake surfaces of the tools used at cutting speed vc = 350 (m/min) and feed per rev f = 0.1 (mm/rev): a) FV 450; b) FC 300 (Mo+RG); c) FC 300 (Mo); d) FC 250. Page 27/30 Page 27/30 Figure 12 Figure 12 Average roughness profile of the machined surface. Average roughness profile of the machined surface. Average roughness profile of the machined surface. Page 28/30 Page 28/30 Effects of the cutting speed in the Ra. Page 29/30 Page 29/30 Figure 14 Fi 14 Figure 14 Effect of the material's parameters on the roughness of the machined surface (a) Pearlite interlayer spacing; (b) Ultimate tensile strength; (c) Microhardness of the pearlite; (d) Brinell hardness. Effect of the material's parameters on the roughness of the machined surface (a) Pearlite interlayer spacing; (b) Ultimate tensile strength; (c) Microhardness of the pearlite; (d) Brinell hardness. Page 30/30
https://openalex.org/W3175004591	https://www.businessperspectives.org/images/pdf/applications/publishing/templates/article/assets/15134/BBS_2021_02_Christiani.pdf	English	null	Proposed changes to the Bank Indonesia law as a solution to the impact of the COVID-19 spread on banking in Indonesia	Banks and bank systems	2,021	cc-by	7,654	businessperspectives.org “Proposed changes to the Bank Indonesia law as a solution to the impact of the COVID-19 spread on banking in Indonesia” AUTHORS Theresia Anita Christiani ARTICLE INFO Theresia Anita Christiani (2021). Proposed changes to the Bank Indonesia law as a solution to the impact of the COVID-19 spread on banking in Indonesia. Banks and Bank Systems, 16(2), 127-136. doi:10.21511/bbs.16(2).2021.12 DOI http://dx.doi.org/10.21511/bbs.16(2).2021.12 RELEASED ON Tuesday, 15 June 2021 RECEIVED ON Monday, 07 December 2020 ACCEPTED ON Friday, 28 May 2021 LICENSE This work is licensed under a Creative Commons Attribution 4.0 International License JOURNAL "Banks and Bank Systems" ISSN PRINT 1816-7403 ISSN ONLINE 1991-7074 PUBLISHER LLC “Consulting Publishing Company “Business Perspectives” FOUNDER LLC “Consulting Publishing Company “Business Perspectives” NUMBER OF REFERENCES 35 NUMBER OF FIGURES 0 NUMBER OF TABLES 1 © The author(s) 2021. This publication is an open access article. “Proposed changes to the Bank Indonesia law as a solution to the impact of the COVID-19 spread on banking in Indonesia” AUTHORS Theresia Anita Christiani ARTICLE INFO Theresia Anita Christiani (2021). Proposed changes to the Bank Indonesia law as a solution to the impact of the COVID-19 spread on banking in Indonesia. Banks and Bank Systems, 16(2), 127-136. doi:10.21511/bbs.16(2).2021.12 DOI http://dx.doi.org/10.21511/bbs.16(2).2021.12 RELEASED ON Tuesday, 15 June 2021 RECEIVED ON Monday, 07 December 2020 ACCEPTED ON Friday, 28 May 2021 LICENSE This work is licensed under a Creative Commons Attribution 4.0 International License JOURNAL "Banks and Bank Systems" ISSN PRINT 1816-7403 ISSN ONLINE 1991-7074 PUBLISHER LLC “Consulting Publishing Company “Business Perspectives” FOUNDER LLC “Consulting Publishing Company “Business Perspectives” NUMBER OF REFERENCES 35 NUMBER OF FIGURES 0 NUMBER OF TABLES 1 © The author(s) 2021. This publication is an open access article. “Proposed changes to the Bank Indonesia law as a solution to the impact of the COVID-19 spread on banking in Indonesia” “Proposed changes to the Bank Indonesia law as a solution to the impact of the COVID-19 spread on banking in Indonesia” © The author(s) 2021. This publication is an open access article. © The author(s) 2021. This publication is an open access article. businessperspectives.org businessperspectives.org Banks and Bank Systems, Volume 16, Issue 2, 2021 Theresia Anita Christiani (Indonesia) JEL Classification K12, K22, O24 JEL Classification K12, K22, O24 BUSINESS PERSPECTIVES www.businessperspectives.org LLC “СPС “Business Perspectives” Hryhorii Skovoroda lane, 10, Sumy, 40022, Ukraine www.businessperspectives.org LLC “СPС “Business Perspectives” Hryhorii Skovoroda lane, 10, Sumy, 40022, Ukraine Abstract Every amendment to the Bank Indonesia Law is caused by a situation that requires changes to the Law regulating the Central Bank in Indonesia as a solution. The spread of COVID-19 in Indonesia has also led to proposals to amend the Bank Indonesia Law. The purpose of the study is to find answers to the relevance of the proposed Amendment to Bank Indonesia Law to address the spread of COVID-19 to banking institutions in Indonesia. This type of research methods is normative legal research. In normative legal analysis, secondary data are used, consisting of primary and secondary legal materials. They are obtained from applicable regulations in Indonesia. The study results show that every change is always based on events that prove the weak imple- mentation of existing rules with a regulatory and conceptual approach. The spread of COVID-19 is a situation, that has no practical basis and requires amendments to the Bank Indonesia Law as an alternative solution. Also, the proposed amendments are not yet relevant to address the impact of COVID-19 on banks because they have not yet realized and achieved the legal goals of providing benefits to the community. Received on: 7th of December, 2020 Accepted on: 28th of May, 2021 Published on: 15th of June, 2021 Received on: 7th of December, 2020 Accepted on: 28th of May, 2021 Published on: 15th of June, 2021 © Theresia Anita Christiani, 2021 Theresia Anita Christiani, PhD, Lecturer, Faculty of Law, Universitas Atma Jaya Yogyakarta, Indonesia. This is an Open Access article, distributed under the terms of the Creative Commons Attribution 4.0 International license, which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited. Conflict of interest statement: Author(s) reported no conflict of interest Keywords alternative, change, problems, regulation, qualitative, weak alternative, change, problems, regulation, qualitative, weak http://dx.doi.org/10.21511/bbs.16(2).2021.12 Theresia Anita Christiani, PhD, Lecturer, Faculty of Law, Universitas Atma Jaya Yogyakarta, Indonesia. 1. LITERATURE REVIEW ing the activity is seen from whether it is good or not (Niesen, 2019). In this case, the Government must increase the happiness that the community will enjoy. The best-known phrase from Bentham is “The greatest happiness for the greatest numbers.” The purpose of Law is to achieve happiness for the majority of people. Utilitarianism theory is asso- ciated with legislators or legislators, and laws are a concrete form of Law. Legislators or legislators must try to make the Law bring happiness to the community. Jeremy Bentham’s concept of utilitar- ianism is the basis for the philosophy of law for- mation used by Alanah Josey (2019) and Ekeløve- Slydal (2018) in their research. The purpose of seeking profit is used for the business’s continuity and the company and benefits many people. If this is not the case, it can return the State’s duty to reg- ulate the purpose of seeking profit according to its nature. Law serves as a tool for social engineering, it is at the forefront of societal development. The function of Law as a social engineering tool was introduced by a thinker from the Sociological Jurisprudence school (Stone, 1966). As Roscoe Pound stated, there is a relevance of the function of Law. There is a relationship between legal norms and their func- tion in controlling community activities (Barry, 1989). Community activity in question is an eco- nomic activity that requires the presence of Law (Tholl et al., 2020). Likewise, economic activities requiring banking institutions require a company to have a law guaranteeing the fulfillment of cer- tainty of legal protection (Riles, 2021). A transpar- ent and healthy banking institution is needed to develop the economic sector. Natural law theory can be used most in answering the real purpose of the Law itself. With regard to the meaning of Law, one can discuss the development of thoughts about the Law from the Greek era to the present. Aristotle was the first thinker to give his views on the purpose of Law. He said that humans are social beings, or the term given is the Zoon Politocoon. Aristotle said that the purpose of Law is to achieve a better life. He also distinguished between pos- itive Law and Natural Law. Thomas Aquinas ar- gues that the Law has a duty that lies in the heart (Mokriski, 2020). The legal objective is the legal objective put forward by Jeremy Bentham. INTRODUCTION The spread of COVID-19 has led the Indonesian Government to is- sue Law No. 2 of 2020 Concerning Law No. 2 of 2020 Stipulation of Government Regulation in Lieu of Law No. 1 of 2020 concerning State Financial Policy and Financial System Stability for Handling the Corona Virus Disease 2019 (Covid-19) Pandemic and/or In Facing Threats That Endanger the National Economy and/or Financial System Stability Becomes Law. Law No. 2 of 2020 provides a basis for the Government, Financial Services Authority, Bank Indonesia and related parties to overcome the impact of the spread of COVID-9 on Indonesia’s economy. Based on Law No. 2 of 2020, a proposal was made to amend the Bank Indonesia Law at a hearing with the Legislative Body of the House of Representatives of the Republic of Indonesia in the current economic situation in Indonesia. The pro- posed amendments are the banking supervision function from the Financial Services Authority to Bank Indonesia, changes in the con- cept of Bank Indonesia independence, changes in the Formulation of Bank Indonesia’s Goals, and the Proposal to Establish a Monetary Board. One of the significant issues for the Amendment of the Bank Indonesia Law that has emerged is to return bank supervision to Bank Indonesia. Based on Law No. 21 of 2011, micro-prudential regulation and supervision have been transferred to Financial Service Authority 127 http://dx.doi.org/10.21511/bbs.16(2).2021.12 Banks and Bank Systems, Volume 16, Issue 2, 2021 from Bank Indonesia. Returning micro-prudential regulation and supervision from the Financial Services Authority to Bank Indonesia as one of the proposed amendments to the Bank Indonesia Law and other proposed amendments is an urgent legal issue for discussion, due to, inter alia, the transfer of micro-prudential regulatory and supervisory duties from Bank Indonesia to the Financial Services Authority from a period and practice that had just occurred. Amending the Law is not only a legal but a political process. Therefore, the legal problem is formulated: Are the proposed amendments to the Bank Indonesia Law relevant as a solution to the impact of the spread of COVID-19 on banking institutions in Indonesia? The purpose of the study to find answers to the question of whether the proposed amend- ments to the Bank Indonesia Law are relevant as a solution to the impact of the spread of COVID-19 on banking institutions in Indonesia. 1. LITERATURE REVIEW Bank Indonesia’s position as an independent Central Bank has also experienced developments in exist- ing regulations. The mandate of the Indonesian Constitution could only be realized in the 1999 Bank Indonesia Law. The provision of independ- ence in the Bank Indonesia Law was due to a situ- ation of monetary crisis that forced the regulation of the independence of Bank Indonesia as the Central Bank. Regulatory renewal is a form of re- form in dealing with banking problems, stated by James R. Bath (Barth et al., 2004). The meaning of independence of Bank Indonesia has also under- gone changes and developments from one regula- tion to the next. The meaning of independence changed before and after the Financial Services Authority Act was also changed with various new regulations related to the Central Bank. Law as a social engineering tool is manifested by the issu- ance of several regulations facing problems that can arise in the future. Also, at this time, the spread of the coronavirus caused the Indonesian Government to issue Law No. 2 of 2020 Concerning the Stipulation of Government Regulations in place of Law No. 1 of 2020. That Law on State Financial Policy and Financial System Stability for Handling the Corona Virus Disease 2019 Pandemic (COVID-19) and the Context of Facing Threats That Endanger the Economic and Financial System Stability enters into Law. That Law Number 2 of 2020 provides a basis for the Government, Financial Services Authority, Bank Indonesia, and related parties to take steps to over- come the impact of the spread of the COVID-19 pandemic on banks and the economy. This law is- suance demonstrates the legal function’s imple- mentation as a social engineering tool as mandat- ed by Roscoe Pound (Pound, 1940). Previous re- search uses the legal concept of legal function con- ducted by Mgr Alexander (Szpojankowski, 2019). The concept of legal functions as a tool for social engineering is also used in other legal research by Jedidiah (Kroncke, 2012) and Wirawan and Saputra (2020). The objective of the Central Bank is to keep currency exchange rates stable. Many factors influence the implementation of these tasks. The Governor’s factor can be a determining factor for a Central Bank’s competence, such as the results of research from Ozili (2020). 1. LITERATURE REVIEW Jeremy Bentham argues that something is considered ac- ceptable or not measured by the consequences of that action. Jeremy Bentham is a thinker whose teachings are known as Utilitarianism (Cello, 2021). According to Bentham, the utility princi- ple is a principle that approves or rejects any ac- tion that increases or decreases the party’s happi- ness affected by the move. Approving and reject- Amendments to the Bank Indonesia Law are ef- forts made by the Government to achieve legal ob- jectives in regulating economic activities and pro- viding protection and benefits as, in essence, Jeremy Bentham said. Roscoe Pound’s opinion that the Law is a tool for social engineering is used as a basis for analyzing whether the proposed amendments to the Bank Indonesia Law can real- ize the legal function of bringing about change in society. Meanwhile, the opinion of Jeremy Bentham that the purpose of Law is to provide benefits and happiness to the community is used in analyzing whether the proposed amendments to the Bank Indonesia Law will benefit the com- munity. Based on those conceptual understand- ing, the basis for Bank Indonesia’s formation as the Central Bank is mandated in the 1945 Constitution as the supreme Constitution in Indonesia, associated with a Central Bank in 128 http://dx.doi.org/10.21511/bbs.16(2).2021.12 Banks and Bank Systems, Volume 16, Issue 2, 2021 Indonesia as stated in Article 23 D of the 1945 Constitution. This article confirms that “the State has a Central Bank whose composition, Law regu- lates authority, responsibility, and independence.” The Central Bank’s independence can be seen from three criteria: First, the Governor’s appoint- ment must be approved by the Parliament. Thus, the Central Bank’s legal independence is meas- ured by the Governor’s position with the Parliament’s approval. Second, the Central Bank’s issue of accountability is responsible to Parliament, not to the President. Third, in determining mone- tary policy, an independent central bank has the power to decide monetary policy (Azikin, 2015; Cottarelli, 1994). Regulations in Indonesia have regulated the position of independence according to the three criteria. Some studies measure the Central Bank’s independence from the inflation rate, the independence from the high and low in- flation rates (Ferr’e & Manzano, 2020). Concerning the independence of the Central Bank, Abdulkader Alquer conducted research related to the effect of the inflation target concerning the level of Central Bank’s independence and transparency in deter- mining monetary policy (Abdelkader, 2018). 1. LITERATURE REVIEW Although Yessy Andriani and Prasanna Gai’s analysis states that the Central Bank’s independence will posi- tively affect the inflation target, the Governor’s change does not show any significance with the inflation target in Indonesia (Andriani & Gai, 2013). Changing a law must have a background and urgency. Likewise, the proposed amendment to the Bank Indonesia Law is an effort so that the Law can become an instrument that helps the State in achieving happiness for the community. Several arguments are strong enough for the Bank Indonesia Law to be amended immediately. Since Law on Bank Indonesia in 1999 has been issued, there have been dynamics in developing other laws that have influenced the Bank Indonesia Law’s provisions. These laws are the Financial Services Authority Law, the Currency Law, and the Law of Prevention and Management of Financial System Crisis. The last one is related to the existence of Law No. 2 of 2020. It is based on the principle of lex posterior derogat legi priori (Sudikno Mertokusumo, 2016), which implies that the new Law will defeat the old Law. The enact- ment of the Financial Services Authority Act in Indonesia has juridical consequences for Bank Indonesia’s duties. The juridical consequence was that The FSA law transferred the supervisory re- Indonesia as stated in Article 23 D of the 1945 Constitution. This article confirms that “the State has a Central Bank whose composition, Law regu- lates authority, responsibility, and independence.” The Central Bank’s independence can be seen from three criteria: First, the Governor’s appoint- ment must be approved by the Parliament. Thus, the Central Bank’s legal independence is meas- ured by the Governor’s position with the Parliament’s approval. Second, the Central Bank’s issue of accountability is responsible to Parliament, not to the President. Third, in determining mone- tary policy, an independent central bank has the power to decide monetary policy (Azikin, 2015; Cottarelli, 1994). Regulations in Indonesia have regulated the position of independence according to the three criteria. Some studies measure the Central Bank’s independence from the inflation rate, the independence from the high and low in- flation rates (Ferr’e & Manzano, 2020). Concerning the independence of the Central Bank, Abdulkader Alquer conducted research related to the effect of the inflation target concerning the level of Central Bank’s independence and transparency in deter- mining monetary policy (Abdelkader, 2018). 2. RESEARCH METHODS The type of research is normative legal research. The normative legal analysis uses secondary data consisting of primary and secondary legal materi- als (Suhartini et al., 2019). They are sourced from applicable regulations in Indonesia. Hierarchical regulation will become a source of Law. The Law adheres to the Civil Law System. The primary legal materials will complement secondary legal mate- rials consisting of expert opinions and related ar- ticles from journals and dictionaries. Data analy- sis begins with collecting data, separating the da- ta according to its relevance to the problem, then describing and analyzing the data using qualita- tive techniques. The data used is secondary data. Qualitative data are used to support qualitative analysis (Suradiyanto, 2019). The approach used is the statutory approach (Sudika, 2021) and con- ceptual approach (Sudiarawan et al., 2020). The regulatory approach is used to study existing legal problems by reviewing the Bank Indonesia Law and other related laws. The conceptual approach is used to analyze legal issues using legal function and legal objectives. Also, deductive conclusion techniques are used. 1. LITERATURE REVIEW Central banking thinking development requires adjust- ments to the Central Bank’s objectives to achieve and maintain the rupiah value’s stability. Based on this, the adjustment of the Central Bank’s duties will support the role of the Central Bank to pro- mote the achievement of objectives. Indonesia’s economy and financial system, integrated with the global economic system, demand Bank Indonesia’s participation in creating financial sys- tem stability (Gaganis et al., 2021). The concept of the function of Law as a tool for social engineering has become the basis of arguments for the urgency of changing Bank Indonesia. The purpose of Law for the benefit of most society will be also used as the basis of conceptual framework and theory. There is a connection between the origin of argu- mentation with one another. The point is that the basis for this argument calls for resolution in the form of Amendments to the Bank Indonesia Law. The concept of the legal function and legal objec- tives will be used as a basis for analysis in discuss- ing whether the proposed amendments to the Bank Indonesia Law are the answer to banking problems in overcoming the impact of the COVID-19 pandemic. The proposed amendments to the Bank Indonesia Law realize the legal func- tion as a social engineering tool (Roscoe Pound) if the amendments can substantially change Bank Indonesia in terms of position, duties, goals and institutions for the better. If this change for the better occurs, then the legal objective to benefit the community, as stated by Jeremy Bentham, will be achieved. The regulatory approach is used as this study uses various regulations related to the proposed amendment to the Bank Indonesia Law. The conceptual approach is used because several banking legal concepts are used to analyze the problem of proposed changes to the Bank Indonesia law in terms of its relevance as a solu- tion to the impact of the spread of COVID-19 on banking institutions in Indonesia. 1. LITERATURE REVIEW Bank Indonesia’s position as an independent Central Bank has also experienced developments in exist- ing regulations. The mandate of the Indonesian Constitution could only be realized in the 1999 Bank Indonesia Law. The provision of independ- ence in the Bank Indonesia Law was due to a situ- ation of monetary crisis that forced the regulation of the independence of Bank Indonesia as the Central Bank. Regulatory renewal is a form of re- form in dealing with banking problems, stated by James R. Bath (Barth et al., 2004). The meaning of independence of Bank Indonesia has also under- gone changes and developments from one regula- tion to the next. The meaning of independence changed before and after the Financial Services Authority Act was also changed with various new regulations related to the Central Bank. Law as a social engineering tool is manifested by the issu- ance of several regulations facing problems that can arise in the future. Also, at this time, the spread of the coronavirus caused the Indonesian Government to issue Law No. 2 of 2020 Concerning the Stipulation of Government Regulations in place of Law No. 1 of 2020. That Law on State Financial Policy and Financial System Stability for Handling the Corona Virus Disease 2019 129 http://dx.doi.org/10.21511/bbs.16(2).2021.12 Banks and Bank Systems, Volume 16, Issue 2, 2021 Indonesia still has the task and authority to regu- late and supervise the macroprudential sector. This is stipulated in Article 55, paragraph 2 of the Financial Services Authority Law, elucidation of article 7 in conjunction with article 40 of the Financial Services Authority Law). In detail, it can be stated that apart from the Financial Services Authority Law, there were juridical factors that led to changes to the Bank Indonesia Law. These ju- ridical factors include the issuance of Law No. 9 of 2016 on the Prevention and Management of Financial System Crises, the Currency Law, and the existence of Law No. 2 of 2020. The last Law is a law formed in an economic situation affected by the spread of COVID-19. The Bank Indonesia Law must adapt to the changes in the world of rules and regulations in Indonesia, which have caused several laws and regulations in the Bank Indonesia Law to become irrelevant in accordance with the legal function as a social engineering tool. 3. RESULTS AND DISCUSSION The objective of returning the supervisory duties of banking institutions to Bank Indonesia is to make macroeconomic stability more effective. It means that replacing the tasks and supervisory authority of banking institutions with Bank Indonesia will support macroeconomic stability. It is a proposal for changes and improvements in macroeconomic management. The goal is that the Bank Indonesia and the Government synergy will help achieve and maintain economic stability. amendment is a proposal to return the banking supervision function from the Financial Services Authority to Bank Indonesia. In Indonesia today, The Financial Services Authority performs super- vising banking institutions in the macro-pruden- tial field under the Financial Services Authority Law. Bank Indonesia has the task of regulating and supervising banking institutions in the mac- roprudential sector. Supervision of the micro-pru- dential Financial Services Authority before the Financial Services Authority’s existence was the task and Authority of Bank Indonesia. The transfer of supervisory duties from Bank Indonesia to the Financial Services Authority is based on Article 34 of the Bank Indonesia Law. The Bank Indonesia Law’s proposed amendment is to return regu- lating and supervising banks from the Financial Services Authority to Bank Indonesia, both on mi- cro-prudential and macroprudential matters. The objective of returning the supervisory duties of banking institutions to Bank Indonesia is to make macroeconomic stability more effective. It means that replacing the tasks and supervisory authority of banking institutions with Bank Indonesia will support macroeconomic stability. It is a proposal for changes and improvements in macroeconomic management. The goal is that the Bank Indonesia and the Government synergy will help achieve and maintain economic stability. The data above shows that there are 1,340 financial institutions operating in the non-banking finan- cial industry. In terms of data, the number of su- pervised financial institutions can be analyzed as the number of responsibilities for these tasks, which may interfere with the implementation of the Financial Services Authority’s duties with the available human resources. Not to mention, there are many unlicensed investment cases in public circulation, which will disturb public confidence in financial institutions and the Financial Services Authority itself. There is a limited number of hu- man resources for the provision of financial ser- vices. In addition, the debate on the concept of Article 34 is still recorded in the Constitutional Court Decision. It became challenging to agree to this proposal. 3. RESULTS AND DISCUSSION There are at least 12 (twelve) proposals for amend- ments to articles and two recommendations for deletions proposed in the amendments to the Bank Indonesia Law. The submitted amended ar- ticles include Article 4, 7, 10, 11, 34, 43, 55, 56, 62, and 75. The proposed elimination is addressed in article 9 and article 58 of the Bank Indonesia Law. The following will describe four proposals for amendments to that Law. The proposed first 130 http://dx.doi.org/10.21511/bbs.16(2).2021.12 http://dx.doi.org/10.21511/bbs.16(2).2021.12 Banks and Bank Systems, Volume 16, Issue 2, 2021 Table 1. Number of institutions operating in the non-banking financial industry Source: Financial Services Authority (2020). Financial institution September 2020 Total Conventional Sharia Insurance 139 13 152 Financial institution 235 9 244 Pension fund 216 4 220 Specialized financial institutions 112 5 117 Non-bank financial industry 228 0 228 Micro finance institution 146 77 223 Fintech 145 11 156 TOTAL 1,221 119 1,340 Table 1. Number of institutions operating in the non-banking financial industry Source: Financial Services Authority (2020). 5,936 rural bank offices throughout Indonesia. The capital market is an institution that is the domain of supervision and regulation of the Financial Services Authority. The data above shows that 123 securities companies are operating in the capital market. However, various other parties/compa- nies are involved in capital market activities under the regulation and supervision of the Financial Services Authority. These companies, among oth- ers, include securities administration bureaus, se- curities rating companies, and trustees. The num- ber of companies is enormous. Many companies operating in the non-bank financial industry un- der the Financial Services Authority will be pre- sented below. amendment is a proposal to return the banking supervision function from the Financial Services Authority to Bank Indonesia. In Indonesia today, The Financial Services Authority performs super- vising banking institutions in the macro-pruden- tial field under the Financial Services Authority Law. Bank Indonesia has the task of regulating and supervising banking institutions in the mac- roprudential sector. Supervision of the micro-pru- dential Financial Services Authority before the Financial Services Authority’s existence was the task and Authority of Bank Indonesia. The transfer of supervisory duties from Bank Indonesia to the Financial Services Authority is based on Article 34 of the Bank Indonesia Law. The Bank Indonesia Law’s proposed amendment is to return regu- lating and supervising banks from the Financial Services Authority to Bank Indonesia, both on mi- cro-prudential and macroprudential matters. http://dx.doi.org/10.21511/bbs.16(2).2021.12 3. RESULTS AND DISCUSSION Bank Indonesia’s current objectives state that Bank Indonesia aims to attain and maintain the rupiah value’s stability. Bank Indonesia shall implement monetary policy in a sustainable, consistent and transparent manner and consider the Government’s general guidelines in the economic sector. At the same time, Bank Indonesia’s objec- tives can be achieved by carrying out the three tasks of Bank Indonesia as set out in Article 8. To accomplish the Bank Indonesia objectives referred to in Article 7 of the Law, Bank Indonesia has the following tasks: to establish and implement mone- tary policy, regulate and maintain the smooth op- eration of the payment system, and regulate and supervise banks. The aim of Indonesia’s proposed changes to the Bank is only to maintain macroe- conomic stability and promote economic growth and the creation of sustainable employment op- portunities. Proposed tasks for Bank Indonesia are contained in Article 6. The proposed change in the objectives and tasks of Bank Indonesia follows on from the proposal to return micro-prudential banking duties to Bank Indonesia from the Financial Services Authority. The fourth proposed amendment is to establish a Monetary Board. The existence of the Monetary Board is not currently regulated in Indonesia. The provisions of the Monetary Board existed during the Old Order pe- riod. In the Regulatory Period in the Indonesian Old Order, provisions were governing the Monetary Board’s existence. This provision has the consequence that the position between the Central Bank and the Government is not firm. This provision had an adverse effect on Bank Indonesia’s independence because Bank Indonesia was chaired by a Monetary Board, one of the Ministers of Finance. So it was not legalized in the existing Law of Bank Indonesia. The establish- ment of a Monetary Board is proposed in accord- ance with Articles 9A, 9B, and C. Article 9B, para- graph 1, provides for the establishment of a Monetary Board. The Minister of Finance will chair the Monetary Board. Monetary Board mem- bers consist of the Minister of Finance, one Minister in charge of the economy, Governor of Bank Indonesia, Senior Deputy Governor of Bank Indonesia, and a Chairman of the Board of Commissioners of the Financial Services Authority. This Council has the function of being able to lead, coordinate, and direct under the authority of Bank Indonesia. 3. RESULTS AND DISCUSSION The goal is that Bank Indonesia’s poli- cies align with general guidelines in the Government’s field, the Authority of the Government. The goal is for the Bank Indonesia to have independence to coordinate with the Government in developing policies to maintain economic stability. There is a relationship between the determination of monetary policy and a coun- try’s financial stability (Nair & Anand, 2020). This explanation shows that the experience of the harmful consequences of having a Monetary Board in the Bank Indonesia Act demands the elimination of this institution. This means that if, stitutions, there has not been any significant prob- lem that has prompted the Financial Service Authority’s duties to Bank Indonesia. The second proposed amendment concerns the position of in- dependence of Bank Indonesia. Proposed changes related to the consequences of changing the mean- ing of Bank Indonesia independence are contained in the Proposed Amendments to the Bank Indonesia Law, Articles 4 and 7, the abolition of Article 9, items 9A, 9B, 9C, and 10. The proposed amendments to Article 4 emphasize that Bank Indonesia is an independent central bank, but Article 4 adds the phrase “cooperate with the gov- ernment.” This phrase is a proposed amendment to the Bank Indonesia Law. The term disturbs the meaning of Bank Indonesia’s independence in the existence of the Bank Indonesia Law. In concept, the measure of a central bank’s independence is the central bank authority in determining mone- tary policy (Moutot, 2020). If the proposal re- moves the Central Bank’s Authority in deciding economic policy, then the request is contrary to its independence concept. Furthermore, the proposal to abolish Article 9 of the Bank Indonesia Law, which stipulates the prohibition of intervening in the implementation of Bank Indonesia’s duties, further obscures the meaning of independence in the Law of Bank Indonesia. Suppose a change in the definition of independence is used to justify the Bank Indonesia Law Amendment. It is ques- tionable whether the empirical facts show that Bank Indonesia independence is the root cause of the current economic stability problem. The pan- demic is the cause, not because of the independ- ence of Bank Indonesia. This proposed change would hamper the Law’s goal of benefiting society, as Jeremy Bentham argued. The third proposed amendment is to replace the formulation of Indonesia’s Bank objectives and tasks. 3. RESULTS AND DISCUSSION The process of shifting regulatory and supervisory tasks from Bank Indonesia to the Financial Services Authority requires a lot of time, effort and cost to implement. From the perspec- tive of regulation and supervision of banking in- The number of financial institutions under the Financial Services Authority can be obtained by integrating the financial institutions’ regulation and supervision. The number of banks is 110 com- mercial banks and 1,517 rural banks, as well as 123 securities companies (FSA, 2021). The number of commercial bank offices is 30,837, and there are 131 Banks and Bank Systems, Volume 16, Issue 2, 2021 stitutions, there has not been any significant prob- lem that has prompted the Financial Service Authority’s duties to Bank Indonesia. The second proposed amendment concerns the position of in- dependence of Bank Indonesia. Proposed changes related to the consequences of changing the mean- ing of Bank Indonesia independence are contained in the Proposed Amendments to the Bank Indonesia Law, Articles 4 and 7, the abolition of Article 9, items 9A, 9B, 9C, and 10. The proposed amendments to Article 4 emphasize that Bank Indonesia is an independent central bank, but Article 4 adds the phrase “cooperate with the gov- ernment.” This phrase is a proposed amendment to the Bank Indonesia Law. The term disturbs the meaning of Bank Indonesia’s independence in the existence of the Bank Indonesia Law. In concept, the measure of a central bank’s independence is the central bank authority in determining mone- tary policy (Moutot, 2020). If the proposal re- moves the Central Bank’s Authority in deciding economic policy, then the request is contrary to its independence concept. Furthermore, the proposal to abolish Article 9 of the Bank Indonesia Law, which stipulates the prohibition of intervening in the implementation of Bank Indonesia’s duties, further obscures the meaning of independence in the Law of Bank Indonesia. Suppose a change in the definition of independence is used to justify the Bank Indonesia Law Amendment. It is ques- tionable whether the empirical facts show that Bank Indonesia independence is the root cause of the current economic stability problem. The pan- demic is the cause, not because of the independ- ence of Bank Indonesia. This proposed change would hamper the Law’s goal of benefiting society, as Jeremy Bentham argued. The third proposed amendment is to replace the formulation of Indonesia’s Bank objectives and tasks. http://dx.doi.org/10.21511/bbs.16(2).2021.12 3. RESULTS AND DISCUSSION Bank Indonesia’s current objectives state that Bank Indonesia aims to attain and maintain the rupiah value’s stability. Bank Indonesia shall implement monetary policy in a sustainable, consistent and transparent manner and consider the Government’s general guidelines in the economic sector. At the same time, Bank Indonesia’s objec- tives can be achieved by carrying out the three tasks of Bank Indonesia as set out in Article 8. To accomplish the Bank Indonesia objectives referred to in Article 7 of the Law, Bank Indonesia has the following tasks: to establish and implement mone 132 http://dx.doi.org/10.21511/bbs.16(2).2021.12 Banks and Bank Systems, Volume 16, Issue 2, 2021 House of Representatives. The Government focus- es on fighting COVID-19, economic recovery, and maintaining financial system stability. The most important thing for the Government is the divi- sion of tasks and responsibilities, the check and balance mechanism of the institutions involved in achieving stability of the financial system. Financial system stability is urgently needed in the situation of the spread of COVID-19 in Indonesia. From a monetary perspective, it is im- perative to have good coordination between the Government and Bank Indonesia. When inter- preting arguments by analogy (Sudikno Mertokusumo, 2016), any proposed changes should be based on events that may indicate weak- nesses in the current Financial Services Authority’s supervision and regulation. The proposed amend- ments to the Bank Indonesia Law are not yet able to demonstrate the legal function as a social engi- neering tool, since the proposed amendments have not been able to bring changes to Bank Indonesia for the better. It can be said that the pro- posed amendments to the Bank Indonesia Law al- so failed to realize the legal objective of providing benefits to the community. The proposed amend- ments to the Bank Indonesia Law to address the impact of COVID-19 in Indonesia are not yet relevant. in its development, the Monetary Board’s propos- al is considered relevant again in the proposed amendments to the Law on Bank Indonesia, the negative impact of the implementation experience in the previous period should be taken into con- sideration. The establishment of the Monetary Board will disrupt the position of Bank Indonesia’s independence. That proposal is irrelevant in solv- ing current banking problems. The common situ- ation with economic stability can be achieved from many perspectives of the related institutions. 3. RESULTS AND DISCUSSION The associated institutions are Bank Indonesia as a Monetary Authority, the Financial Services Authority as an Authority for all financial institu- tions in Indonesia, the Deposit Insurance Corporation as a party that guarantees customer deposits, and the government party that organizes economic activities. The Government provides an opinion on the proposed changes. The Government believes that the Government’s position on Bank Indonesia’s role in maintaining economic stability is that the monetary policy of Bank Indonesia must be credible, effective, and independent (CNBC, 2020). It can be analyzed that the Government has not changed the importance of changing the current Bank Indonesia Law. Addressing efforts are linked to the impact of COVID-19. As it is known, the proposed amend- ments to laws in Indonesia are proposals from the http://dx.doi.org/10.21511/bbs.16(2).2021.12 CONCLUSION Based on the results and discussion above, it can be concluded that the proposed amendments to the Bank Indonesia Law are in essence the right of the Indonesia People’s Representative Council. Indonesia’s People’s Representative Council asks for alternative solutions to address the impact of the spread of COVID-19 on Indonesia’s banks and economy. The Amendment to the Law requires chang- es to the Bank Indonesia Law. In essence, however, the proposed amendments to the Indonesian Bank Law concerned the return of the banking supervision function from the Financial Services Authority to Bank Indonesia. Changes in the concept of Bank Indonesia independence, changes in the formulation of Bank Indonesia’s objectives, and the proposal to establish a Monetary Board are not appropriate until empirical facts prove weaknesses in implementing regulatory and supervisory mechanisms of Financial Services Authority for Banking. The spread of COVID-19 is a situation that has not provided a practical basis that requires amendments to the Bank Indonesia Law as an alternative solution. Therefore, the proposed amendment to the Law of Bank Indonesia is irrelevant in delivering solutions to the impact of COVID-19 in Indonesia. The proposed amendments to the Bank Indonesia Law cannot demonstrate the legal function as a social engineering tool. The proposed amendments to the Bank Indonesia Law have also failed to fulfill the lawful objective of providing benefits to the community, so they are not yet relevant as a solution to the impact of the spread of COVID-19 in Indonesia. A limitation of this study is that it focuses on four proposed changes to the Bank Indonesia Law. There are several proposals for amendments to the Law on Bank Indonesia that can be the subject of further research. 133 Banks and Bank Systems, Volume 16, Issue 2, 2021 AUTHOR CONTRIBUTIONS Conceptualization: Theresia Anita Christiani. Conceptualization: Theresia Anita Christiani. Data curation: Theresia Anita Christiani. Formal analysis: Theresia Anita Christiani. Funding acquisition: Theresia Anita Christiani. Investigation: Theresia Anita Christiani. Methodology: Theresia Anita Christiani. roject administration: Theresia Anita Christiani. Resources: Theresia Anita Christiani. Supervision: Theresia Anita Christiani. Validation: Theresia Anita Christiani. Visualization: Theresia Anita Christiani. Writing – original draft: Theresia Anita Christiani. Writing – reviewing & editing: Theresia Anita Christiani. ACKNOWLEDGMENT Thanks to Universitas Atma Jaya Yogyakarta, Indonesia, for providing funding for research and publication. 5. Barth, J. R., Caprio, G., & Levine, R. (2004). Bank regulation and supervision: what works best? Journal of Financial Intermediation, 13(2), 205-248. https://doi. org/10.1016/j.jfi.2003.06.002 REFERENCES www.legal-tools.org/doc/76b5aa/ pdf/ 6. Cello, L. (2021). Jeremy Bentham’s vision of international order. Cambridge Review of International Affairs, 34(1), 46-64. https://doi.or g/10.1080/09557571.2020.1722610 1. Aguir, A. (2018). Central Bank Credibility, Independence, and Monetary Policy. Journal of Central Banking Theory and Practice, 3, 91-110. https://doi. org/10.2478/jcbtp-2018-0025 10. Ferr´e, M., & Manzano, C. (2020). Independent Central Bank: Low Inflation at No Cost? A Model with Fiscal Policy. 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https://openalex.org/W3013356715	https://birmingham.elsevierpure.com/files/95825387/er.5347.pdf	English	null	High performance cooling of a HVDC converter using a fatty acid ester‐based phase change dispersion in a heat sink with double‐layer oblique‐crossed ribs	International journal of energy research	2,020	cc-by	15,540	General rights U l li General rights Unless a licence is specified above, all rights (including copyright and moral rights) in this document are retained by the authors and/or the copyright holders. The express permission of the copyright holder must be obtained for any use of this material other than for purposes permitted by law. •Users may freely distribute the URL that is used to identify this publication. 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High performance cooling of a HVDC converter using a fatty acid ester‐based phase change dispersion in a heat sink with double‐layer oblique‐crossed ribs Li, Qi; Fischer, Ludger; Qiao, Geng; Mura, Ernesto; Li, Chuan; Ding, Yulong DOI: 10.1002/er.v44.7 License: Creative Commons: Attribution (CC BY) Document Version Publisher's PDF, also known as Version of record Citation for published version (Harvard): Li, Q, Fischer, L, Qiao, G, Mura, E, Li, C & Ding, Y 2020, 'High performance cooling of a HVDC converter using a fatty acid ester‐based phase change dispersion in a heat sink with double‐layer oblique‐crossed ribs', International Journal of Energy Research, vol. 44, no. 7, pp. 5819-5840. https://doi.org/10.1002/er.v44.7 Citation for published version (Harvard): Li, Q, Fischer, L, Qiao, G, Mura, E, Li, C & Ding, Y 2020, 'High performance cooling of a HVDC converter using a fatty acid ester‐based phase change dispersion in a heat sink with double‐layer oblique‐crossed ribs', International Journal of Energy Research, vol. 44, no. 7, pp. 5819-5840. https://doi.org/10.1002/er.v44.7 Link to publication on Research at Birmingham portal University of Birmingham R E S E A R C H A R T I C L E R E S E A R C H A R T I C L E Summary Summary The paper concerns with a high performance cooling method for a HVDC con- verter using fatty acid ester-based phase change dispersion (PCD) in a heat sink with double-layer oblique-crossed ribs. Thermo-physical properties of PCDs were first characterized under both solid and liquid states, and the cooling per- formance of the heat exchanger was then experimentally examined, by heating two copper blocks clamped closely to the aluminium heating surfaces. A three- dimensional Euler-Euler multiphase approach was further performed to evalu- ate the thermal performance under different operating conditions including heating power, flowrate and PCD concentration. The results showed that the viscosity of PCD can be a 100 times that of water, but the increased pumping power was only ~17.01% on average. The use of the PCD achieved a lower tem- perature of heat sink and fluid than that of water under the same set of condi- tions due to the latent heat of the PCM, thus enabling a safer and cooler environment for temperature-sensitive HVDC components such as insulated gate bipolar transistors (IGBT). An optimal set of working conditions was pro- posed and a flowrate of 8 L/min under a heating power of 1.1 kW and a PCM concentration of 25% was recommended for industrial cooling operations. 2Lucerne University of Applied Sciences and Arts, Horw, Switzerland 3Global Energy Interconnection Research Institute Europe GmbH, Berlin, Germany Qi Li1 \| Ludger Fischer2 \| Geng Qiao3 \| Ernesto Mura3 \| Chuan Li1 \| Yulong Ding1 1Birmingham Centre for Energy Storage (BCES) & School of Chemical Engineering, University of Birmingham, Birmingham, UK 2Lucerne University of Applied Sciences and Arts, Horw, Switzerland 3Global Energy Interconnection Research Institute Europe GmbH, Berlin, Germany 1Birmingham Centre for Energy Storage (BCES) & School of Chemical Engineering, University of Birmingham, Birmingham, UK Highlights g g • A novel cooling method for HVDC converter using a fatty acid based PCD presented. • A 3D Euler-Euler modelling performed and compared with experiments. • Effects of operating conditions and PCM loading on cooling behaviour discussed. • Three calculating methods of heat transfer coefficients compared. • Optimal PCD cooling condition given based on overall performance evaluation. • Optimal PCD cooling condition given based on overall performance evaluation. K E Y W O R D S Take down policy Take down policy While the University of Birmingham exercises care and attention in making items available there are rare occasions when an item has been uploaded in error or has been deemed to be commercially or otherwise sensitive. If you believe that this is the case for this document, please contact UBIRA@lists.bham.ac.uk providing details and we will remove access to the work immediately and investigate. Download date: 24. Oct. 2024 Received: 24 December 2019 Revised: 23 February 2020 Accepted: 28 February 2020 Received: 24 December 2019 Revised: 23 February 2020 Accepted: 28 February 2020 DOI: 10.1002/er.5347 DOI: 10.1002/er.5347 Correspondence Chuan Li and Yulong Ding, Birmingham Centre for Energy Storage (BCES) & School of Chemical Engineering, University of Birmingham, UK B15 2TT. Email: c.li.4@bham.ac.uk (C. L.) and y. ding@bham.ac.uk (Y. D.) Funding information tate Grid Corporation of China and Global Energy Interconnection Research Institute Europe GmbH, Grant/Award Number: SGRIWLZXQT[2017]882 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. g p p y © 2020 The Authors. International Journal of Energy Research published by John Wiley & Sons Ltd Int J Energy Res. 2020;44:5819–5840. High performance cooling of a HVDC converter using a fatty acid ester-based phase change dispersion in a heat sink with double-layer oblique-crossed ribs Qi Li1 \| Ludger Fischer2 \| Geng Qiao3 \| Ernesto Mura3 \| Chuan Li1 \| Yulong Ding1 1 \| INTRODUCTION 5820 This could lead to system thermal runaway because silicon as workhorse material could not bear the crucial operational conditions.7 To avoid the thermal runaway, new semiconductor materials such as SiC or GaN have been developed.7,8 However, applica- tions of such materials are limited by available power module packages,9 package agents,10 peripheral compo- nents11 and economic considerations. Moreover, persis- tent heat still generates in the presence of new material because multiple processing by power electronics con- verters before the end use. These reasons push the rapid development of novel thermal management technologies. Additionally the smaller the heat sinks leads to a higher volumetric power density,12 which requires a more advanced and efficient cooling method. The two most commonly used technologies in cooling electronic devices are air cooling and water cooling. The air based cooling systems, classified as natural or forced air flux by the way air flows, are normally suffice for lower power electronic devices with a power dissipation rate below ~1500 W.13 The introduction of a liquid but water, which is often a dielectric, a high cooling effi- ciency or lower/stabilized temperature can be achieved. Among various coolants, phase change material (PCM) offers more isothermal operation through storing heat at a high energy density within a small temperature change.14-18 Further thermal performance enhancements can be real- ized by dispersing and transporting PCM in a thermally active fluid that is immiscible with the PCM, the so-called phase change dispersion (PCD). An incredible number of PCM slurries have been developed by combining various proportions of PCMs, modifiers, nucleation agents, etc. This allows customizing thermal-physical properties with an adaptability that is hardly possible for other class of sol- vents. 1 \| INTRODUCTION 5820 LI ET AL. LI ET AL. 5820 To ensure long-term lifespan in a high voltage envi- ronment, the PCD component is best to be electrically insulating for cooling HVDC converters. As a type of organic dielectric material, paraffin wax have been widely used as PCM due to the widely studied interaction between paraffin and classical emulsifier.23-25 However, paraffin often yields different crystalline phases during phase change, which may result in contamination on the wall of heat exchanger and require strong solvents for cleaning at a high temperature. The low thermal conduc- tivity, unpleasant odour and flammability make them even harder for industrial applications. The long-chain fatty acid ester from renewable sources offers an alterna- tive PCM to paraffin.26 Such a material normally works in the ambient-to-moderate temperature range27 (20-100C), which is suitable for HVDC electronic devices cooling28 (around 60C). PCD emulsions are often fabri- cated in a high energy environment provided by a high- shear mixer or an ultrasound generator. The multiphase suspension shows Newtonian behaviour at low emulsion concentrations, but becomes highly non-Newtonian at an emulsion concentration, with a clear demarcation hard to be determined empirically. For example, a 30 w/w % tetradecane emulsion29 was found to be Newtonian, whereas a paraffin/water emulsion30 showed pseudo- plastic behaviour with a mass fraction between 15% and 50%. The viscosity of emulsions can be 2-550 times that of water,31,32 yet the pressure drop has been found not as big as the viscosity increase and hence a small increase in the pumping work.33,34 As a result, a detailed material characterization of the PCD under both solid and liquid conditions of the PCM is required to enable an accurate prediction of thermal performance of the heat sink using PCDs. Rapid growing demand for electric power and continuous effort of miniaturization of high-voltage direct current (HVDC) devices drive heat flux to the megawatt range.1,2 In a high power transmission system, devices such as HVDC converters that transform alternating current (AC) to direct current (DC) and vice versa via controlla- ble electric switches, high power dissipation can be up to 9.72 kW, which is nearly 30% of the total HVDC power loss.3,4 Under such a high dissipation, sensitive power semiconductor devices such as insulated gate bipolar transistor (IGBT) in the HVDC converter could experi- ence a heat flux over 2 MW/m25 and a temperature increase to 90C,6 depending on the source voltage and switching frequency. K E Y W O R D S Euler-Euler multiphase modelling, fatty acid ester, HVDC converter cooling, multi-stream heat sink, oblique-crossed ribs, phase change dispersion wileyonlinelibrary.com/journal/er 5819 wileyonlinelibrary.com/journal/er 5 Int J Energy Res. 2020;44:5819–5840. 5821 showed a decreased junction temperature up to 5 K for a 40 W heating power with air cooling, but the fanning fric- tion factor was 19 times higher than a straight channel at high Re numbers. In industrial practical applications, the series and parallel arrangement of heat sinks can amplify the increased pressure drop. To avoid unnecessary energy consumption but keep the advantage of thermal perfor- mance enhancement, a multi-stream plate heat exchanger can be used due to compact structure, high efficiency, low cost and ease of handling multiple streams.40-42 Radwan et al.43 developed a monolithic double-layer microchannel heat sink for concentrated photovoltaic cooling under par- allel and counter flows, and found a single-phase-liquid parallel flow could effectively cool the PV at a higher flowrate. et al.51 studied ice slurry (<25 v/v %) in horizontal, vertical and 90 elbow pipes using the Euler-Euler model, and found a relative errors of ±20% with respect to the mea- surements. Ma et al.20 investigated the hydraulic and heat transfer characteristics of PCD in a circular smooth pipe using the Euler-Euler model, and showed a good agree- ment with previous experimental results. However, the use of the Eulerian-Eulerian model to study heat sinks is still limited due to large computation resource demand. Another noteworthy fact of heat sink is that there is still lack of unified standard of temperatures used for heat transfer coefficient calculation. The wall temperature can either be derived from wetted wall or outer wall of the heat sink, and the fluid temperature is either the bulk mean temperature or average value between inlet and outlet. The temperature difference used to drive the convective heat transfer coefficient can be calculated by taking the differ- ence between the wall and fluid temperature,36,52 or from log mean temperature difference.53,54 A large deviation between the two can occur at small temperature differ- ences under a high heat flux. Thus, an accurate and more convenient calculation method is needed. Metals such as aluminium alloys and copper are widely used to manufacture heat sinks, due to their light- weight and high thermal conductivity.44-46 In the evalua- tion of heat sink performance, heat transfer across a contact intermediate formed by any two solid surfaces is accompanied by a measurable temperature drop, because there exist interfacial thermal contact resistance (ITCR) to heat flows. The ITCR is caused by the imperfect heat transfer surfaces at the joint, which only give a small fraction of the apparent contact area while rest is filled with air or other media. The complex geometrical and thermo-physical parameters in such a case depend on many factors, such as surface micro-topography, micro- hardness, contact pressure, temperature level of the inter- face, solid thermal conductivities and type of substance in the interstitial gaps. The ITCR between aluminium alloys and copper has received lots of attention due to their wide use in practical applications. Yüncü47 found that the thermal contact conductance of Al–Cu varies between 10.3 and 30.88 kW/(m2 K), depending on the contact pressure and micro-hardness. Shi et al.48 evalu- ated the influence of temperature on ITCR between alu- minium nitride and copper at cryogenic conditions. The ITCR was found to decrease with increasing interface temperature and contact pressure, and ranging between 5 × 10−4 and 3 × 10−3 m2W/K. Depending on the contact surface condition, the thermal conductance of ground and milled Al–Cu was found to be 42-56 kW/(m2 K) and 12-22 kW/(m2 K),49 respectively. In this work, an electrically insulting fatty acid ester- based PCD is used for efficient HVDC converter cooling through a heat exchanger with double-layer oblique- crossed ribs. The PCD was characterized and the heat transfer performance of the heat exchanger was experi- mentally examined, by heating two copper blocks clamped closely to the aluminium heating surfaces. Such a device is able to disrupt thermal and hydraulic bound- ary layers, providing a higher temperature gradient near the wall and rapid removal of thermal energy. A 3D Euler-Euler multiphase approach was then used to model the PCD cooling performance under different heating powers, flowrates and PCD concentrations. The complex geometry of heat sink brings about difficulties in the eval- uation of heat transfer coefficient, thus three analysis methods based on temperatures of the heating wall, the wetted wall and the inlet/outlet fluid were compared. An optimal set of working conditions was proposed and rec- ommended for industrial cooling operations. The present work provides a high-performance and economic cooling method, using a novel coolant inside a heat sink with interrupted hydraulic and thermal boundary layers. Due to the complexity of performing local concentra- tion and velocity measurements of PCD in an insulated pipe, numerical simulations have therefore been widely used for the study. In particular, the Euler-Euler model has been shown to be a fruitful approach to dealing with two- phase mixtures. Göktepe et al.50 compared Eulerian- Eulerian and Eulerian-mixture model to study heat transfer coefficients and friction factors, and recommended the for- mer for description of the two-phase interaction. Wang LI ET AL. 1 \| INTRODUCTION 5820 Under a targeted set of operational conditions, the PCD can be designed to enhance thermal performance of heat sink with a uniform temperature distribution and a small temperature change.19-22 The relative low thermal conductivity of the organic PCD reduces the cooling efficacy, leading to challenges in enhancing and optimizing the thermal management of PCD based heat sinks.35 A great deal of efforts has been devoted to the design of enhanced heat transfer surfaces to lower the convective heat transfer resistance.36 An intro- duction of a large number of fins in the cross-sectional direction can reduce the thermal and hydraulic boundary layers, leading to higher temperature and velocity gradients at the wetted wall for an efficient removal of thermal energy.29 Similarly the use of metal foams could enhance heat transfer.37 However, a significant increase in the pres- sure drop may offset the enhanced heat transfer by an increase in the pumping power. Chai et al.38 studied the heat transfer in microchannel containing offset ribs, with rectangular, backward triangular, isosceles triangular, for- ward triangular and semicircular shapes. Kanargi et al.39 introduced the cross-connected alternating converging- diverging channel heat sink to disrupt boundary layers and 5822 (BCES) of the University of Birmingham. Table 1 summa- rizes the results. Each measurement was repeated at least three times to ensure reproducibility. The solution den- sity was measured using an Anton-Paar DMA 4100 M density meter (UK). The thermal conductivity was derived from laser flash measurements with a Netzsch LFA 427 (Netzsch, German).55 The specific heat capacity, melting point and phase change enthalpy of melting were determined using a DSC (Differential Scanning Calorime- ter, DSC2, Mettler Toledo, USA). The specific heat data are shown in Figure 1 for different PCDs with a PCM volume fraction (ϕ) between 5% and 25%. One can see that the emulsifier is also a PCM, which is therefore multifunctional in this application. The PCD stability against creaming, sedimentation and coalescence was also studied by an optical centrifuge of LUMiSizer, and the results demonstrated the sufficient stability of the proposed PCD even experienced more than 200 cycling times. ensure safety with a long lifespan in a high voltage envi- ronment and a high heat flux over a temperature range of ~320.65-323.15 K. The melting range of the target PCM should align with the temperature range, taking into account the effect of the emulsifier for producing the emulsion (PCD). An extensive screening and formulation exercise was performed to produce the PCD using deionised water and a mixture of commercial fatty acid esters based PCM (50 w/w % Crodatherm 53 + 50 w/w % Crodatherm 47, Croda International PLC, UK). An emul- sifier, consisting of a long chain and a short chain ethoxylated fatty alcohols (75 w/w % Steareth-100 + 25 w/w % Steareth-2, KLK Oleo, Germany), and a small por- tion (1.6 w/w %) of glycerol, were added to the formula- tion to ensure the PCD stability. An optimal PCD was found to be containing 16 w/w % of PCM, 4 w/w % of emulsifier, 0.5 w/w % nucleation agents in continuous phase, which was used in this work. The dispersion was manufactured by dispersing PCM phase into deionized water using a Polytron 10-35 GT lab rotor-stator at the Lucerne University of Applied Sciences and Arts, Switzerland. The rheological behaviour of PCD was measured with a rotational MCR 502 rheometer (Anton Paar, UK). The influences of shear rate and temperature on PCM and emulsifier viscosities are illustrated in Figure 2A. 2.1 \| PCD preparation and properties measurements To meet the cooling requirements of a HVDC converter, the PCD should have low electrical conductivity to LI ET AL. LI ET AL. 5823 0 50 100 150 5 200 10 15 20 200 250 300 350 400 450 μ, mPas γ, 1/s Emulsifier, 328.15 K Emulsifier, 323.15 K Emulsifier, 328.15 K PCM, 328.15 K PCM, 323.15 K 330 332 334 336 338 19 20 21 300 350 400 μ, mPas K T, PCM Emulsifier (A) (B) (C) (D) 0 50 100 150 200 1 10 100 1000 10000 γ, 1/s μ, mPa•s 298.15 K 318.15 K 323.15 K 328.15 K 295 300 305 310 315 325 320 330 335 1 2 3 4 5 6 T, K μ, mPa•s φ=10% φ=20% φ=25% FIGURE 2 A, Rheology behaviour of pure PCM and emulsifier under various shear rates and temperature; B, temperature dependence f viscosity at a shear rate of 100 seconds−1; C, rheological behaviour of PCD under various shear rates and temperatures with ϕ = 20%; nd D, temperature dependence of viscosity of the PCD at γ = 100 s−1 [Colour figure can be viewed at wileyonlinelibrary.com] 0 50 100 150 5 200 10 15 20 200 250 300 350 400 450 μ, mPas γ, 1/s Emulsifier, 328.15 K Emulsifier, 323.15 K Emulsifier, 328.15 K PCM, 328.15 K PCM, 323.15 K (A) (C) 330 332 334 336 338 19 20 21 300 350 400 μ, mPas K T, PCM Emulsifier (B) (D) (A) (B) (D) (C) 0 50 100 150 200 1 10 100 1000 10000 γ, 1/s μ, mPa•s 298.15 K 318.15 K 323.15 K 328.15 K 295 300 305 310 315 325 320 330 335 1 2 3 4 5 6 T, K μ, mPa•s φ=10% φ=20% φ=25% FIGURE 2 A, Rheology behaviour of pure PCM and emulsifier under various shear rates and temperature; B, temperature dependence of viscosity at a shear rate of 100 seconds−1; C, rheological behaviour of PCD under various shear rates and temperatures with ϕ = 20%; and D, temperature dependence of viscosity of the PCD at γ = 100 s−1 [Colour figure can be viewed at wileyonlinelibrary.com] The size distribution of the PCD was measured using the dynamic light scattering method with a Zetasizer Nano-ZS (Malvern, UK) in BCES of University of Birmingham, and the results are shown in Figure 3. The particle size distribu- tion could be fitted by the Rosin-Rammler (R-R) distribution (given in the inset), Yd = e−d=d ð Þ n , with Yd the cumulative PCD volume fraction having a diameter larger than d; n = 1.276 the size distribution parameter; d = 3.0 μm the particle size and size constant; and dpmin = 0.065 μm and dpmax = 6.5 μm, corresponding to 99.9% and 0.1% of the cumulative fraction for the R-R distribution,58 respec- tively. The average deviation between the measurements and fitted correlation is 12.89%. resolution.56,57 An increase in the shear rate to ~25 seconds−1 leads to the viscosity of almost constant for a given temperature, exhibiting the Newtonian behaviour. Such a rheological behaviour is also confirmed by the lin- ear relationship between viscosity and temperature under a constant shear rate of 100 seconds−1 (Figure 2B). Figure 2A and B also indicate that viscosity of the emulsifier is signifi- cantly higher than that of PCM by a factor of 20. resolution.56,57 An increase in the shear rate to ~25 seconds−1 leads to the viscosity of almost constant for a given temperature, exhibiting the Newtonian behaviour. Such a rheological behaviour is also confirmed by the lin- ear relationship between viscosity and temperature under a constant shear rate of 100 seconds−1 (Figure 2B). Figure 2A and B also indicate that viscosity of the emulsifier is signifi- cantly higher than that of PCM by a factor of 20. Unlike the PCM and the emulsifier, the rheological behaviour of the PCD follows a non-Newtonian charac- teristic, as shown in Figure 2A and B. With increasing shear rate, the PCD viscosity decreases regardless of the PCM at the solid or liquid status, and the viscosity of the PCD can reach up to 10 000 mPas at a shear rate below ~ < 25 seconds−1 even when PCD is melted (323.15 K and 328.15 K); see Figure 2C. Increasing the PCM con- centration increases the PCD viscosity (Figure 2D). The PCD viscosity is lower when the PCM is at the liquid sta- tus as shown in Figure 2D, but the decrease is rather smaller compared to the low shear rate values. At a shear rate lower than ~25 seconds−1, an irregular fluctua- tion of the viscosity occurs mainly due to low torque The thermo-physical properties of PCD were mea- sured at the Birmingham Centre for Energy Storage TABLE 1 Thermo-physical properties of water, PCM and PCD under both solid and liquid status of the PCM ρ [kg/m3] k [W/(m K)] cp [kJ/(kg K)] Tmelt [C] Δhpc [kJ/kg] Working Temperature [K] 298.15 328.15 298.15 328.15 298.15 328.15 - - Water 998.2 988.4 0.61 0.651 4.18 4.18 - - PCM 902 827.2 0.231 0.206 2.78 2.38 47.13 193.38 Emulsifier 1011.4 1005.3 0.293 0.301 2.28 2.21 49.72 141.53 PCD 983.9 980.5 0.529 0.561 3.82 3.78 50.95 26.11 300 310 320 330 340 -4 0 4 8 Heat Flow, W/g T, K PCM Emusifier 300 310 320 330 340 3000 6000 9000 12000 T, K Cp, J/kg•K φ = 5% φ = 10% φ = 20% φ = 25% (A) (B) FIGURE 1 A, DSC measurement of PCM and emulsifier, and B, Cp variation with temperature of different PCM concentration, TABLE 1 Thermo-physical properties of water, PCM and PCD under both solid and liquid status of the PCM ρ [kg/m3] k [W/(m K)] cp [kJ/(kg K)] Tmelt [C] Δhpc [kJ/kg] Working Temperature [K] 298.15 328.15 298.15 328.15 298.15 328.15 - - Water 998.2 988.4 0.61 0.651 4.18 4.18 - - PCM 902 827.2 0.231 0.206 2.78 2.38 47.13 193.38 Emulsifier 1011.4 1005.3 0.293 0.301 2.28 2.21 49.72 141.53 PCD 983.9 980.5 0.529 0.561 3.82 3.78 50.95 26.11 300 310 320 330 340 -4 0 4 8 Heat Flow, W/g T, K PCM Emusifier 300 310 320 330 340 3000 6000 9000 12000 T, K Cp, J/kg•K φ = 5% φ = 10% φ = 20% φ = 25% (A) (B) FIGURE 1 A, DSC measurement of PCM and emulsifier, and B, Cp variation with temperature of different PCM concentration, ϕ = 5%, 10%, 20% and 25% [Colour figure can be viewed at wileyonlinelibrary.com] sical properties of water, PCM and PCD under both solid and liquid status of the PCM ABLE 1 Thermo-physical properties of water, PCM and PCD under both solid and liquid status of the PCM 300 310 320 330 340 -4 0 4 8 Heat Flow, W/g T, K PCM Emusifier (A) 300 310 320 330 340 3000 6000 9000 12000 T, K Cp, J/kg•K φ = 5% φ = 10% φ = 20% φ = 25% (B) (B) Heat Flow, W/g FIGURE 1 A, DSC measurement of PCM and emulsifier, and B, Cp variation with temperature of different PCM concentration, ϕ = 5%, 10%, 20% and 25% [Colour figure can be viewed at wileyonlinelibrary.com] LI ET AL. 2.2 \| Experimental rig of cooling performance measurements The performance of the PCD for the HVDC converter cooling was performed by using a heat sink with double- LI ET AL. 5824 PT100 1/3 DIN wire sensors (Roth +Co AG, Switzerland) were placed to measure the temperatures of one of the blocks with a measurement uncertainty less than ±0.1 K; see late for more details. The heating section was thermally insulated by an aluminium foil together with PIR shells (Swisspor, Switzerland). The pressure drop was measured by a Deltabar S PMD75 device (Endress + Hauser, Switzer- land), with a range of 0-40 bar and an accuracy of 0.075%. 2 0 6 4 8 10 0.0 0.2 0.4 0.6 0.8 1.0 1 0.1 10 0 1 2 3 4 5 6 φ, % Yd dp, μm dp, μm Experiment Rosin-Rammler fitting y=exp(-(0.333x)^1.276) 2 0 6 4 8 10 0.0 0.2 0.4 0.6 0.8 1.0 1 0.1 10 0 1 2 3 4 5 6 φ, % Yd dp, μm dp, μm Experiment Rosin-Rammler fitting y=exp(-(0.333x)^1.276) FIGURE 3 Relationship between particle volume fraction and particle diameter. Inset shows the fitting of Rosin–Rammler distribution [Colour figure can be viewed at wileyonlinelibrary.com] Figure 4C illustrates a schematic of the experimental rig. The fluid was driven by an impeller pump (Zuwa NIROSTAR/V 2000-B/PT, Germany) into the system. A Coriolis flow meter (Promass F83, Endress + Hauser AG, Switzerland) and a by-pass valve were used to control the mass flowrate of the flow with an uncertainty of ±0.004 _m, where _m is the mass flow rate. To ensure a stable and safe operation in the electrical environment, the PCD went through a deionization unit filled with a resin, Amberjet UP 6150, purchased from Rohm & Haas, USA. A nylon filter with a mesh size of 200 μm was used to remove any unwanted impurity before the PCD entering the system. To remove the heat added to the fluid in the heat section for recirculation, a thermostat (TYA-201, JUMO Co Ltd, UK) was used to cool the fluid before re- entering the testing rig. All measurements were recorded at the steady state (often reached after ~10 minutes opera- tion). To determine heat loss of the system, the setup was first tested with water under various _m and Pel. 2.2 \| Experimental rig of cooling performance measurements The heat loss, identified as the difference between Pel and water heating from inlet to outletQloss = Pel −_m CpTout−CpTin , was found to be 20 W on average, which was used for the PCD calculations. FIGURE 3 Relationship between particle volume fraction and particle diameter. Inset shows the fitting of Rosin–Rammler distribution [Colour figure can be viewed at wileyonlinelibrary.com] layer oblique-crossed ribs, manufactured by Mersen Co Ltd, Shanghai. The heat sink was made of AlMgSi0.5, which has heat and electrical conductivities of 185 W/ (m K) and 28.6 × 106 S/m, respectively. Figure 4A shows a snapshot of the heat sink. Two identical circular heating surfaces with a diameter of 135 mm were made in close contact with the top and bottom surfaces of the heat sink. The cooling section was formed by overlapping two web layers within the heat sink to increase the heat transfer area and fluid turbulence. The angle between the ribs of upper and bottom layer was 30. The fluid channel was formed between the rib gaps with a cross-section dimension of 3.6 mm (Wc) × 4 mm (Hc) and porosity of 0.576. The cooling fluid was pumped into the circular inlet (internal diameter, ID, = 9 mm), exchanging heat with the cooling section before exiting at the outlet (with the same ID as the inlet). The cooling section had width of 130 mm and length (refers to the distance between inlet and outlet centre) of 118 mm. 2.3 \| Data analysis Temperatures measured by T03 to T06 (Figure 4C) were from the copper block side rather than from the heat sink. To accurately describe the thermal performance of the heat sink, thermal resistance within the copper (Rth1) and ITCR between the aluminium heat sink and copper block (Rth2) should be estimated, as illustrated in Figure 4D. The copper resistance is a function of thermal conductivity, k, distance, s, and heating area, A: The cooling experiment was conducted at Lucerne Uni- versity of Applied Science and Arts (Switzerland). The cir- cular heating surfaces were provided by two copper blocks powered by electrical heating as shown in Figure 4B. The blocks and the heating surfaces of the heat sink were tightly clamped by torque wrenches. Each block was equipped with six heating cartridges so that a constant heat flux boundary condition can be maintained, to simulate the power dissipation of a HVDC converter thyristor. The maximum heating power per cartridge was 200 W, giving a total power of 1.5 kW per side. All 12 cartridges were powered by a thyristor power controller (TYA-201, JUMO Co Ltd, UK), allowing a precise regulation of the heat input with an uncertainty of ±0.02 Pel (Pel is the electrical power). At a distance of 2.5 mm from the interface, four Rth1 = s=kA ð1Þ ð1Þ which yields a constant value of 4.48 × 10−4 K/W. The relationship between ITCR and thermal conductance (hj) is defined as Rth2 = 1 hjA ð2Þ ð2Þ LI ET AL. LI ET AL. 2.3 \| Data analysis 5825 FIGURE 4 A, A snapshot of the heat sink, B, a snapshot of the heating section, C, a schematic diagram of the experimental rig (1 – Deionisation; 2 – Filter; 3 – Converter; 4 – Re-dispersion unit; 5 – Re-cooling unit; 6 – Pump; 7 – By-pass valve; 8 – Throttle valve), and D, schematic of the thermal resistance between heating surface and copper block [Colour figure can be viewed at wileyonlinelibrary.com] FIGURE 4 A, A snapshot of the heat sink, B, a snapshot of the heating section, C, a schematic diagram of the experimental rig (1 – Deionisation; 2 – Filter; 3 – Converter; 4 – Re-dispersion unit; 5 – Re-cooling unit; 6 – Pump; 7 – By-pass valve; 8 – Throttle valve), and D, schematic of the thermal resistance between heating surface and copper block [Colour figure can be viewed at wileyonlinelibrary.com] where ωX is the uncertainty of variable X and ωXn is the uncertainty of parameter xn. The maximum uncertainty of temperature derived is therefore ±0.35 K. Since both metal surfaces were planar and finely polished, the thermal conductance was chosen as hj = 12-22 kW/(m2 K). This gives to Rth2 a range between 3.18 × 10−3 K/W and 5.82 × 10−3 K/W for the present work. The temperature difference between heating sur- face and T3456 (as shown in Figure 4D) can thus be deter- mined by ΔT = P(Rth1 + Rth2), where P is the heating power. 5826 LI ET AL. temperature; and g0 is the radial distribution func- tion defined as64: where the equation of motion is solved for each of particles, the Euler granular model solves only one conservation equation for the solid phase. Thus it can be used for concen- trated fluid in a relative large computational domain. The k-ω turbulence model with shear stress transport (SST) model was adopted, which uses the k-ε model in the core of the flow and switches to the k-ω model in the near-wall regions. The rationale for the use of such a model lies in their proven accuracies in solving mixture problems in the near-wall region.59,60 The detailed turbulence kinetic energy and specific dissipation rate can be referred to Menter.61 g0 = 1−αs=αs,max ð Þ1=3 h i−1 ð9Þ ð9Þ where αs, max is the maximum packing fraction and equals to 0.62 for monodisperse spheres.65 The granular temperature θs is used to describe the fluctuating particle motion, derived from the fluctuating energy balance equation as follows: and specific dissipation rate can be referred to Menter.61 3 2 ∂ ∂t αsρsθs ð Þ + r αsρs v ! sθs = τ = s −Ps I = rv ! s + r kθsrθs ð Þ + φsl −γθs ð10Þ • Continuity equation for ith phase: ð10Þ ∂ ∂t αiρi ð Þ + r αiρi v ! i = 0 ð4Þ ð4Þ where kθs , φsl and γθs represent the diffusion coefficient, interphase energy exchange and collisional dissipation of energy, respectively. The solid pressure, Ps, follows the work of Gidaspow66 and given by: where the subscript i = l, s, representing the liquid phase or solid phase, respectively; α, ρ and v ! denote respectively the volume concentration, density and velocity of the differ- ent phase. Ps = αsρsθs 1 + 2g0αs 1 + ess ð Þ ½ ð11Þ ð11Þ • Momentum conservation equation: • Momentum conservation equation: The granular temperature at the inlet is given by67: Ts = 0:004 Us ð Þ2 ð12Þ ∂ ∂t αiρi v ! i + r αiρi v ! i v ! i = −αirP + r τ = i + αiρi g ! + F ! D + F ! L + F ! td + F ! 3.1 \| Mathematical model The measurement uncertainty is defined as the uncer- tainty of a derived parameter, X, caused by the uncer- tainties of individual measured variables, can be calculated by the following expression43: With the Euler-Euler method, the PCM particles and glycerol-water solution are treated as interpenetrative con- tinua, coupled through the interphase momentum and heat balance. The PCD is assumed to be incompressible and in the turbulent flow regime, and the PCM particles are assumed to be smooth, inelastic and spherical during phase change. Compared to the Eulerian-Lagrangian method ωX = ∂X ∂x1 2 ω2 X1 + ∂X ∂x2 2 ω2 X2 + … + ∂X ∂xn 2 ω2 Xn ð3Þ ð3Þ LI ET AL. 3.2 \| Computational domain and boundary conditions ð16Þ A symmetric computational domain with the same dimension as the experimental heat sink was used and shown in Figure 5A. To avoid poor quality meshed grids, the geometry was slightly simplified by removing the round angles and pilot holes as given in Figure 5B. The simplification has been numerically proved to barely affect the temperature distribution of the heat sink. The fluid domain and the nearby zone were meshed with tet- rahedron grids while the rest solid domain with hexahe- dron grids. Figure 5C gives a side view of the computational grids on cross-section A. The average mesh size of the fluid domain varies between 0.1 and 0.35 mm for a grid-dependence analysis, and the 0.2 mm mesh size was found to be sufficient for an accurate cal- culation without consuming too much computational resource. Mesh sizes larger than 0.25 mm was found to result in temperature deviation higher than 3.83% com- pared to the experimental value thus not recommended. The selected mesh yielded a dimensionless wall distance y + near 1 in the sublayer, and average solid domain size of 0.48 mm. The mesh count was approximately 5 million for the whole solution domain, with 43.19% tetrahedron and 56.81% hexahedron. A periodic meshed fluid domain was given in Figure 5D for easily understand by the readers. where CL is the lift force coefficient and taken as 0.25 for a spherical particle. The particle distribution is domi- nated by the turbulent dispersion force when the size of turbulent eddies are larger than the particle size69: F ! td,1 = Ctdγsl μt,1 σsl rαs αs −rαl αl ð17Þ ð17Þ where Ctd is the dispersion coefficient and determined to be 1. The energy conservation equation for ith phase can be formulated as: ∂ ∂t αiρiHi ð Þ + r αiρi v ! iHi = r λe,irTi ð Þ + τ= i v ! i −hsl Ti −Tq ð18Þ ð18Þ where hsl is the particle–liquid heat transfer coefficient and given by70: hsl = 6αsλl d2 h ð7 + 10αl + 5α2 l Þð1 + 0:7Re0:2 s Pr1=3Þ + ð1:33−2:4αl + 1:2α2 l ÞRe0:7 s Pr1=3i ð19Þ ð19Þ The effective thermal conductivities in the main flow and near-wall regions have been given by Zehner and Schlünder71 and Legawiec and Ziólkowski,72 respectively. 5827 F ! L,l = 2CLv0:5ρdij ρsd dlkdkl ð Þ0:25 v ! s −v ! l ð16Þ VM ð5Þ ð12Þ ð5Þ Among the interfacial forces, the dominate drag force is described by Syamlal and O'Brien63 model which takes the following form: where τ = l represents the stress-strain tensor of the ith phase: F ! D = CDResαl 24v2 s ð13Þ τ = i = αiμi rv ! i + rv ! i T − ζi μi −2 3 r v ! i I ! ð6Þ ð13Þ ð6Þ where μi, ζi and I ! are the shear viscosity, bulk viscosity and unit vector, respectively; F ! D,l , F ! L,l ,F ! td,l , F ! VM are respectively the drag force, lift force, turbulent dispersion force and virtual mass force between the two phases. The solid phase bulk viscosity, ζs, and shear viscosity, μs, are given by Lun et al.62 and Syamlal and O'Brien,63 respectively: where CD is the drag force coefficient expressed as: CD = 0:63 + 4:8 ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ Res=vs p !2 ð14Þ ð14Þ A virtual mass would occur when the solid phase accel- erates relative to the carrier phases. The inertia of the carrier-phase mass encountered by the accelerating parti- cles exerts the virtual mass force on particles, given as: ζs = 4αsρsdg0 1 + ess ð Þ ﬃﬃﬃﬃ θs p 3 ﬃﬃﬃπ p ð7Þ ð7Þ 0αs 2 ð8Þ F ! VM = 0:5αsρl dlv ! l−dsvs ! dt ! ð15Þ g0 ﬃﬃﬃﬃ θs p + 10ρsd ﬃﬃﬃﬃﬃﬃﬃ πθs p 96αs 1 + ess ð Þg0 1 + 4 1 + ess ð Þg0αs 5 2 ð8Þ F ! VM = 0:5αsρl dlv ! l−dsvs ! dt ! ð15Þ F ! VM = 0:5αsρl dlv ! l−dsvs ! dt ! ð15Þ μs = 4αsρsd 1 + ess ð Þg0 ﬃﬃﬃﬃ θs p 5 ﬃﬃﬃπ p + 10ρsd ﬃﬃﬃﬃﬃﬃﬃ πθs p 96αs 1 + ess ð Þg0 1 + 4 1 + ess ð Þg0αs 5 2 ð8Þ ð15Þ ð8Þ In a non-uniform or swirling flow, the dispersed par- ticles experience a lift force perpendicular to the relative velocity vector68: where ess is the particle–particle restitution coeffi- cient and chosen to be 0.9; θs is the granular LI ET AL. LI ET AL. 3.2 \| Computational domain and boundary conditions In the near-wall region, no slip boundary is assumed for the liquid phase while a partial slip boundary is assumed for solid phase following Johnson-Jackson equations73 with the shear force given as: The 3D simulations were conducted using a CFD software under the FLUENT 18.2 (ANSYS Inc.) envi- ronment, which employs the Euler-Euler model for two-phase mixtures. The water properties were incor- porated as a polynomial function with coefficients listed in Table 2, so does the measured thermos- physical properties of the PCD. The inlet mass flowrate varied from 4 to 10 L/min, and the atmospheric pres- sure (101 325 Pa) was assigned to the sink outlet. Con- stant velocity of the two phases (Uin = 1.05-2.62 m/s) and solid volume fraction (ϕ = 5-25%) were applied at the heat sink inlet. A uniform heat flux was assigned to the circular heating surface, which ranges from 0.5 to 2.5 kW. The rest walls of the heat sink were set to be adiabatic at the room temperature of 298.15 K. In addi- tion, as mentioned earlier, a no-slip boundary condi- tion was given to the water phase and the Johnson- Jackson partial-slip condition was adopted for the PCM phase at the wall. The governing equations for both fluid and particle phases of the PCD were discretized using the finite-volume method with a second order upwind scheme. The pressure-velocity coupling corre- lation was solved with the phase-coupled SIMPLE (Semi-Implicit Method for Pressure Lined Equation) algorithm.75 The time step was selected to be τsw = − ﬃﬃﬃ 3 p ρsg0αsφ ﬃﬃﬃﬃ θs p ν ! sw 6αs,max ð20Þ ð20Þ and the fluctuating energy expressed by: qsw = ﬃﬃﬃ 3 p ρsg0αsφ ﬃﬃﬃﬃ θs p ν ! sw ν ! sw 6αs,max − ﬃﬃﬃ 3 p ρsg0αsφ 1−e2 w θ3=2 s 4αs,max ð21Þ ð21Þ where φ and ew represent the specularity coefficient and particle–wall restitution coefficient, respectively. The value of φ and ew are adjusted to be between 0.9-0.99 and 0.0001-0.001, respectively, to ensure mass balance for various conditions. The discrepancy of solid distribution and velocity caused by coefficient differences has been proven to be very small.74 LI ET AL. 4.1 \| Pressure drop comparison between PCD and water Figure 6A compares the pressure drop of water and PCD derived from both experiment and simulation for Q = 1-14 L/min. Inlet temperature of 313.15 K and 333.15 K are tested for water. The discrepancy of experimental pressure drop caused by inlet temperature is small, with an average deviation of 5.82% under all flow rates. One can see that the Euler-Euler model agrees well with experimental measure- ments within 6.12%. A change in the particle size (1-7 μm) barely influences the pressure drop (deviation within 0.11%), thus the medium particle size, d = 3.5 μm, was employed for the rest simulations. 5829 0.0001 second and the convergence criterion was set to be 10−4 for all variables. the viscosity of PCD can be 10 times that of water during phase change, the pressure drop only increased by 17.01% on average under the studied conditions. Chen et al.32 showed a PCD (d = 51 μm, 30 wt. %) pressure drop increase by up to 35.71% in a straight pipe with the vis- cosity of the PCD 5.57 times that of water. Similarly Alvarado et al.33 proved that the increased pressure cau- sed by PCD barely affected the pumping work even though the viscosity was 3.5 times higher than water. 828 5828 RE 5 A, Symmetric computational domain and boundaries, B, grid system, C, Side views of computational grids on cross- A, and D, grid of the a periodic web [Colour figure can be viewed at wileyonlinelibrary.com] TABLE 2 Thermal and transport properties of water as a function of temperature A1 A2 A3 A4 A5 /m3] 1.351E+03 −2.422 6.32E-03 −6.989E-06 - /(mK)] −2.946E-01 4.757E-03 −5.712E-06 - - (k K)] 6 515E 01 6 166E 01 2 299E 03 3 755E 06 2 276E 09 LI ET AL 5828 FIGURE 5 A, Symmetric computational domain and boundaries, B, grid system, C, Side views of computational grids on cross- section A, and D, grid of the a periodic web [Colour figure can be viewed at wileyonlinelibrary.com] TABLE 2 Thermal and transport properties of water as a function of temperature TABLE 2 Thermal and transpor properties of water as a function of temperature ; A1 A2 A3 A4 A5 ρ [kg/m3] 1.351E+03 −2.422 6.32E-03 −6.989E-06 - k [W/(mK)] −2.946E-01 4.757E-03 −5.712E-06 - - cp [J/(kgK)] 6.515E+01 −6.166E-01 2.299E-03 −3.755E-06 2.276E-09 μ [kg/(ms)] 3.738E-02 −3.093E-04 9.609E-07 −1.320E-09 6.746E-13 Note: ;(T) = A1 + A2T + A3T2 + A4T3 + A5T4 with T in Kelvin. Note: ;(T) = A1 + A2T + A3T2 + A4T3 + A5T4 with T in Kelvin. Note: ;(T) = A1 + A2T + A3T2 + A4T3 + A5T4 with T in Kelvin. LI ET AL. 830 5830 LI ET AL. LI ET AL. ð23Þ ρfluid,avg = Ð n 0 ρfluid,idvi Ð n 0 dvi ð26Þ Ppump = mfΔP ρavg,fluid ð23Þ ð26Þ ð23Þ where Dhyd is the hydraulic diameter of channel; ρavg, fluid and Uavg represent the volumetric-averaged fluid density and velocity, respectively; N is the channel num- ber of the two-layer web; W is the width of cooling section and α is the angle of web against the side wall (75). Dhyd in Equation (22) is defined by the channel height (Hc = 4.0 mm) and width (Wc = 3.6 mm) as: Figure 6B plots the fFanning and Ppump against the volumetric-averaged velocity at Q = 4-10 L/min. The inlet velocity under such mass flowrate range varies within 1.05-2.62 m/s, which is markedly higher than the Uavg (0.35-0.89 m/s). The Uavg of the PCD is smaller than that of water by 3.04% on average, due to higher viscosity as discussed earlier. Compared to water, the fFanning of the PCD shows a steeper decreasing trend with an aver- age value of 19.40% higher than that of water. Addition- ally, PCD requires a higher pumping power than that of water (by 16.19% on average), especially at a high Uavg. Dhyd = 4A P = 2HCWc HC + Wc ð24Þ ð24Þ and ρavg, fluid and Uavg are given respectively by: and ρavg, fluid and Uavg are given respectively by: The effect of PCM mass fraction on pressure perfor- mance is also evaluated under Q = 8 L/min, as given in Figure 6C. Both the fFanning and Ppump increase with the growth of mass fraction. 4.1 \| Pressure drop comparison between PCD and water It is noteworthy that although To evaluate the Fanning friction fFanning and the pumping power Ppump, the following equations are used: • Averaged Fanning friction factor • Averaged Fanning friction factor fFanning = 2ΔPDhydsinα Nρavg,fluidUavg2W ð22Þ • Pumping power fFanning = 2ΔPDhydsinα Nρavg,fluidUavg2W ð22Þ ð22Þ 2 4 6 8 10 12 14 0.0 0.2 0.4 0.6 0.8 1.0 ∆P, bar Q, L/min PCD, exp, Tin = 323.15 K Water, exp, Tin = 313.15 K 0.3 0.4 0.5 0.6 0.7 0.8 Water, exp, Tin = 333.15 K PCD, sim Water, sim 0.9 0.40 0.45 0.50 0.55 0.60 fFanning Uavg, m/s 0 2 4 6 8 10 12 Ppump, W water, Fanning friction PCD, Fanning friction water, pumping power PCD, pumping power (A) (B) (C) FIGURE 6 A, Experimental and numerical pressure drop as a function of flow rate for water and PCD, Q = 4, 6, 8, 10 L/min; B, Fanning friction factor and pumping power as a function of flow rate, Q = 4, 6, 8, 10 L/min, P = 1.1 kW; C, Pumping power, fanning friction and volumetric average velocity as a function of PCM mass fraction, Q = 8 L/min and P = 1.1 kW [Colour figure can be viewed at wileyonlinelibrary.com] 2 4 6 8 10 12 14 0.0 0.2 0.4 0.6 0.8 1.0 ∆P, bar Q, L/min PCD, exp, Tin = 323.15 K Water, exp, Tin = 313.15 K 0.3 0.4 0.5 0.6 0.7 0.8 Water, exp, Tin = 333.15 K PCD, sim Water, sim 0.9 0.40 0.45 0.50 0.55 0.60 fFanning Uavg, m/s 0 2 4 6 8 10 12 Ppump, W water, Fanning friction PCD, Fanning friction water, pumping power PCD, pumping power (A) (B) (A) (B) (C) (C) FIGURE 6 A, Experimental and numerical pressure drop as a function of flow rate for water and PCD, Q = 4, 6, 8, 10 L/min; B, Fanning friction factor and pumping power as a function of flow rate, Q = 4, 6, 8, 10 L/min, P = 1.1 kW; C, Pumping power, fanning friction and volumetric average velocity as a function of PCM mass fraction, Q = 8 L/min and P = 1.1 kW [Colour figure can be viewed at wileyonlinelibrary.com] 5831 section (L = 118 mm). The experimental heating surface represents the temperature directly derived from copper block, and the deviation caused by thermal resistance and temperature uncertainty is ±1.82 K for P = 1.1 kW. Under flow rates of Q = 4 and 8 L/min (shown in Figure 7A and B), the numerical water temperatures of the inlet and outlet are almost identical to experiment measurement. The fluid temperature difference (ΔTfluid = Tout - Tin) decreases markedly with the increas- ing flow rate, from 8.09 K at Q = 4 L/min to 3.94 K at Q = 8 L/min. On the heating surface, high temperature are spotted near the outlet side from simulation while the experiment found it near the centre. The difference may be attributed to the non-uniform temperature distribu- tion of six heating cartridges in the copper block. Never- theless, the simulation predictions fall into the range of experimental measurement. The surface temperature of becomes 5.52% from ϕ = 5% to ϕ = 25%. The decreasing Uavg at high mass fraction attributed to the increasing PCD viscosity. However, the influence of PCM concentra- tion on Uavg is very limited as the growth is only 3.05% from ϕ = 0 to ϕ = 25%. 830 Taken fFanning as an example, the increment is 13.47% from ϕ = 0 to ϕ = 5% but Ufluid,avg = Ð n 1 Uidvi Ð n 0 dvi ð25Þ ð25Þ 0.0 0.2 0.4 0.6 0.8 320 324 1.0 328 332 336 l/L P = 1.1 kW T, K Q = 4.0 L/min Simulation heating surface Experimental heating surface Simulation water Experimental water .0 0 0.2 .0 4 0.6 .0 8 320 1.0 324 328 332 Q = 8.0 L/min T, K l/L P = 1.1 kW Simulation heating surface Experimental heating surface Simulation water Experimental water .0 0 0.2 .0 4 0.6 .0 8 1.0 320 324 328 332 T, K Q = 4.0 L/min l/L P = 1.1 kW Simulation heating surface Experimental heating surface Simulation PCD Experimental PCD .0 0 0.2 .0 4 0.6 .0 8 320 324 1.0 328 332 T, K Q = 8.0 L/min l/L P = 1.1 kW Simulation heating surface Experimental heating surface Simulation PCD Experimental PCD (C) (D) (A) (B) IGURE 7 Comparison of water temperature on the heating surface and fluid from experiment and simulation, A, Q = 4 L/min, = 1.1 kW; B, Q = 8 L/min, P = 1.1 kW; Comparison of PCD temperature on the heating wall and fluid from experiment and simulation, C, Q = 4 L/min, P = 1.1 kW; D, Q = 8 L/min, P = 1.1 kW [Colour figure can be viewed at wileyonlinelibrary.com] (A) .0 0 0.2 .0 4 0.6 .0 8 320 1.0 324 328 332 Q = 8.0 L/min T, K l/L P = 1.1 kW Simulation heating surface Experimental heating surface Simulation water Experimental water (D) (B) 0.0 0.2 0.4 0.6 0.8 320 324 1.0 328 332 336 l/L P = 1.1 kW T, K Q = 4.0 L/min Simulation heating surface Experimental heating surface Simulation water Experimental water (C) (A) (B) T, K 3 (D) (C) .0 0 0.2 .0 4 0.6 .0 8 320 324 1.0 328 332 T, K Q = 8.0 L/min l/L P = 1.1 kW Simulation heating surface Experimental heating surface Simulation PCD Experimental PCD ( ) .0 0 0.2 .0 4 0.6 .0 8 1.0 320 324 328 332 T, K Q = 4.0 L/min l/L P = 1.1 kW Simulation heating surface Experimental heating surface Simulation PCD Experimental PCD FIGURE 7 Comparison of water temperature on the heating surface and fluid from experiment and simulation, A, Q = 4 L/min, P = 1.1 kW; B, Q = 8 L/min, P = 1.1 kW; Comparison of PCD temperature on the heating wall and fluid from experiment and simulation, C, Q = 4 L/min, P = 1.1 kW; D, Q = 8 L/min, P = 1.1 kW [Colour figure can be viewed at wileyonlinelibrary.com] LI ET AL. 830 LI ET AL. 4.3 \| Parameters influences of PCD Having verified the confidence of numerical model, the influences of flow rate, heating flux and PCM fraction on heat transfer as well as the overall performance were dis- cussed in detail. The inlet temperature of PCD was cho- sen just below the PCM solidus point and kept at Tin = 320.15 K. Figure 8 shows the 3D temperature distri- bution of the heat sink surface under various PCD flow rates (Q = 4-10 L/min), where PCD was pumped into the heat sink from left entrance. High temperature can always be spotted near the outlet region on the heating surface. The highest surface temperature at Q = 4 L/min reaches 353 K but decreases to 339 K at Q = 10 L/min, and a more evenly distributed temperature surface is obtained at high flow rate. The uniform temperature Similar comparison are also conducted for PCD at Q = 4 and 8 L/min and P = 1.1 kW, as shown in Figure 7C and D. The outlet fluid temperature from simulation is slightly lower than the experiment mea- surements, by 0.8 K at Q = 8 L/min. The ΔTfluid of PCD decreases from 3.6 K at Q = 4 L/min to 1.8 K at Q = 8 L/min. The predicted surface temperature fol- lows the trend of experiment, with the highest temper- ature located nearly 1/4 L distance from outlet. The average surface temperature of Q = 8 L/min is 1.73 K lower than that of Q = 4 L/min. 5832 LI ET AL. LI ET AL. features of PCD can now be confidently studied using the Euler-Euler model. Q = 4 L/min is significantly higher than that of Q = 8 L/ min under the same P, by 4.37 K on average from simula- tion. The discrepancies of heat sink and fluid tempera- tures caused by different PCM particle sizes (1-7 μm) are very small (less than ±0.05 K), and the medium particle size d = 3.5 μm will be used for the following thermal analysis. 4.2 \| Temperature comparison between PCM and water Both the measurement uncertainty (± 0.35 K) and ther- mal resistance (3.628 × 10−3 K/ Both the measurement uncertainty (± 0.35 K) and ther- mal resistance (3.628 × 10−3 K/ Both the measurement uncertainty (± 0.35 K) and ther- 3 W < Rth < 6.268× 10−3 K/W) are considered when ana- lysing the temperature heating sink. On Figure 11, the temperatures of heating surface and fluid are plotted against dimensionless distance, which is defined as the ratio of distance from inlet centre and length of cooling FIGURE 8 Comparison of temperature distribution on the heat sink surface under various PCD flow rates with A, Q = 4 L/min, B, Q = 6 L/min, C, Q = 8 L/min and D, Q = 10 L/min at the conditions of P = 2.2 kW, ϕ = 20% and T = 325-353 K [Colour figure can be viewed at wileyonlinelibrary.com] FIGURE 8 Comparison of temperature distribution on the heat sink surface under various PCD flow rates with A, Q = 4 L/min, B, Q = 6 L/min, C, Q = 8 L/min and D, Q = 10 L/min at the conditions of P = 2.2 kW, ϕ = 20% and T = 325-353 K [Colour figure can be viewed at wileyonlinelibrary.com] guarantees the safety working condition of sensitive HVDC converter components. Large temperature gradient can be observed at the surrounding area of heating surface, reaching temperature difference of 6 K within 2-3 mm. Similarly, the temperature on other walls of the heat sink decrease with the growth of flow rates. The non-uniformity of the heat sink temperature, defined as θ = THS,max −THS,min ð Þ q , yields θ = 1.3e-04 at Q = 4 L/min and θ = 7.8e-05 at Q = 10 L/min. The low non-uniformity presents a more evenly distributed temperature field inside the heat sink. temperature. The Tfluid, cal shows the same trend of Tfluid, sim, with average deviation of 0.828%. The temperature of average wetted wall (TWW,cal), which refers to the liquid-solid coupled surface, is employed to cal- culate the heat sink temperature36,76 and given as TWW,cal = THS,surf −Rcond HSQ ð27Þ ð27Þ where the 1D thermal conduction resistance of the heat sink is calculated as The volumetric-averaged fluid/solid temperature can be directly determined by Tfluid,sim = Ð n 0 Tidvi Ð N 0 dvi from simula- The volumetric-averaged fluid/solid temperature can be directly determined by Tfluid,sim = Ð n 0 Tidvi Ð N 0 dvi from simula- tion. However, it is difficult to measure the value from experiment and the connection between Tfluid, sim and Tin/Tout is unclarified for the heat sink with complex geometry. Figure 9A compares the PCD Tfluid, sim and cal- culated temperature, Tfluid,cal = Tin + Tout 2 , under different flow rates and heating powers. Overall, high heat power P and low flow rate Q favour the growth of PCD Rcond HS = HHS kHSAHS = 0:032 m 185 W=mK 3:14 × 0:0675 × 0:0675 ð Þm2 = 0:0121 K W ð28Þ tion. However, it is difficult to measure the value from experiment and the connection between Tfluid, sim and Tin/Tout is unclarified for the heat sink with complex geometry. Figure 9A compares the PCD Tfluid, sim and cal- culated temperature, Tfluid,cal = Tin + Tout 2 , under different flow rates and heating powers. Overall, high heat power P and low flow rate Q favour the growth of PCD ð28Þ where HHS is the average distance between the heating surface and wetted wall and THS, surf is the average tem- perature of the heating surface. 4.3 \| Parameters influences of PCD 5833 4.3 \| Parameters influences of PCD Overall, the proposed model yields accurate results in terms of pressure and temperature, and the hydro- and thermo-dynamic 0.5 1.0 1.5 2.0 2.5 320 322 324 326 T, K P, kW Q = 10 L/min, Tfluid,cal Q = 4 L/min, Tfluid,cal Q = 6 L/min, Tfluid,cal Q = 8 L/min, Tfluid,cal Q = 10 L/min, Tfluid,sim Q = 4 L/min, Tfluid,sim Q = 6 L/min, Tfluid,sim Q = 8 L/min, Tfluid,sim 0.5 1.0 1.5 2.0 2.5 324 328 332 336 340 344 T, K P, kW Q= 4L/min, THS,sim Q= 6L/min, THS,sim Q= 8L/min, THS,sim Q= 10L/min, TWW,cal Q= 10L/min, THS,sim Q= 4L/min, TWW,cal Q= 6L/min, TWW,cal Q= 8L/min, TWW,cal 0.5 1.0 1.5 2.0 319 2.5 320 321 322 323 324 325 T, Κ P, kW water, Q = 8 L/min, Tfluid,sim PCD, Q = 8 L/min, Tfluid,sim water, Q = 8 L/min, Tfluid,cal PCD, Q = 8 L/min, Tfluid,cal 0.5 1.0 1.5 2.0 322 324 2.5 326 328 330 332 334 T, Κ P, kW water, Q = 8 L/min, THS,sim PCD, Q = 8 L/min, THS,sim water, Q = 8 L/min, TWW,cal PCD, Q = 8 L/min, TWW,cal (A) (B) (C) (D) FIGURE 9 Temperature of PCD A, and HS B, from simulation and calculation, Q = 4-10 L/min, P = 0.5-2.5 kW, ϕ = 20%; C, volumetric-averaged fluid temperature; D, volumetric-averaged heat sink and wetting wall from simulation at Q = 8 L/min, P = 0.5-2.5 kW, ϕ = 20% [Colour figure can be viewed at wileyonlinelibrary.com] 0.5 1.0 1.5 2.0 2.5 320 322 324 326 T, K P, kW Q = 10 L/min, Tfluid,cal Q = 4 L/min, Tfluid,cal Q = 6 L/min, Tfluid,cal Q = 8 L/min, Tfluid,cal Q = 10 L/min, Tfluid,sim Q = 4 L/min, Tfluid,sim Q = 6 L/min, Tfluid,sim Q = 8 L/min, Tfluid,sim (A) (C) 0.5 1.0 1.5 2.0 2.5 324 328 332 336 340 344 T, K P, kW Q= 4L/min, THS,sim Q= 6L/min, THS,sim Q= 8L/min, THS,sim Q= 10L/min, TWW,cal Q= 10L/min, THS,sim Q= 4L/min, TWW,cal Q= 6L/min, TWW,cal Q= 8L/min, TWW,cal (B) (D) (A) (B) (C) (D) 0.5 1.0 1.5 2.0 322 324 2.5 326 328 330 332 334 T, Κ P, kW water, Q = 8 L/min, THS,sim PCD, Q = 8 L/min, THS,sim water, Q = 8 L/min, TWW,cal PCD, Q = 8 L/min, TWW,cal 0.5 1.0 1.5 2.0 319 2.5 320 321 322 323 324 325 T, Κ P, kW water, Q = 8 L/min, Tfluid,sim PCD, Q = 8 L/min, Tfluid,sim water, Q = 8 L/min, Tfluid,cal PCD, Q = 8 L/min, Tfluid,cal FIGURE 9 Temperature of PCD A, and HS B, from simulation and calculation, Q = 4-10 L/min, P = 0.5-2.5 kW, ϕ = 20%; C, volumetric-averaged fluid temperature; D, volumetric-averaged heat sink and wetting wall from simulation at Q = 8 L/min, P = 0.5-2.5 kW, ϕ = 20% [Colour figure can be viewed at wileyonlinelibrary.com] LI ET AL. LI ET AL. 5834 PCD is better due to the lower fluid and heat sink tem- perature under the same heat dissipation from HVDC converter. volumetric-averaged heat sink temperature of THS,sim and TWW,cal under the same condition of fluid. Similarly, higher heat sink temperature can be found at high P and low Q. The TWW,cal is slightly higher than that of THS,sim by 1.55% on average. As a result, the temperature of fluid (Tfluid, cal) and heat sink (TWW,cal) derived from outlet and heating surface respectively can be used to evaluate the thermal performance. When it comes to evaluate the thermal performance using heat transfer coefficient (h = q/ΔT), various temp differences (ΔT) driving the convective heat transfer are employed. It is noteworthy that small deviation of ΔT may result in large oscillation of h under high heat flux, leading to a contrary analysis of water and PCD. To ensure reliability of the evaluation, three calculation methods of heat transfer coefficient are compared: hsim based on volumetric-averaged temperature, hcal from temperature of wetted wall and fluid inlet/outlet and hΔT based on log mean temp difference: Figure 9C compare the temperature of fluid and heat sink for water and PCD under the same condition. The temperature of water is higher than that of PCD by 1.42 K for Tfluid, sim and 1.53 K for Tfluid, cal. The temp dif- ference between water and PCD is more obvious at high heating power, reaching 2.25 K at P = 2.5 kW for Tfluid, cal. It is attributed to the latent heat absorption of fatty acid ester, which introduces a more isothermal fluid envi- ronment for the cooling process. Compared to the fluid, the heat sink temp difference between water and PCD is slight larger, with average value of 2.18 K for THS,sim and 1.55 K for TWW,cal. Figure 9B shows the 0.5 1.0 1.5 2.0 2.5 10000 12000 14000 16000 18000 20000 P, kW hcal, W/(m 2•K) 0.5 1.0 1.5 2.0 2.5 10000 12000 14000 16000 18000 20000 22000 Water, Q = 4 L/min PCD, Q = 4 L/min Water, Q = 8 L/min PCD, Q = 8 L/min P, kW h∆T, W/(m 2 •K) Water, Q = 4 L/min PCD, Q = 4 L/min Water, Q = 8 L/min PCD, Q = 8 L/min 0.5 1.0 1.5 2.0 2.5 10000 15000 20000 25000 30000 hsim, W/(m 2•K) P, kW Water, Q = 4 L/min PCD, Q = 4 L/min Water, Q = 8 L/min PCD, Q = 8 L/min (A) (B) (C) FIGURE 10 Heat transfer coefficient using three calculation methods for Q = 4 and 8 L/min, P = 0.5-2.5 kW, ϕ = 20% [Colour figure can be viewed at wileyonlinelibrary.com] 0.5 1.0 1.5 2.0 2.5 10000 12000 14000 16000 18000 20000 P, kW hcal, W/(m 2•K) Water, Q = 4 L/min PCD, Q = 4 L/min Water, Q = 8 L/min PCD, Q = 8 L/min 0.5 1.0 1.5 2.0 2.5 10000 15000 20000 25000 30000 hsim, W/(m 2•K) P, kW Water, Q = 4 L/min PCD, Q = 4 L/min Water, Q = 8 L/min PCD, Q = 8 L/min (A) (B) (A) (B) 0.5 1.0 1.5 2.0 2.5 10000 15000 20000 25000 30000 hsim, W/(m 2•K) P, kW Water, Q = 4 L/min PCD, Q = 4 L/min Water, Q = 8 L/min PCD, Q = 8 L/min (A) 0.5 1.0 1.5 2.0 2.5 10000 12000 14000 16000 18000 20000 P, kW hcal, W/(m 2•K) Water, Q = 4 L/min PCD, Q = 4 L/min Water, Q = 8 L/min PCD, Q = 8 L/min (B) 0.5 1.0 1.5 2.0 2.5 10000 12000 14000 16000 18000 20000 22000 P, kW h∆T, W/(m 2 •K) Water, Q = 4 L/min PCD, Q = 4 L/min Water, Q = 8 L/min PCD, Q = 8 L/min (C) 0.5 1.0 1.5 2.0 2.5 10000 12000 14000 16000 18000 20000 22000 P, kW h∆T, W/(m 2 •K) Water, Q = 4 L/min PCD, Q = 4 L/min Water, Q = 8 L/min PCD, Q = 8 L/min (C) (C) FIGURE 10 Heat transfer coefficient using three calculation methods for Q = 4 and 8 L/min, P = 0.5-2.5 kW, ϕ = 20% [Colour figure can be viewed at wileyonlinelibrary.com] Therefore, the cooling performance of hsim = q THS,sim −Tfluid,sim ð29Þ hcal = q TWW,cal −Tfluid,cal ð30Þ ð29Þ ð30Þ 0.5 1.0 1.5 2.0 2.5 0.4 0.8 1.2 1.6 Q = 4 L/min Q = 6 L/min Q = 8 L/min Q = 10 L/min PEC (h∆T) P, kW 0.5 1.0 1.5 2.0 2.5 0.6 0.8 1.0 1.2 1.4 1.6 Q = 4 L/min Q = 6 L/min Q = 8 L/min PEC (hcal) Q = 10 L/min P, kW 0.5 1.0 1.5 2.0 2.5 0.8 1.2 1.6 2.0 Q = 4 L/min Q = 6 L/min Q = 8 L/min PEC (hsim) P, kW Q = 10 L/min (A) (B) (C) FIGURE 11 PEC comparison using three calculation methods for Q = 4-10 L/min, P = 0.5-2.5 kW, and ϕ = 20% [Colour figure can be viewed at wileyonlinelibrary.com] 0.5 1.0 1.5 2.0 2.5 0.6 0.8 1.0 1.2 1.4 1.6 Q = 4 L/min Q = 6 L/min Q = 8 L/min PEC (hcal) Q = 10 L/min P, kW (B) 0.5 1.0 1.5 2.0 2.5 0.6 0.8 1.0 1.2 1.4 1.6 Q = 4 L/min Q = 6 L/min Q = 8 L/min PEC (hcal) Q = 10 L/min P, kW 0.5 1.0 1.5 2.0 2.5 0.8 1.2 1.6 2.0 Q = 4 L/min Q = 6 L/min Q = 8 L/min PEC (hsim) P, kW Q = 10 L/min (A) (B) 0.5 1.0 1.5 2.0 2.5 0.8 1.2 1.6 2.0 Q = 4 L/min Q = 6 L/min Q = 8 L/min PEC (hsim) P, kW Q = 10 L/min (A) (A) (B) (C) 0.5 1.0 1.5 2.0 2.5 0.4 0.8 1.2 1.6 Q = 4 L/min Q = 6 L/min Q = 8 L/min Q = 10 L/min PEC (h∆T) P, kW (C) 0.5 1.0 1.5 2.0 2.5 0.4 0.8 1.2 1.6 Q = 4 L/min Q = 6 L/min Q = 8 L/min Q = 10 L/min PEC (h∆T) P, kW (C) FIGURE 11 PEC i i th l l ti th d f Q 4 10 L/ i P 0 5 2 5 kW d ϕ 20% [C l fi 0.5 1.0 1.5 2.0 2.5 0.4 0.8 1.2 1.6 Q = 4 L/min Q = 6 L/min Q = 8 L/min Q = 10 L/min PEC (h∆T) P, kW (C) FIGURE 11 PEC comparison using three calculation methods for Q = 4-10 L/min, P = 0.5-2.5 kW, and ϕ = 20% [Colour figure can be viewed at wileyonlinelibrary.com] ϕ = 5 % ϕ = 10 % ϕ = 20 % ϕ = 25 % 0 5 10 15 20 25 10000 15000 20000 25000 30000 φ, % h, W/(m 2⋅K) hsim hcal 5 h∆T 0 1 15 20 25 1.0 1.2 1.4 1.6 1.8 2.0 hsim hcal h∆T φ, % PEC (A) (B) (C) (E) (F) (D) E 12 Comparison of temperature distribution on the heating surface under various PCM mass fraction with A, 5%, B, % and D, 25% at the conditions of Q = 8 L/min, P = 2.2 kW, T = 326-343 K; E, heat transfer coefficient and F, PEC as a 5835 LI ET AL. The corresponding P of intersection point between water and PCD at Q = 8 L/min is P = 2.25 kW for hsim, but becomes P = 1.63 kW for hΔT. The favorable work- ing condition of PCD is narrower at low Q, and the cooling performance of PCD is always worse than that of water at Q = 4 L/min for hΔT. To clarify the influence of PCM mass fraction on ther- mal performance, the 2D temperature distribution from a top view are compared under ϕ = 5-25% as given in Figure 12A-D. The PCD enters the heat sink from top left and leaves at a higher temperature on the bottom left. Higher temperature can still be detected on the heating sur- face near outlet side, but no remarkable improvement can be observed under all ϕ. The average heating surface tem- perature of ϕ = 5% is higher than that of ϕ = 25% by 0.71 K. The advantage of PCD is resulted from the latent heat of PCM, but the influence of latent heat reduces when large transport heat applied, where sensible heat becomes dominated. Thus, the superiority of PCD over water is more apparent at small heating power. Mean- while, the high fluid temperature caused by low Q is beyond the latent heat range. For example, Tfluid, sim reaches 326 K at Q = 4 L/min for P = 2.5 kW, but decreases to 322 K at Q = 10 L/min under the same condition. The Cp of PCD at 322 K is nearly 7000 J/ (kg K) but only around 4000 J/(kg K) at 326 K. There- fore, the thermal performance of PCD may be worse than that of water at low flow rates. A quantitative analysis of heat transfer coefficient and PEC under different PCM mass fractions is given in Figure 12E and F. The thermal transfer performance improves with the increasing of PCM concentration. The highest heat transfer coefficient can be found at ϕ = 25%, which is around 1.42 times of water regardless of the calcu- lation method. Taken pressure drop penalty into consider- ation, the best performance can still be observed at the highest PCD concentration, because the enhanced thermal transfer is able to offset the increased pressure drop caused by PCM particles. The PEC results from hcal and hΔT are very close, but lower than that of hsim by 29.88% on average. As a conclusion, the best working condition of the devel- oped fatty acid ester-based PCD is Q = 8 L/min, P = 1.1 kW and ϕ = 25% under the present study range. In order to evaluate the heat transfer enhancement and pressure drop penalty, a performance evaluation criteria (PEC) proposed by Webb77 was used: PEC = Nup,avg=Nuw,avg fp,avg=fw,avg 1=3 ð32Þ ð32Þ LI ET AL. 5835 ϕ = 5 % (A) ϕ = 10 % (B) (A) (B) (C) ϕ = 20 % (C) (E) (D) ϕ = 25 % (F) (D) (E) (F) 0 5 10 15 20 25 10000 15000 20000 25000 30000 φ, % h, W/(m 2⋅K) hsim hcal h∆T (E) 5 0 1 15 20 25 1.0 1.2 1.4 1.6 1.8 2.0 hsim hcal h∆T φ, % PEC FIGURE 12 Comparison of temperature distribution on the heating surface under various PCM mass fraction with A, 5%, B, 10%, C, 20% and D, 25% at the conditions of Q = 8 L/min, P = 2.2 kW, T = 326-343 K; E, heat transfer coefficient and F, PEC as a function of mass fraction at the conditions of Q = 8 L/min and HF = 1.1 kW [Colour figure can be viewed at wileyonlinelibrary.com] LI ET AL. 5836 LI ET AL. Nuavg = havgDhyd λTmean,fluid ð33Þ ð33Þ ð31Þ A comparison of PEC using the three calculation methods are given in Figure 11. The trend predicted by hsim and hcal is very similar but the overall PEC based on hsim is larger. Thus, a wider application range where PCD behaves better than water (PEC > 1) is derived by hsim. The maxi- mum PEC can be obtained at P = 1.1 kW under all flowrates, due to the thermal enhancement. The influence of flow rates on PEC predicted from hcal is less significant than that of hsim. For example, PEC (hsim) and PEC (hcal) are almost identical at Q = 4 L/min, but the difference of the two PECs may reach upto 24.98% at Q = 8 L/min. The peak of PEC (hΔT) can be found at a lower heating power P = 0.5 kW, because the thermal enhancement could not offset the pressure drop penalty at P = 1.1 kW with this method. It is understandable because the hsim increased by 68.99% from P = 0.5 kW to P = 1.1 kW at Q = 8 L/min, while the increment is only 21.30% for hΔT. Considering the representativeness of hsim as a volumetric-averaged parame- ter, the hcal method is recommended for future experimen- tal evaluation due to small deviation. where THS, avg is the average temperature of the heating sur- face. Using expressions (30) and (31), the measured tem- peratures of heating surface and fluid inlet/outlet can be integrated for thermal evaluation. Figure 10 com- pares the heat transfer coefficients of water and PCD using the three methods. The volumetric-averaged tem- perature always yields a higher value (hsim) than the other two methods for both fluids. Increasing Q favours the growth of heat transfer coefficient for water. Under a given Q, the heat transfer coefficient of water shows a nearly linear increasing relationship of heating power P. However, a peak can be spotted at P = 1.1 kW for PCD, and the maximum value increases with increas- ing Q. For P < 1.1 kW, PCD shows better cooling per- formance than water especially at high Q. Exceeding this point, the heat transfer coefficient of PCD decreases until lower than that water, which implies that the working condition is no longer favourable. The corresponding P of intersection point between water and PCD at Q = 8 L/min is P = 2.25 kW for hsim, but becomes P = 1.63 kW for hΔT. The favorable work- ing condition of PCD is narrower at low Q, and the cooling performance of PCD is always worse than that of water at Q = 4 L/min for hΔT. where THS, avg is the average temperature of the heating sur- face. Using expressions (30) and (31), the measured tem- peratures of heating surface and fluid inlet/outlet can be integrated for thermal evaluation. Figure 10 com- pares the heat transfer coefficients of water and PCD using the three methods. The volumetric-averaged tem- perature always yields a higher value (hsim) than the other two methods for both fluids. Increasing Q favours the growth of heat transfer coefficient for water. Under a given Q, the heat transfer coefficient of water shows a nearly linear increasing relationship of heating power P. However, a peak can be spotted at P = 1.1 kW for PCD, and the maximum value increases with increas- ing Q. For P < 1.1 kW, PCD shows better cooling per- formance than water especially at high Q. Exceeding this point, the heat transfer coefficient of PCD decreases until lower than that water, which implies that the working condition is no longer favourable. fatty acid ester-based phase change dispersion (PCD) in a heat sink with double-layer oblique-crossed ribs. Both experimental investigation and numerical modelling were performed and the following conclusions are obtained: CL lift coefficient cp specific heat capacity, (kJ/kg K) Ctd dispersion coefficient d droplet size, (μm) Dhyd hydraulic diameter, (m) ess particle–particle restitution coefficient ew particle–wall restitution coefficient f average fanning friction factor F force, (N) g0 radial distribution function h heat transfer coefficient, (W/m2 K) hsl particle–liquid heat transfer coefficient, (W/m2 K) H height, (m) k thermal conductivity, (W/m K) kθs diffusion coefficient Δh latent heat, (kJ/kg) n size distribution parameter P heating power, (W) Pel electrical power, (W) Ps pressure, (Pa) qsw Johnson–Jackson fluctuating energy Q volume flow rate, (L/min) R thermal resistance, (K/W) s distance, (m) T temperature, (K) v ! velocity vector, (m/s) Uavg average velocity, (m/s) W width, (m) Yd cumulative volume fraction 1. Although the viscosity of the PCD was several times higher than that of water, the increased pumping power was only 17.01% on average. An addition of a small amount of PCM particles significantly increased the friction factor from ϕ = 0% to 5%, but the extent of the increase reduced with further increase in the ϕ from 5% to 25%. 2. The temperature of both heat sink and fluid under water cooling were higher than that of PCD under the same set of working conditions, due to the latent heat of the PCM. Thus, the PCD could achieve a safer and cooler environment for sensitive HVDC converter components like IGBT. 3. The hsim method based on volumetric-averaged tem- perature yielded a larger value than the other two cal- culation methods, which were derived from temperatures of heating surface, wetted wall and fluid inlet/outlet. The thermal performance of PCD was only enhanced at a suitable working range, where heating power, fluid flowrate and PCM concentrations all played important roles. For all three methods, a peak of heat transfer coefficient can be observed at P = 1.1 kW, where average fluid temperature was more close to PCM melting temperature and higher Cp presented. Increasing fluid flowrate favoured the growth of heat transfer coefficient. 4. Considering the fact that the operational condition of industrial heat exchange is usually at turbulent flow regime, the PCD is greatly competitive than water as energy transport HTF. 5 \| CONCLUSIONS where Nuw and fw refers to the Nusselt number and fric- tion factor of water, and the averaged Nu number can be calculated based heat transfer coefficient: The work detailed in this paper concerns with a high- performance cooling method for a HVDC converter using 5837 LI ET AL. ACKNOWLEDGEMENTS The research is partially supported by State Grid Corpo- ration of China and Global Energy Interconnection Research Institute Europe GmbH under Project No. SGRIWLZXQT[2017]882 (Cooling of converters by using phase change materials). 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https://openalex.org/W4288025024	https://hal.science/hal-02416097/document	Latin	null	French contribution to polar gravimetry	HAL (Le Centre pour la Communication Scientifique Directe)	2,019	cc-by	1,701	French contribution to polar gravimetry Anthony Mémin, Yves Rogister, Roger L. Hothem, Jyri Näränen, Matt Amos, J. O’Brien, P. Gentle, Terry Wilson, Alessandro Capra, Jean-Daniel Bernard, et al. French contribution to polar gravimetry Anthony Mémin, Yves Rogister, Roger L. Hothem, Jyri Näränen, Matt Amos, J. O’Brien, P. Gentle, Terry Wilson, Alessandro Capra, Jean-Daniel Bernard, et al. To cite this version: Anthony Mémin, Yves Rogister, Roger L. Hothem, Jyri Näränen, Matt Amos, et al.. French contri- bution to polar gravimetry. Rencontres scientifiques et techniques RESIF 2019, Nov 2019, Biarritz, France. , 2019. hal-02416097 Distributed under a Creative Commons Attribution 4.0 International License HAL Id: hal-02416097 https://hal.science/hal-02416097v1 Submitted on 20 Jan 2020 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Sites AG sites we occupied at least once in 2009, 2011, 2015 or 2018 are located at McMurdo Station (McM, station THIEL), Scott Base (SB, station SBG-1), Cape Roberts (CR, station ROB4) and Mario Zucchelli Station (MZS, stations IAGS and TNB AB) in the region of Ross Sea and Terra Nova Bay. In 2000, 2006 and 2019 we also performed ground gravity measurements at Dumont d’Urville Station (DdU) in Adélie Land. In the french southern lands (Amsterdam, Crozet and Kerguelen) AG sites were occupied twice, in 2003 and 2005. At Ny-Alesund, 13 AG measurements were made, 7 using french FG5-X. Gravity variations measured in Antarctica and the french southern lands 1 Gal 90 100 Southern lands 0 10 20 30 40 50 60 70 80 90 100 1996 1998 2000 2002 2004 2006 2008 2010 2012 2014 2016 2018 2020 µGa Site References values (μGal) Amsterdam 980 092 592.31 Crozet 980 964 480.27 Kerguelen B1 981 061 035.53 Kerguelen Sacristie 981 059 352.02 Southern lands Antarctica Site References values (μGal) Amsterdam 980 092 592.31 Crozet 980 964 480.27 Kerguelen B1 981 061 035.53 Kerguelen Sacristie 981 059 352.02 Southern lands Ground gravity measurements • The ground gravity variation !g is related to the space gravity variation ∆g by !g = ∆g + u . ∇g, where u is the displacement of the ground and ∇g is the gradient of the static average gravity field. • The ground gravity variation !g is related to the space gravity variation ∆g by !g = ∆g + u . ∇g, where u is the displacement of the ground and ∇g is the gradient of the static average gravity field. • The ground displacement u can be measured by precise geodetic positioning techniques, such as GPS. • The ground displacement u can be measured by precise geodetic positioning techniques, such as GPS. • The ratio ! g/! u is approx. 0.15-0.20 μGal/mm for the viscoelastic post-glacial rebound subsequent to the last deglaciation that occurred 20 000-10 000 years ago. • The ratio ! g/! u is approx. 0.15-0.20 μGal/mm for the viscoelastic post-glacial rebound subsequent to the last deglaciation that occurred 20 000-10 000 years ago. • The ratio !g/!u is approx. 0.26 μGal/mm for the elastic deformation that accompanies the present-day ice melting. • The ratio !g/!u is approx. 0.26 μGal/mm for the elastic deformation that accompanies the present-day ice melting. HAL Id: hal-02416097 https://hal.science/hal-02416097v1 Submitted on 20 Jan 2020 Distributed under a Creative Commons Attribution 4.0 International License French contribution to polar gravimetry Mémin A.1, Rogister Y.2, Hothem L.3, Näränen J.4, Amos, M.5, O’Brien J.5, Gentle P.5, Wilson T.6, Capra A.7, Bernard J.-D.2, Hinderer J.2 1- Université Côte d’Azur, Géoazur, Valbonne, France; 2- Ecole et Observatoire des Sciences de la Terre, Strasbourg, France; 3- US Geological Survey, Reston, VA, USA; 4- Finnish Geospatial Research Institute, Masala, Finland; 5- Land Information New Zealand; 6- School of Earth Sciences, Ohio State University, OH, USA; 7- Dipartimento di Ingegneria Meccanica e Civile, Università di Modena e Reggio Emilia, Italy; French contribution to polar gravimetry 1- Université Côte d’Azur, Géoazur, Valbonne, France; 2- Ecole et Observatoire des Sciences de la Terre, Strasbourg, France; 3- US Geological Survey, Reston, VA, USA; 4- Finnish Geospatial Research Institute, Masala, Finland; 5- Land Information New Zealand; 6- School of Earth Sciences, Ohio State University, OH, USA; 7- Dipartimento di Ingegneria Meccanica e Civile, Università di Modena e Reggio Emilia 1- Université Côte d’Azur, Géoazur, Valbonne, France; 2- Ecole et Observatoire des Sciences de la Terre, Strasbourg, France; 3- US Geological Survey, Reston, VA, USA; 4- Finnish Geospatial Research Institute, Masala, Finland; 5- Land Information New Zealand; 6- School of Earth Sciences, Ohio State University, OH, USA; 7- Dipartimento di Ingegneria Meccanica e Civile, Università di Modena e Reggio Emilia Finnish Geospatial Research Institute, Masala, Finland; 5- Land Information New Zealand; USA; 7- Dipartimento di Ingegneria Meccanica e Civile, Università di Modena e Reggio Emilia, Italy; Gravity variations estimated from GRACEa (GRGSb RL4v1) between 2002 and 2016 Svalbard Ny-Alesund 0 10 20 30 40 50 60 70 80 90 100 1996 1998 2000 2002 2004 2006 2008 2010 2012 2014 2016 2018 2020 µGal TNB AB (Mario Zucchelli) THIEL (McMurdo) Scott Dumont d’Urville Cape Roberts IAGS (Mario Zucchelli) Satgrav (McMurdo) Arctic Antarctica Gravity variations measured at Ny-Alesund, Svalbard Gravity variations measured in Antarctica and the french southern lands Site References values (μGal) MZS (TNB AB) 982 865 600 MZS (IAGS) 982 855 230 MCM (Satgrav) 982 972 700 MCM (Thiel-1) 982 970 200 SB 982 977 550 DdU 982 387 100 CR 982 905 600 Reference value 983017045.30 μGal Site References values (μGal) Amsterdam 980 092 592.31 Crozet 980 964 480.27 Kerguelen B1 981 061 035.53 Kerguelen Sacristie 981 059 352.02 Southern lands Antarctica 0 10 20 30 40 50 60 70 80 90 100 1996 1998 2000 2002 2004 2006 2008 2010 2012 2014 2016 2018 2020 µGal TNB AB (Mario Zucchelli) THIEL (McMurdo) Scott Dumont d’Urville Cape Roberts IAGS (Mario Zucchelli) Satgrav (McMurdo) Dumont D’Urville site Gravity variations estimated from GRACEa (GRGSb RL4v1) between 2002 and 2016 Svalbard Ny-Alesund Arctic Antarctica Gravity variations measured at Ny-Alesund, Svalbard Gravity variations estimated from GRACEa (GRGSb RL4v1) between 2002 and 2016 Arctic Antarctica Gravity variations estimated from GRACEa (GRGSb RL4v1) between 2002 and 2016 Gravity variations estimated from GRA Svalbard Ny-Alesund Arctic Acknowledgment We gratefully acknowledge financial and logistical support from (alphabetical order) the Academy of Finland, Antarctica New Zealand (ANZ), the Institut Polaire Fran¸cais Paul-Émile Victor (IPEV - Program 337 GRAVITE), the Italian Programma Nazionale di Ricerche in Antartide (PNRA), Land Information New Zealand (LINZ), the international POLENET program, and United States Antarctic Program (USAP) managed by the National Science Foundation (NSF). Goals Gravity depends on the distribution of the masses inside the Earth, which varies with time. AG measurements are necessary to: Gravity variations measured at Ny-Alesund, Svalbard Gravity variations measured at Ny-Alesund, Svalbard y y , Gravity variations measured in Antarctica and the french southern lands Reference value 983017045.30 μGal in Antarctica and the french southern lands Reference value 983017045.30 μGal • build models of the geoid • study the long-term gravity variations, which are mainly caused by the post-glacial rebound and present-day ice melting • discriminate between the different observations provided by space gravimetry. • study the long-term gravity variations, which are mainly caused by the post-glacial rebound and present-day ice melting • discriminate between the different observations provided by space gravimetry. They are complementary to precise positioning observations to constrain both the ice-mass variations over the polar regions and the post-glacial rebound. Abstract CE (GRGS RL4v1) between 2002 and 2016 Antarctica Four campaigns of absolute gravity (AG) measurements were conducted with FG5 and FG5-X absolute gravimeters in the region of Ross Sea and Terra Nova Bay in Antarctica in 2009, 2011, 2015 and 2018. The campaigns resulted from collaborations between French, Finnish, Italian, New Zealand and US agencies and institutes, the 2009, 2011 and 2015 campaigns having been supported by the international Polar Earth Observing Network (POLENET) program. Moreover, absolute gravity was also measured in Adélie Land in 2000, 2006 and 2019. We show the gravity variations at various stations, taking also into account AG measurements previously made in the 1990s. We also show the two AG measurement campaigns made in the french southern lands and the one made in Arctic, at Ny-Alesund, Svalbard. a GRACE : Gravity Recovery And Climate Experiment b GRGS : Groupe de Recherche de Géodésie Spatiale a GRACE : Gravity Recovery And Climate Experiment b GRGS : Groupe de Recherche de Géodésie Spatiale Conclusions & Perspectives 0 10 20 30 40 50 60 70 80 90 100 1996 1998 2000 2002 2004 2006 2008 2010 2012 2014 2016 2018 2020 µGal Antarctica Over Ross Sea, Terra Nova Bay and Adélie Land, as well as at Ny- Alesund in the Arctic, small gravity variations observed between over the last 20 to 30 years both on the ground and from space are of order of a few μGal/yr. In Antarctica, the 24-year trend can be a small gravity decrease or a small gravity increase. It requires a long-term monitoring for an accurate determination. In Ny- Alesund the rapid decrease of the gravity is directly due to past and present-day ice mass change and requires a regular monitoring. The next campaign is planed for 2021. 0 10 20 30 40 50 60 70 80 1996 1998 2000 µGal Dumont D’Urville site Dumont D’Urville site TNB AB (Mario THIEL ( Dumon Ca IAGS (Mario Satgrav ( 02 2004 2006 2008 2010 2012 2014 20 TNB AB (Mario Zucchelli) THIEL (McMurdo) Scott Dumont d’Urville Cape Roberts IAGS (Mario Zucchelli) Satgrav (McMurdo) Zucchelli) McMurdo) Scott d’Urville e Roberts Zucchelli) McMurdo) Site References values (μGal) MZS (TNB AB) 982 865 600 MZS (IAGS) 982 855 230 MCM (Satgrav) 982 972 700 MCM (Thiel-1) 982 970 200 SB 982 977 550 DdU 982 387 100 CR 982 905 600 6 2018 2020 In the french southern lands, the 2020 campaign will provide a 16-year trend. Unit : 1 μGal = 10−8 m/s2
https://openalex.org/W4311957669	https://preprints.ru/files/942	Russian	null	Электромагнетизм и гравитация - производная структуры частицы	null	2,022	cc-by	6,636	Аннотация Аннотация Концепция, согласно которой все частицы находятся в непрерывной связи между собой, в статье дополнена концепцией развития структуры связанного состояния от простого к сложному. Концепция, согласно которой все частицы находятся в непрерывной связи между собой, в статье дополнена концепцией развития структуры связанного состояния от простого к сложному. Показано, как в рамках модели последовательное усложнение структуры связанного состояния гипотетических мнимых частиц приводит к появлению материальных частиц с всевозможными формами взаимодействия и созданному частицами сложному пространству. При этом оказалось возможным объединить электрические, магнитные и силы гравитации. Масса является производной от структуры частицы, и вместе со скоростью образуют компоненты импульса. Релятивистская Масса есть следствие процесса устранения внутреннего конфликта частицы, возникшего из-за взаимодействия с другой частицей. Фотон также материален, как и Электрон, плоскость поляризации Фотона образована суммарным вектором магнитной составляющей. Взаимодействие между частицами отвечает принципу квантовой нелокальности (информация о локализации частицы проходит со скоростью выше скорости света, следовательно, свойства частиц не определенны до взаимодействия). Abstract. The concept according to which all particles are in continuous communication with each other is supplemented in the article with the concept of the development of the structure of a bound state from simple to complex. It is shown how, within the framework of the model, the sequential complication of the structure of the bound state of hypothetical imaginary particles leads to the appearance of material particles with all possible forms of interaction and a complex space created by particles. In this case, it turned out to be possible to combine electric, magnetic and gravitational forces. Mass is a derivative of the structure of the particle, and together with the velocity form the components of the momentum. Relativistic Mass is a consequence of the process of eliminating the internal conflict of a particle that has arisen due to the interaction with another particle. Photon is a material particle, like an Electron, the plane of polarization of a Photon is formed by the vector of the magnetic component. The interaction between particles corresponds to the principle of quantum nonlocality (information about the localization of a particle passes at a speed higher than the speed of light, therefore, the properties of particles are not determined before the interaction). Ключевые слова: масса, заряд, магнитное поле, сечение взаимодействия, материализация, спин, поляризация. Key words: mass, charge, magnetic field, interaction cross section, materialization, spin, polarization. Электромагнетизм и гравитация - производная структуры частицы Электромагнетизм и гравитация - производная структуры частицы ру ур М.В. Чеснаков ОАО «Концерн «Созвездие», 394018 г. Воронеж, ул. Плехановская,14, E-mail: chesnakov47@mail.ru УДК 539 времени и пространстве образований, обладающую собственным, независимым от других существованием. Интервалы пространства мнимые частицы, понятие Массы для них не используется. Интервалы пространства имеют два разнополярные полюса (условно названных положительным и отрицательным), связанные осью симметрии и соответствующим уровнем потенциальной энергии (действия) между полюсами. Действие направлено на сближение разнополярных полюсов. Физический смысл разной полярности полюсов в том, что поток энергии и его изменение могли происходить только в одном направлении, от одного полюса к другому через ось симметрии. Поэтому уровень энергии свободных Интервалов пространства не мог измениться самостоятельно. Некоторые свойства Интервала пространства становятся более понятными и наглядными, если ось симметрии будет представлена в виде вектора, у которого длина определяет уровень энергии. Направление перераспределения потока энергии совпадает с условным направлением вектора. Интервалы пространства могли различаться только уровнем энергии и относительной направленностью осей симметрии. Нулевое и бесконечное энергетические состояния считаются запрещенными. Понятие «длины» для Интервалов пространства неприемлемо и его следует заменить понятием Уровень энергии (действия). «Длина» в отношении к свободным Интервалам пространства величина несуществующая. Понятие длины, вместе с ним изменение длины, возникнет только тогда, когда Интервалы пространства образуют взаимосвязанную структуру и в этой структуре становится возможным процесс обмена энергией. 1. Основные свойства Интервалов пространства Согласно гипотезе, элементарные частицы, в том числе и Фотоны, не являются элементарными частицами в прямом смысле, а являются вторичными образованиями, созданными в системе частиц, называемыми Интервалами пространства, и связаны между собой Интервалами пространства. В гипотезе было сделано предположение, что Интервалы пространства представляют собой простую частицу, не структурированную, неделимую и неуничтожимую, не содержащую локальных во времени и пространстве образований, обладающую собственным, независимым от других существованием. Интервалы пространства мнимые частицы, понятие Массы для них не используется. времени и пространстве образований, обладающую собственным, независимым от других существованием. Интервалы пространства мнимые частицы, понятие Массы для них не используется. 2. Образование мнимого и действительного Пространства Первоначально гипотетический Мир Интервалов пространства это сгусток частиц, в котором не существовали границы, направления, размеры. Информация, как мера различия, была минимальной, ограничиваясь фактом самостоятельного существования Интервалов пространства, относительной Состояние, в котором находились Интервалы пространства до образования Узлов, неопределенно. Некоторой аналогией (по крайней мере, по двум параметрам) является внутриядерное пространство. Действительно, вполне возможно предположить, что Интервалы пространства, в условиях малых расстояний (максимальной энергии Интервала) и большой плотности внутри ядра, переходят в пространственно-определенную фазу (известно, что взаимодействие внутри ядра является прямым, энергия связи является максимально большой и не зависит от расстояния между нуклонами). В какой-то момент времени, используя два своих полюса, под действием неких Сил Интервалы пространства связались своими однополярными полюсами (положительными или отрицательными), образовав Узлы и систему связанных между собой Узлов. Каждый Узел в системе оказался связанным с другим Узлом одним Интервалом пространства, полюса каждого Интервала пространства оказались включенными в состав того или иного Узла. Свойство Интервалов пространства к отдельному, самостоятельному существованию делало невозможным слияние двух частиц в одну, то есть переход в состояние, неразличимое для каждой частицы. Это свойство проявило себя в процессе образования Узлов, как принцип Паули, согласно которому не разрешалось двум одинаково направленным интервалам пространства связывать два одинаковых Узла, или, то же самое, нельзя двум Интервалам пространства находиться в одном фазовом состоянии. В результате в образовании всех Узлов приняло участие одинаковое количество Интервалов пространства. Таким образом, в природе появились два вида Узлов, разных по знаку, но одинаковых по структуре. Это, например, в полной мере объясняет факт отсутствия дробного заряда, величина заряда определена числом Интервалов в Узле, равное во всех Узлах. Одинаковая структура Узлов объясняет одинаковое для всех частиц ограничение максимальной относительной скорости C, и одинаковое значение постоянной Планка h, и некоторые другие константы. Энергия Интервалов пространства, связывающих Узлы, будучи положительной, направлена на сближение Узлов. Одновременно система связанных между собой Узлов создала Пространство с мнимой и действительной частью, Комплексное пространство. Мнимую часть образовали Интервалы пространства за пределами Узла. Мнимость означает, что Интервалы пространства находятся в таком фазовом состоянии, при котором всё возможное фазовое пространство, кроме самих Узлов, одновременно принадлежит всем Интервалам пространства. Такое состояние следует назвать пространственно- неопределенным. Под фазовым пространством понимается всевозможный набор значений уровней энергии и относительной направленности осей симметрии. Узлы, будучи локальными образованиями, самим фактом своего существования отрицающими пространственную неопределенность, образовали действительное пространство. Не смотря на то, что любая точка фазового пространства принадлежит всем Узлам, тем не менее, Узел получил собственное, независимое от других Узлов, Существование, возник эффект пространственной определенности самого Узла. 2. Образование мнимого и действительного Пространства Интервалы пространства первичны, порожденное ими Мнимое пространство, как и Действительное, вторичны и реальны. 3. Образование электрического Поля 3. Интервалы пространства за пределами Узла являются его неотделяемой мнимой частью, его продолжением в мнимом пространстве и, с учетом знака, представляют собой положительный или отрицательный Заряд Узла (иначе Поле, электрическое Поле). Около каждого Узла образовалось Поле (с тем или иным знаком). Будучи принадлежностью мнимого пространства, Заряды могли перекрывать друг друга, исключая сами Узлы. В связи с этим следует различать пространственную определенность самого Узла, и пространственную неопределенность Заряда. Из реальных частиц такой Заряд имеет, например, Электрон, Заряд которого не прерывается, не равен нулю в любой точке пространства. После окончательного формирования Узлов принцип Паули стал проявлять себя как сила отталкивания между однополярными Узлами, в отличие от силы притяжения между разнополярными Узлами. В результате возникли Электрические силы притяжения и отталкивания. Пространственная неопределенность Интервалов пространства предопределила обратную квадратичную пропорциональность сил притяжения (отталкивания) от расстояния. Кажется, что Силы притяжения можно было бы назвать гравитационными Силами, а Силы отталкивания между одноименными зарядами можно назвать анти гравитационными силами. Такое предположение противоречит опыту. Как известно, гравитация действует как сила притяжения между любыми зарядами, независимо от их знака. Дальнейший процесс усложнения структуры взаимосвязанной системы Узлов изменит Электрическую Силу притяжения и отталкивания, изменит фазовое состояние Узлов, в результате этих изменений возникнет гравитационная Сила. Электрические силы можно представить в виде силовых линий электрического поля, подразумевая под каждой силовой линией Интервал пространства. Векторный характер силовой линии условный и определен направлением силы, действующей на пробный заряд, помещённый в Поле. Кроме того, векторный характер изначально определён тем, что поток энергии и его изменение в Интервале пространства мог происходить только в одном направлении, от одного полюса к другому через ось симметрии. 4. Действительные и материальные объекты Действительный объект Появившиеся в результате связанного состояния Узлы условно можно представить как дырки или пучности, существующие в Мнимом пространстве. Являясь локальными образованиями, Узлы самим своим существованием отрицают фундаментальное свойство пространственной неопределенности, и как нечто, отрицающее это свойство, Узлы становятся Действительными объектами. Действительное пространство и существующие в нем объекты есть Эффект, появившийся в результате связанного состояния мнимых частиц. Принципиально новый феномен, Действительный объект, появился не на пустом месте, а в результате усиления свойства Интервалов пространства к отдельному существованию, вызванного их огромной плотностью в составе Узла. Все Узлы самим процессом образования созданы действительными объектами, но еще не веществом. Структура действительного объекта слишком проста (например, не имеет геометрических размеров), и не может участвовать в сложных процессах анализа и синтеза. Пространство, построенное только из действительных объектов, не имеет шансов на развитие. В Узле свойства Интервалов пространства существенно отличаются от свойств свободных Интервалов пространства не только по причине огромной плотности. Полюса Интервалов пространства, связанные в Узлах, уже не были свободны друг от друга. . Пространство действительных объектов представляет собой некое компактное образование. Узлы максимально стянуты между собой электрическими силами притяжения (электрическими Полями). Это нечто, похоже на пространство внутри ядра. В таком пространстве нет движения, нет относительных скоростей, нет линейных размеров, но уже есть локальные образования и способность передавать возмущения внутри системы Узлов. Внутренние напряжения в системе Узлов, возникшие из-за взаимно связанного состояния, в итоге через анализ и синтез должны привести к некой стабильной конструкции. Сущность внутреннего конфликта заключается в том, что каждый Интервал пространства обладает своим уровнем потенциальной энергии. Соответственно этому уровню возникает разная сила притяжения между Узлами. Это приводит к появлению сил, пытающихся разрушить Узел. Законы физики не накладывают ограничений на скорость передачи возмущения «внутри» Интервала пространства, поэтому вполне логично предположить, что возмущение передаётся быстрее скорости света. Это объясняет многие эффекты, в том числе и ограничение относительной скорости. Чтобы устранить конфликт Природа дополнительно должна пройти несколько этапов развития. Прежде всего, реальный объект должен пройти этап нелинейных преобразований, так называемый процесс материализации энергии, что и было сделано на следующем шаге развития. образование, вектор, расположенный ортогонально оси симметрии и способный особым образом оказывать влияние на Заряды. образование, вектор, расположенный ортогонально оси симметрии и способный особым образом оказывать влияние на Заряды. Термин псевдо означает, что локальное образование не отрицает пространственную неопределённость. Локальное образование, оставаясь принадлежностью конкретного Интервала пространства, тем не менее, может существовать в любой точке мнимого пространства. При этом локальность, наличие полюсов и принцип Паули не позволяют размещать элементарные векторы случайным образом, независимо друг от друга. Единственно возможным способом исключить конфликты около движущегося Узла является системное расположение элементарных векторов в виде окружностей в плоскости, ортогональной направлению перемещения. Таким образом, около Узла образуется Поле с особыми свойствами, названное магнитным. Каждый элемент магнитного Поля представляет собой часть Интервала пространства, преобразованную в пространственно-определенную форму. Элементарные векторы магнитного Поля (как и электрическое Поле) существуют во всем мнимом пространстве, но взаимодействие будет происходить с наибольшей вероятностью в области псевдоОболочки. При этом, чем ближе к Узлу, тем «плотность» Интервалов пространства больше, следовательно, сильнее должно быть и магнитное Поле. Очевидно, с ростом скорости движения Узла энергия элементарного вектора магнитного Поля также увеличится, соответственно увеличится длина вектора, как мера его потенциальной энергии. В начале статьи было показано, на предельно малых расстояниях (соответствующих максимальной энергии связи) Интервалы пространства могут существовать в пространственно-определенной фазе. На существование этого эффекта указывают и особенности внутриядерных сил (силы притяжения не зависят от расстояния между нуклонами, взаимодействие прямое). Возникает вполне логичное предположение, что в ближней зоне, на критически малых расстояниях от Узла, плотность электрической и магнитной составляющей достигнет такого предела, за которым материализация должна привести псевдо локальные образования (в данном случае элементарные векторы магнитного Поля), к локальной форме, с выраженной пространственной определенностью. В результате материализации центральная часть Узла приобретает геометрические размеры, находящиеся в прямой зависимости от достигнутой в результате ускорения скорости (точно такие же свойства в мире реальных частиц имеет Сечение взаимодействия). При материализации возникли два противоположных процесса. С одной стороны материализация привела к появлению псевдоОболочки. Этот процесс гигантский по масштабам. При ускорении бесконечная область существования и взаимодействия вдруг сжалась во вполне измеряемую область, причем, эта область тем меньше, чем больше потенциальная энергия Узла. С другой стороны в фазовом пространстве Узла появилось такое образование, как Сечение взаимодействия, мизерное по масштабам, но с прямой зависимостью от внутренней (потенциальной) энергии Узла. Требуется объяснить, откуда Узел «знает», что именно он движется, а не его партнер на противоположном конце общей оси? Ответ простой. Векторный характер Интервала пространства однозначно определяет направление изменения энергии (условно по вектору). Изменение энергии порождает магнитное Поле. Материализация и магнитное поле Сущность предполагаемого процесса материализации в общем случае заключается в том, что некоторая часть Интервала пространства, находящаяся в пространственно–неопределенной фазе, при ускорении Узла переходит в пространственно-определенную фазу. Пространственно- определенная часть Интервала пространства располагается ортогонально направлению перемещения (оси симметрии), и сохраняется в этом состоянии до следующего взаимодействия. В связи с отложенным характером использования (до момента последующего взаимодействия), энергия, заключенная в пространственно определенной части Интервала пространства, является потенциальной. Из наблюдений следует, что процесс материализации затрагивает, прежде всего, Поле. Проявляется это в формировании около Узла своеобразной области, определенной в классической физике как область наиболее вероятного существования и взаимодействия. Причем, чем больше внутренняя энергия Узла, тем меньше эта область. Примером служит Фотон, у которого с ростом энергии уменьшается длина волны. То есть процесс материализации начинается с уменьшения пространственной неопределенности (увеличения пространственной определенности) области около Узла, названной в гипотезе псевдоОболочкой. Безусловно, это побочный, интегративный эффект, вызванный тем, что каждый Интервал пространства, затронутый перемещением Узла, подвергся процессу материализации. В результате материализации некоторая часть каждого Интервала трансформировалась в псевдо локальное Спиновая масса В общем смысле, любое ускоренное движение (линейное или в форме момента количества движения) придает объекту в разной степени выраженную пространственную определенность. Линейного перемещения в системе действительных объектов не существовало. Поэтому процесс трансформации действительных объектов в материальные начался с внутреннего момента количества движения, с образования Спина. Физически момент количества движения в форме Спина обеспечивает частице начальную постоянную (неустранимую) минимальную пространственную определенность, и, таким образом, вещественность в действительном пространстве. Механизм, обеспечивающий стабильное существование Спина и его направление, не выяснен. Тем не менее, в природе нет частиц, не имеющих Спина. Благодаря этому и Фотон, не просто действительный объект, но, одновременно, и вещественный объект, как и Электрон, и, тем самым, получает возможность взаимодействовать с ним. Отсутствие у Фотона Массы покоя не является существенным, аналогом Массы покоя у Фотона является энергетический компонент момента импульса (mS), спиновая Масса. Спиновая Масса (суммарный вектор магнитной составляющей), очевидно, расположена в плоскости, ортогональной оси перемещения. Условный вектор, определяющий направление импульса, создаваемого Спином, лежит на оси перемещения Фотона. На это указывают опыты по давлению света на вещество. Спин выполнил две задачи. Спин, прежде всего, обеспечил стабильное существование Узла. Кроме того, из-за блокировки части энергии Интервала пространства между Узлами появились расстояния, соответствующие величине потенциальной энергии Спина. Соответственно, появились линейные скорости. Начался взрывной процесс материализации. образование, вектор, расположенный ортогонально оси симметрии и способный особым образом оказывать влияние на Заряды. В противоположном направлении (против вектора) изменение энергии невозможно, следовательно, «сзади» движущегося Узла магнитное Поле не образуется. Элементарный вектор магнитного поля может появиться в любой условной точке Интервала пространства. Но системно, учитывая интегрирующие свойства Узла и взаимовлияние других элементарных векторов магнитного Поля, все элементарные магнитные векторы сконцентрируются в пределах движущегося Узла, образуя комплексное Поле. Некоторые сложности возникают с объяснением направления силовых линий магнитного Поля. Возможно, это определено Спином. Спиновая масса Оболочечная Масса С появлением Спина часть энергии Интервалов пространства оказалась заблокированной в качестве энергии связи Узлов. Таким образом, начался процесс, который условно можно назвать «остыванием». Дальнейшее больше похожее на процесс кристаллизации со снижением температуры, то есть на «Большое похолодание», а вовсе не на «Большой взрыв», разметавший Материю по Вселенной. В пространстве, возникшем после материализации внутреннего углового момента количества движения, баланс сил притяжения определил направление линейного перемещения для каждого Узла. При линейном перемещении статический конфликт Интервалов пространства в Узле, вызванный их разной силой притяжения, перешёл в динамическую форму. С этого момента началась линейная материализация. Процесс идёт с ускорением до момента, когда Узел достигнет максимальной относительной скорости (есть основания полагать, что эта скорость были выше скорости света). При достижении этого уровня материализация достигнет максимума. Линейная материализация ускорила «Великое похолодание». Трансформация статических сил притяжения через динамический процесс материализации в потенциальную энергию Узла (внутреннюю энергию) происходила под контролем защитного механизма.. Это выразилось в изменении состояния всех интервалов пространства Узла в виде одинаковых порций, квантов действия, в которых энергия находится в связанном состоянии с временем её передачи. Квант действия получил название постоянная Планка. Квант - производная интегративной функции Узла, которая очевидно имеет одинаковое значение для всех Узлов. Передача энергии квантами исключает внутренний конфликт в частице при её перемещении в поле сил притяжения. Точно такой же механизм применим и к Спину. Можно предположить, что численное значение постоянной Планка является производной от числа Интервалов пространства, образующих Узел. Процесс материализации Узлов получил своё дальнейшее развитие в появлении Оболочки (возможно, в системе нелинейных уравнений поля найдутся решения, объясняющие этот феномен). Конкретный механизм появления Оболочки неизвестен. Но очевидно, что в этом процессе участвуют Спин, линейная скорость и закон сохранения четности. Сущность такой материализации заключается в том, что все Интервалы пространства, (как перед Узлом, так и сзади Узла) перешли в пространственно-определённую фазу, в потенциальную энергию связи Оболочки. Появилась так называемая Масса Оболочки (пока еще не Масса в привычном смысле). Это принципиально новый момент в образовании свойств материи. Из фазы Заряда почти вся пространственно-неопределенная часть Узла перешла в пространственно-определенную фазу, в фазу Оболочки. И, как следует из наблюдений, энергия связи Оболочки, по какой-то неизвестной причине закрепилась на конкретном уровне (Масса покоя Электронов и Позитронов практически равны). Минимальное значение этой Массы получило название Масса покоя (m0). Как станет ясно далее, Электрон представляет собой комплексную частицу, состоящую из двух Узлов. Одна из составляющих Электрона находится в фазе Заряда, другая в фазе Оболочки, которая содержит в себе минимальный стабильный энергетический компонент (m0), Массу покоя. Оболочечная Масса Оболочки не всех частиц имеют одинаковую с Электроном потенциальную энергию. Например, существуют разные Нейтрино с разной потенциальной энергией связи Оболочки, то есть с разной Массой Оболочки. Очевидно, Оболочка построена из элементарных векторов магнитного поля, предполагая при этом, что все интервалы пространства, образующие Узел, понимаются как электрическое поле вокруг него. Импульс из Оболочки направлен внутрь Частицы. Процесс образования Оболочки происходил в мнимом пространстве, поэтому скорость передачи импульса не меньше скорости света C. Следовательно, импульс равен m0C. Соответственно, формула внутренней энергии Оболочки (оболочечная Масса) получит вид: E=m0C 2. При образовании Оболочек природа позаботилась о том, чтобы он проходил без нарушения закона четности системы. Поэтому всегда без исключений, трансформация положительного заряда Узла в зарядово-нейтральную Оболочку, например Оболочку Частицы, сопровождается трансформацией отрицательного заряда другого Узла также в Оболочку, но в Оболочку антиЧастицы. Первыми реальными частицами, образованными таким образом, является пара Нейтрино и антиНейтрино. Подобное предположение имеет подтверждение в мире реальных частиц, и там это явление рядовое. Наблюдается оно, например, в образовании Электрон – Позитронной пары в потоке Фотонов. Имеются и другие реальные реакции, которые можно объяснить этим процессом. Обратный процесс, аннигиляция Электрона и Позитрона в Фотоны, также проходит с соблюдением закона четности. Кроме того, процесс аннигиляции с двумя реальными частицами, однозначно указывает, что и в образовании Электрон - Позитронной пары также участвуют частицы, но не вакуум. Из общих признанных принципов академической науки следует, что энергия переходит в Массу и наоборот. Но, как следует из гипотезы, энергия является атрибутом Интервала пространства, и не существует вне Интервала. Таким образом, тезис о материализации энергии нельзя понимать буквально. На самом деле изменяется фаза состояния частиц, имеющих определенный уровень энергии. Соответственно, меняется форма представления самой энергии. По определению Интервалы пространства неуничтожаемые частицы. Из чего следует, что нулевое значение энергии, также как и бесконечное, запрещено. После окончания процесса материализации за пределами Оболочки остается некоторая часть Интервала Пространства в пространственно- неопределенной фазе. Во взаимодействиях между Узлами эта часть проявляет себя как весьма незначительная остаточная сила притяжения, пропорциональная уровню материализации. То есть, в остаточной электрической силе притяжения между Узлами имеется составляющая, пропорциональная потенциальной энергии Оболочки. Создаваемый ею гравитационный потенциал описывается формулой Φ=−U r . незначительная остаточная сила притяжения, пропорциональная уровню материализации. То есть, в остаточной электрической силе притяжения между Узлами имеется составляющая, пропорциональная потенциальной энергии Оболочки. Создаваемый ею гравитационный потенциал описывается формулой Φ=−U r . На этапе образования Спина и Оболочки материализация делает Пространство 3-х мерным Евклидовым, но без гравитации и релятивизма. Оболочечная Масса Это было Пространство свободных Зарядов, структура которых искажена присутствием в них материального компонента, и Оболочек. Все относительные скорости достигли максимума, расстояния определялись остаточными силами притяжения. Оболочки отличались инертностью. В отличие от них Заряды были исключительно активными, для них расстояния, как таковые, не существовали. На этом этап подготовки условий для создания полноценных гравитационных сил был закончен. Двуядерные частицы, Монополь Следующий шаг был сделан, когда с противоположной полярностью Заряд и Оболочка создали комплексный, двуядерный Узел. В дальнейшем оказалось удобно вместо понятия Узел использовать обобщённое понятие Монополь. Таким образом, в Природе существуют отрицательный и положительный Монополь в фазе Заряда, и соответствующие им 2 Монополя разной полярности в фазе Оболочки. Соответственно, каждый двуядерный Узел содержит и отрицательный и положительный Монополь. Два разнополярных Монополя в разных фазах в связанном состоянии образуют все без исключения элементарные частицы (Электрон, Позитрон, Фотон). Нейтрино (антиНейтрино) - одиночный Узел в фазе Оболочки. Электрон образуют два Монополя, из которых один в фазе отрицательного Заряда, другой в фазе Оболочки. То же и в отношении Позитрона, но Заряд положительный. Фотон образуют два Монополя в фазе Заряда. Одиночные Монополи в фазе Заряда могут существовать исключительно короткое время, поэтому неизвестны. Частицы, у которых оба Монополя в фазе Оболочки, принцип Паули запрещает, элементарные векторы магнитного поля не могут занимать одинаковые фазовые состояния. В некоторых прикладных случаях, удобно использовать другую терминологию (без упоминания полярности): отрицательный Монополь (Заряд или Оболочка) удобно называть Частицей, а положительный Монополь – антиЧастицей. В обычной практике элементарную частицу Электрон (Позитрон) по полярности Заряда относят к классу Частиц (антиЧастиц), не упоминая при этом полярность Оболочки. Связанное состояние Оболочки и Заряда можно разрушить, но при столкновениях это явление маловероятно. В то же время, реакция трансформации Электрона в Фотон (в паре с Позитроном) обычное явление. Связанное состояние двуядерных частиц, Гравитация формулу силы гравитации можно записать: F=m1 r × m2 r =m1m2 r 2 , где r – коллинеарный интервал. Образование связанного состояния двуядерных частиц породило пространственно-временные отношения (расстояния, относительные скорости и ряд других эффектов). Взаимодействие стало носить строго определенный порядок. Вначале взаимодействие происходит в мнимом пространстве. Результат отражается на движении частиц в действительном пространстве, новые пространственные соотношения между частицами создают новые условия для взаимодействия в мнимом пространстве. И так по кругу. Более того, этот принцип повторен на более высоком уровне развития Материи, в функционировании мозга (от простейших созданий к человеку).. Связанное состояние двуядерных частиц, Гравитация Учитывая обратную зависимость силы притяжения от расстояния (следует из опыта) формулу силы гравитации можно записать: F=m1 r × m2 r =m1m2 r 2 , где r – коллинеарный интервал. Учитывая обратную зависимость силы притяжения от расстояния (следует из опыта) m1 m2 m1m2 Учитывая обратную зависимость силы притяжения от расстояния (следует из опыта) Связанное состояние двуядерных частиц, Гравитация Затем был сделан последний шаг: каждый двуядерный Узел оказался связанным с другим двуядерным Узлом. Связь обеспечивали два коллинеарных, противоположно направленных Интервала пространства. С этого момента в пространстве появились релятивистские эффекты, в том числе и такой, как время. Эвклидово пространство превратилось в Лоренцево, возникли силы гравитации. В Bl Rd Bl С Rd Р А Rd Bl F Рис. 1 На рисунке 1 в центре показана частица «B», связанная с помощью двух Интервалов пространства (двух коллинеарных векторов) с другими частицами «A» и «C». Частицы «A» и «B», являются Электроном, частица «C» Позитрон. Перекрёстные связи не показаны. Каждая двуядерная частица состоит из двух одноядерных частиц, одна из которых в фазе Заряда, другая в фазе Оболочки. Оболочка каждой частицы обозначена толстой линией соответствующего цвета, Заряд обозначен пятном другого цвета. Частица «F» является Фотоном. В Bl Rd Bl С Rd Р А Rd Bl F Рис. 1 Рис. 1 На рисунке 1 в центре показана частица «B», связанная с помощью двух Интервалов пространства (двух коллинеарных векторов) с другими частицами «A» и «C». Частицы «A» и «B», являются Электроном, частица «C» Позитрон. Перекрёстные связи не показаны. Каждая двуядерная частица состоит из двух одноядерных частиц, одна из которых в фазе Заряда, другая в фазе Оболочки. Оболочка каждой частицы обозначена толстой линией соответствующего цвета, Заряд обозначен пятном другого цвета. Частица «F» является Фотоном. Из опытов известно, что частицы трансформируются в условиях строгого соблюдения закона четности, как, например, в реакции образования Электрон – Позитронной пары. Поэтому если один коллинеарный интервал связывает два разнополярных (разноимённых) Заряда, то другой - две Оболочки, если коллинеарный интервал связывает Заряд и Оболочку, то другой непременно связывает противоположные Заряд и Оболочку (у Фотона два Заряда). Создание двуядерных узлов привело к появлению новых эффектов. Пусть два коллинеарных интервала соединяют частицу «A» с частицей «B», и пусть частицы «A» и «B» находятся в относительном покое, то есть Оболочка не содержит дополнительной потенциальной энергии, вызванной относительным перемещением. Расстояние между частицами определено энергией отдельного Интервала пространства за пределами Оболочки (частицы кажутся тем ближе, чем больше сила притяжения). Как следует из текста выше, эта же величина в момент образования получила поправку на пропорциональность потенциальной энергии Оболочки. Поэтому скалярное произведение векторов, представленных коллинеарными противоположно направленными Интервалами пространства за пределами Оболочки, представляет собой силу гравитации между частицами. Минимальная потенциальная энергия Оболочки двуядерной частицы в относительном покое является Массой покоя, m0 . Образование релятивистской массы Из метода формирования оболочки и магнитного поля следует, что состояние коллинеарных интервалов пространства влияет на энергетическое состояние обоих. А именно, увеличение энергии Интервала пространства сопровождается эффектом увеличения фазового пространства Оболочки или усилением магнитного Поля Заряда, что по сути одно и то же. Верно и обратное, то есть состояние Оболочки или изменение энергии магнитного Поля влияет на Интервалы пространства. Эти процессы возникают при разных способах взаимодействия частиц между собой (электрический, магнитный, механический). Процесс взаимодействия всегда начинается с создания временно существующей коллективной Оболочки (показана на рис. 1 пунктирной линией). Пусть, как показано на рисунке 1, частица «В» движется в сторону частицы «А». Передача энергии возможна через Заряд или Оболочку. В первом случае будет образована коллективная псевдоОболочка. Во втором случае будет образована коллективная Оболочка. Любой способ передачи осуществим, если, по крайней мере, одна частица имеет пространственно-определенную Оболочку. Коллективная оболочка необходима для того, чтобы соединить пути перераспределения энергии через оси симметрии между двумя частицами (в свободной частице эти пути всегда открыты) Интервал пространства представлен как вектор, поэтому по закону сложения векторов через коллективную Оболочку внутренняя энергия частицы «В» перераспределится в соответствующей пропорции между осями симметрии всех интервалов пространства, связывающих частицу «А» и «В» со всеми частицами всего мирового пространства. В том числе, перейдет в частицу«A» по пути, направление которого совпадает с направлением вектора, тем самым увеличив её внутреннюю энергию. Между уровнями энергии коллинеарных Интервалов пространства, связывающих частицу «А» и «В» со всеми частицами всего мирового пространства, возникнет неравенство, другими словами диспропорция. Результат будет тот же, если влияние на частицу «А» будет оказано через Заряд внешним электрическим или магнитным Полем. В гипотезе сделано предположение, что Коллинеарные интервалы пространства, находящиеся в пространственно-неопределенной фазе за пределами Оболочки в мнимом пространстве, стремятся устранить диспропорцию. Устранение диспропорции проявляет себя в перераспределении энергии между Оболочкой и магнитным Полем. Устранение диспропорции сопровождается ускорением (торможением) частицы. Обнаружить процесс ускорения (торможения) при ударе визуально невозможно, так как изменяется внутреннее состояние Интервалов пространства, следовательно, со скоростями выше скорости света. Будет виден только результат движения частиц в действительном пространстве (в виде новых векторов равномерной скорости или покоя). Вместе с исчезновением диспропорции энергия коллективной Оболочки также перестанет преобладать над внутренней энергией частиц, и исчезнет. Конечным результатом устранения диспропорции являются новые скорости, направления, новый уровень потенциальной энергии Оболочки и магнитного Поля с соответствующим сечением взаимодействия (геометрические размеры Оболочки). Потенциальную энергию, которая создана диспропорцией, удобнее назвать релятивистской Энергией. Образование релятивистской массы Численное значение потенциальной релятивистской Энергии на одной из осей симметрии не будет иметь никакого отношения к значениям на направлениях, определяемых другими осями симметрии, то есть релятивистская Энергия всегда относительна (в отличие от внутренней Энергии). В современной физике принято вместо релятивистской Массы использовать понятие релятивистской Энергии. С физической точки зрения это обосновано. Однако там, где используется импульс, приходится обозначать и объяснять его компоненту, Массу, имеющую обозначение m. По способу образования m0 (масса покоя) и mR (релятивистская масса) являются следствием (результатом) материализации энергии Интервалов пространства. Поэтому нет смысла использовать одну (m0) и не использовать другую (mR). Далее в тексте при необходимости будут использованы и понятие релятивистской Массы и релятивистской Энергии. Вывод формулы 4-х мерного импульса Релятивистский механизм заключается в устранении диспропорции между энергией Оболочки и энергией магнитного Поля частицы в условиях закона сохранения импульса. Интервалы пространства в процессах взаимодействия изначально представлены как векторы. Д A 2 2 C 2 Интервалы пространства в процессах взаимодействия изначально представлены как векторы. Длины векторов находятся из условия A 2+B 2=C 2 2 2 2 Интервалы пространства в процессах взаимодействия изначально представлены Длины векторов находятся из условия A 2+B 2=C 2 Длины векторов находятся из условия A 2+B 2=C 2 Если, B 2=0то возникает равенство(m C) 2=(mV ) 2. Если, B 2=0то возникает равенство(m C) 2=(mV ) 2. Здесь величина mC есть импульс, генерируемый коллективной Оболочкой, C – вектор скорости изменения энергии Оболочки в мнимом пространстве, m V – приобретенный в результате ускорения импульс частицы, m – релятивистская Масса,V – приобретенный вектор скорости (в действительном Пространстве), реально, скорость перемещения частицы. Величина m одинаковым образом входит в обе части равенства. Если обозначить mC 2=E, а mV=P, то все выражение можно привести к виду (E/C) 2−P 2=0, где E – энергия Оболочки, а P – приобретенный импульс. Это выражение хорошо известно в классической физике, и представляет собой 4-х мерный импульс для частиц, не имеющих массы покоя, например, Фотонов. Как следует из формулы, C=V Фотоны движутся со скоростью C. Если Узел имеет массу покоя, то учитывая, что действие Сил, порожденных Массой покоя, происходит в мнимой области, следует записать выражение для импульса, как m0C, а энергия, соответственно, m0C 2, m0- масса покоя. Таким образом, в действие вступает ещё один вектор m0C. Как ясно из предыдущего изложения, релятивистская Масса m и Масса покоя m0, как результат устранения диспропорции, ближайшие «родственники», следовательно, и по реакции на воздействие внешней Силы не должны отличаться. Поэтому общая формула импульса принимает вид (m C) 2=(mV ) 2+(m0C) 2 Приведенное выше выражение легко преобразуется в известную формулу 4-х мерного импульса в 4-х мерном пространстве (E /C) 2−P 2=m0 2C 2 Величину (E/C) 2 принято называть нулевой компонентой 4-х мерного импульса, E=mC 2 - энергия, m0 2C 2 - внутренний импульс частицы (квадрат импульса). р 0 у р у ц ( др у ) Из той же формулы с помощью элементарного расчета легко получается связь между той же формулы с помощью элементарного расчета легко получается свя m0 : . релятивистской Массой m и Массой покоя m0 : . релятивистской Массой m и Массой покоя m0 Таким образом, для вывода формулы 4-х мерного импульса оказалось достаточным использовать только исходные положения и простой расчет и не потребовалось использовать, например, такие понятия, как Континуум пространства-времени, Изотропность, Однородность и другие свойства Пространства. Но это всего лишь видимость. На самом деле Однородность пространства, которая, как признано в физике, является условием сохранения импульса, неявным образом содержится в свойствах гипотетического мнимого пространства. Гипотетическое мнимое пространство, если не рассматривать ничтожную долю включений в виде действительных объектов, из-за отсутствия в нем локальных образований является априори однородным. В классической физике чаще всего встречается утверждение, что релятивистская Масса есть активное звено механизма, который ограничивает относительную скорость, в конечном итоге не давая превысить некое максимальное значение. С точки зрения гипотезы это не так, релятивистская Масса и скорость в действительном пространстве являются остаточными явлениями действия закона сохранения импульса. Релятивистский механизм есть следствие всеобщего закона сохранения импульса в условиях ограниченности максимальной скорости передачи импульса. Релятивистская Масса, вместе со скоростью, образуются вследствие процесса устранения внутреннего конфликта частицы, возникшего из-за взаимодействия с другой частицей при обмене энергией. 6. Принцип относительности Принцип относительности заключается в том, что процесс в левой части (интервалы пространства обозначены сплошными линиями) может рассматриваться независимо от процесса в правой части (интервалы пространства обозначены пунктирной линией). То есть не имеет значения, что происходит с интервалом пространства, обозначенного толстым пунктиром, если рассматривается изменение интервала пространства, обозначенного толстой сплошной линией. Условно говоря, не имеет значения, что происходит «сзади» частицы «В», если рассматривать её взаимодействие с частицей «А». Последующие рассуждения будут иметь отношение к левой части, и строго вдоль импульса Р (оси симметрии). На рисунке 2 в центре показана частица связанная осями симметрии со всеми остальными частицами. Все пространство разделено плоскостью Z, ортогональной листу бумаги на 2 части (I и II), проходящей через частицу и ортогональной импульсу P, действующего в плоскости бумаги на частицу «А». Возникли два подмножества I и II Рис. 2 Рис. 2 Рис. 2 Пусть Оболочка частицы «А» образована Интервалами пространства, включенными в неё началом вектора. В соответствии с условной предварительной договоренностью, внешний импульс может передать энергию только в те интервалы пространства, которые обращены к импульсу началом. После воздействия на частицу «А» импульса P энергия всех Интервалов пространства всех частиц подмножества I, получит положительное приращение. Возникнет диспропорция с одинаковым знаком для вех коллинеарных Интервалов пространства, соединяющих частицу «А» со всеми частицами в подмножестве I. Вслед за этим возникнет одинаково направленная сила ускорения (результат устранения диспропорции), в результате у всех интервалов пространства подмножества I появится составляющая в виде потенциальной релятивистской Энергии. В подмножестве II все процессы идут с обратным знаком. Соответственно, процесс в левой части «I» (интервалы пространства обозначены сплошными линиями) может рассматриваться независимо от процесса в правой части «II» (интервалы пространства обозначены пунктирной линией). То есть не имеет значения, что происходит с интервалом пространства, обозначенного пунктиром, если рассматривается изменение интервала пространства, обозначенного сплошной линией. Гипотетический принцип относительности отличается от Общей Теории Относительности (ОТО) Эйнштейна именно независимостью процессов в условно левой и правой части. В ОТО процесс в левой части тот же процесс, что и в правой части, но с обратным знаком. Такая относительность была бы реализована в системе частиц, связанных одним интервалом пространства, находящимся не в векторной, а скалярной форме. Гипотетическую относительность, в отличие от классической (скалярной), было бы удобно назвать направленной (векторной). Опыт Фарадея с постоянным магнитом и проводником не находят объяснения в ОТО. В то же время, векторная относительность полностью удовлетворяет опыту Фарадея. Вероятно, в Природе реализована векторная относительность. 7. Релятивистская энергия Фотона В образовании релятивистской Энергии задействованы только половина Интервалов пространства. В образовании Оболочки задействованы все Интервалы пространства (поэтому E=mC 2). Соответственно, релятивистская Масса mR должна иметь коэффициент пропорциональности 1/2. По этой же причине коэффициент пропорциональности в формуле гиромагнитного отношения орбитальных моментов будет равен 1/2. В то же время гиромагнитное отношение спиновых моментов имеет коэффициент пропорциональности в два раза больший, то есть равен 1. Формула импульса будет иметь вид: P=m 2 V . Где V , скорость в действительном пространстве. Энергия, соответственно: E=m V 2 2 . Фотон обычная материальная частица, от других частиц её отличает максимально возможная относительная скорость для действительного пространства, поэтому его энергия с учетом скорости должна выглядеть также E=m C 2 2 , (что не соответствует общепринятому выражению энергии Фотона: E=mC 2) Известно другое выражение для энергии Фотона: E=h ν Приравнивая оба выражения, получим: mC 2 2 =hν , отсюда m=2 hν C 2 Приравнивая оба выражения, получим: mC 2 2 =hν , отсюда m=2 hν C 2 Приравнивая оба выражения, получим: mC 2 2 =hν , отсюда m=2 hν C 2 То есть релятивистская Масса Фотона в два раза больше, чем считалось То есть релятивистская Масса Фотона в два раза больше, чем считалось. 8. Квантовая нелокальность В современных дискуссиях о принципе взаимодействия между частицами, свойства предлагаемой модели явно соответствуют той части неравенств Белла, в которой реализован принцип квантовой нелокальности [1]. То есть, информация о локализации частицы проходит со скоростью выше скорости света, и как следствие, свойства частиц не определены до момента взаимодействия. Это следует из гипотетического механизма образования частиц, благодаря которому у частиц существует действительная и мнимая части. Взаимодействие между частицами происходит в мнимой части комплексного пространства, следовательно, со скоростью, превышающей скорость света, и только затем отражается в действительном пространстве в виде нового взаиморасположения частиц[2]. Так как мнимые части всех частиц распространяются на всё мнимое пространство, следовательно, все частицы всегда неразрывно связаны между собой и всегда находятся в стадии взаимодействия. Современными исследованиями обнаружен ряд эффектов, которые можно объяснить только взаимодействием в мнимом пространстве, например, спутанное состояние частиц [4]. С классических позиций это кажется необъяснимым. Но гипотетически явление должно быть рядовым. Появление пары Электрон – Позитрон в потоке Фотонов невозможно, если нет неразрывной связи между Фотонами. То есть сохранение всеобщего закона чётности выполняется, если есть неразрывная связь между частицами и информация передается быстрее скорости света.. Неразрывная связь и взаимодействие в мнимом пространстве действуют на любом расстоянии, и со «скоростью», всегда превышающей скорость света. Это свойство, вместе с зависимостью длины волны Фотона от расстояния, лежат в основе «красного смещения». Независимо от того, как далеко от Земли находится Фотон, он регулярно передаёт Земле часть своей энергии. Удаленный от Земли фотон имеет весьма малую силу притяжения. Поэтому за один акт может передать некую ничтожно малую величину энергии. Тем не менее, чем дальше объект излучения от Земли, тем больше будет актов передачи энергии, тем больше будут энергетические потери Фотона, тем больше он «покраснеет». Из-за того, что энергия упакована в постоянную Планка, передача малой энергии передается за весьма длительный период (длина волны). То есть, заявленная длина волны Фотона проявляется только в ближней зоне. В самой постановке вопроса о существовании свойств у частиц до взаимодействия, содержится доля механицизма, по крайней мере, в форме неявно присутствующего тезиса о независимом существовании частиц. Из гипотетической структуры элементарных частиц, следует, что в каждой частице присутствует доля другой частицы, и о независимом существовании не может быть речи. Следовательно, неравенство Белла, применительно к гипотезе, содержит расширенный смысл: конкретные свойства частиц возникают только в системе частиц и только в условиях и в момент конкретного Взаимодействия. То есть релятивистская Масса Фотона в два раза больше, чем считалось. Понятие «релятивистская Масса» Фотона содержится некорректность. Фотон представляет собой два Заряда связанных вместе. У Фотона нет массы покоя, нет Оболочки. Такая структура исключает релятивистский механизм. Тем не менее, Фотон обладает способностью хранить и переносить энергию в потенциальной форме. Ответ на этот вопрос лежит на поверхности. Благодаря отсутствию релятивистского механизма Фотон всегда движется с максимальной скоростью. Вокруг двух движущихся противоположных по знаку Зарядов, образуются два магнитных поля, которые взаимно нейтрализуют друг друга во всём пространстве, кроме плоскости, разделяющей Заряды и включающей направление движения. Эта плоскость аналогична Плоскости поляризации в классической физике. Элементарные магнитные векторы обоих Полей в этой плоскости направлены одинаково и могут организоваться единственным образом в виде суммарного вектора, направленного ортогонально оси перемещения (не в виде концентрических силовых линий). Электрон, при торможении, например, в антенне, передаёт свою энергию положительному Заряду Фотона. Отрицательный Заряд Фотона энергию не получает. Но благодаря процессу устранения диспропорции соответствующая часть энергии перейдет в отрицательный Заряд. В плоскости поляризации возникнет суммарный вектор магнитного Поля, пространственное направление которого в плоскости поляризации (условно вверх или вниз) будет однозначно определяться направлением торможения Электрона (ортогонально направлению) и сохранится при движении Фотона в пространстве. Электрон, при последующей встрече с таким Фотоном, получит импульс соответствующего направления. Таким образом, энергию магнитного Поля Фотона с большой натяжкой можно назвать Массой, тем более релятивистской. Абсолютно никакой релятивистской составляющей (зависимости от скорости) в образовании потенциальной энергии магнитного Поля Фотона нет. Если использовать понятие Массы в отношении Фотона (по примеру частиц с Оболочкой), то её следует назвать, например, зарядовой (предполагая магнитный заряд, название «магнитная Масса» - слишком оригинальное). 8. Квантовая нелокальность При этом характер Взаимодействия является определяющим, и операторы взаимодействия активизируют те параметры системы, которые подвержены локальным изменениям (количество движения в разных формах, фаза состояния, состав частицы). Эти параметры системы выступают как свойства частиц. 2. Белинский А.В., «О концепции «волны – пилота» Девида Бома», УФН, 189, №12 (2019). 4. Баргатин И. В., Гришанин Б. А., Задков В. Н. «Запутанные квантовые состояния атомных систем» УФН, 2001.Т. 171, № 6.. 3. Я. П. Терлецкий. Парадоксы теории относительности – М.: Наука, 1966 г. 5. Чеснаков М.В. Электронный научный журнал «Apriori. Серия естественные и технические науки», 2019, №2, «Alternative physical Model of Space Structure». Литература: 1. Белинский А.В., Жуковский А.К., «Квантовая нелокальность» или «нелокальный «реализм»? Пространство, время и фундаментальные взаимодействия. № 3. С. 4-15 (2016). 2. Белинский А.В., «О концепции «волны – пилота» Девида Бома», УФН, 189, №12 (2019). 4. Баргатин И. В., Гришанин Б. А., Задков В. Н. «Запутанные квантовые состояния атомных систем» УФН, 2001.Т. 171, № 6.. 5. Чеснаков М.В. Электронный научный журнал «Apriori. Серия естественные и технические науки», 2019, №2, «Alternative physical Model of Space Structure». 5. Чеснаков М.В. Электронный научный журнал «Apriori. Серия естественные и технические науки», 2019, №2, «Alternative physical Model of Space Structure».
https://openalex.org/W3153780607	https://repositorio-aberto.up.pt/bitstream/10216/135343/2/486284.pdf	English	null	A Critical Review of Obesity in Healthcare Systems in Brazil and Portugal: Pathways, Guidelines and Strategies	The Open public health journal	2,021	cc-by	9,811	* Address correspondence to this author at Department of Food and Nutrition, School of Pharmaceutical Sciences, São Paulo State University (UNESP), Araraquara, São Paulo, Brazil; Tel: +55 61 99397-4141, E-mail: lucianedagdacosta@hotmail.com Conclusion: This study shows that Portugal and Brazil have taken different paths when it comes to the creation and implementation of their strategies to manage obesity. In Brazil, actions aimed at promoting healthy eating have been introduced to children and are implemented by many government agencies. Portugal, on the other hand, has provided greater access to individualized healthcare services and has involved different sectors in addressing these issues. Keywords: Overweight, Primary health care, Healthy eating, Health care, Health policy, Brazil, Portugal. World Health Assembly passed the Global Strategy on Diet, Physical Activity and Health, stressing the importance of lifestyle habits in the obesity equation. The Global Strategy urged all Member States of the United Nations Organization (UNO) to join efforts to implement actions to encourage the development of healthy eating and physical activity habits. In September 2011, the Political Declaration of the High-level Meeting of the General Assembly on the Prevention and Control of Non-communicable Diseases, underlined the importance for Member States to strongly commit to the Global Strategy on Diet, Physical Activity and Health [3]. At the same event, the 2013-2020 Global Action Plan for the A Critical Review of Obesity in Healthcare Systems in Brazil and Portugal: Pathways, Guidelines and Strategies Luciane da Graça da Costa 1,, Adriana Aparecida de Oliveira Barbosa 1, Thabata Koester Weber 2, Flora Correia 3, Isabel Monteiro 4 and Maria Rita Marques de Oliveira 2 1The Graduate Program in Food Science and Nutrition, School of Pharmaceutical Sciences, São Paulo State University (UNESP), Araraquara, São Paulo Brazil 2Department of Human Sciences and Nutrition and Food Sciences, Institute of Biosciences, São Paulo State University (UNESP), Botucatu, São Paulo, Brazil 3Faculty of Nutrition and Food Sciences, Investigator in Infections and Nephrological Diseases University of Porto, Porto, Portugal 4University Institute of Health Sciences and Senior Nutrition Advisor at ACeS Porto Ocidental - ARS Norte – IP, Porto, Portugal Send Orders for Reprints to reprints@benthamscience.net Send Orders for Reprints to reprints@benthamscience.net Send Orders for Reprints to re 1874-9445/21 1874 9445/21 Send Orders for Reprints to reprints@benthamscience.net 206 The Open Public Health Journal Content list available at: https://openpublichealthjournal.com REVIEW ARTICLE A Critical Review of Obesity in Healthcare Systems in Brazil and Portugal: Pathways, Guidelines and Strategies Luciane da Graça da Costa 1,, Adriana Aparecida de Oliveira Barbosa 1, Thabata Koester Weber 2, Flora Correia 3, Isabel Monteiro 4 and Maria Rita Marques de Oliveira 2 1The Graduate Program in Food Science and Nutrition, School of Pharmaceutical Sciences, São Paulo State University (UNESP), Araraquara, São Paulo, Brazil 2Department of Human Sciences and Nutrition and Food Sciences, Institute of Biosciences, São Paulo State University (UNESP), Botucatu, São Paulo, Brazil 3Faculty of Nutrition and Food Sciences, Investigator in Infections and Nephrological Diseases University of Porto, Porto, Portugal 4University Institute of Health Sciences and Senior Nutrition Advisor at ACeS Porto Ocidental - ARS Norte – IP, Porto, Portugal Abstract: Background: Obesity and its associated diseases in the 21 st century has led to new public policies with international commitments. Objective: The objective of this review was to examine public initiatives and policies to tackle obesity in Brazil and Portugal over the past two decades, identifying frameworks, guidelines and strategic actions. Methods: Official documents Brazilian and Portuguese public health policies were analyzed for international guidelines from 1999 to 2019. The documents were organized and analyzed by date. Additionally, they were evaluated for frameworks and actions proposed for individuals, communities, and the population across all levels of healthcare. Conclusion: This study shows that Portugal and Brazil have taken different paths when it comes to the creation and implementation of their strategies to manage obesity. In Brazil, actions aimed at promoting healthy eating have been introduced to children and are implemented by many government agencies. Portugal, on the other hand, has provided greater access to individualized healthcare services and has involved different sectors in addressing these issues. Keywords: Overweight, Primary health care, Healthy eating, Health care, Health policy, Brazil, Portugal. Article History Received: December 10, 2020 Revised: February 7, 2021 Accepted: February 7, 2021 206 The Open Public Health Journal 1. INTRODUCTION The 21 st century began with a global focus on the severity of obesity [1]. In 1997, the WHO warned that special measures were needed to prevent diseases and promote health, drawing attention to the risks of unhealthy eating habits [1]. Morbidity and mortality related to the most prevalent chronic conditions accounted for 60% of all health problems and 47% of Global Burden morbidity in 2002 [2]. In that context, in 2004, the 57 th DOI: 10.2174/1874944502114010206, 2021, 14, 206-217 The Open Public Health Journal, 2021, Volume 14 207 A Critical Review of Obesity in Healthcare Systems Prevention and Control of Non-communicable Diseases was introduced to ensure the implementation of the commitments agreed upon in the Political Declaration of UNO on Non- communicable Diseases [4]. In 2013, deaths associated with diet as a modifiable risk factor totaled 11.3 million people worldwide, while the number of years of healthy life lost amounted to 241.4 million. One-third of both these numbers could have been reduced had obesity control and its prevention measures been adopted [5]. Official documents made available from Brazil’s and Portugal’s Ministries of Health were searched to identify policies aimed at combating obesity and their alignment with global guidelines. The documents were evaluated and fell into three main categories: a) timelines for national public policies, grouped according to the goal (promotion of healthy eating and physical activity; overweight and obesity; severe obesity) b) actions proposed at the individual, community, and national levels across all parts of the healthcare system, also identifying actions and initiatives; and; c) measures of for policy effectiveness. In Brazil, overweight rates have increased across genders and all ages and socioeconomic groups for at least three decades [6]. According to the VIGITEL survey conducted in 2019, 55.4% of Brazilian adults were overweight, 20,3% of them were obese. Obesity prevalence was 21,7% in women and 19,5% in men [7]. In Portugal, 57.1% of adults were overweight in 2018. The prevalence of obesity and pre-obesity was 22.3% and 34.8% respectively in the Portuguese population between 2015 and 2016, as reported in the National Food, Nutrition, and Physical Activity Survey (IAN-AF) of the same period. While obesity was greater in Portuguese women (24.3% vs. 20.1%), the prevalence of pre-obesity was higher in men (38.9% vs. 30.7%) [8]. 3. RESULTS AND DISCUSSION This study sought to identify convergences and divergences in approaches to obesity in the health systems of Brazil and Portugal. Both nations differ in terms of geography, population size, historical and cultural background. Their healthcare systems are also distinct, and it is instructive first to understand these. In 2014, Brazil spent 8.3% of Product Gross Domestic Product (GDP) on healthcare while Portugal spent a 9.5% period [16]. Although the difference between the two figures is small, the difference in terms of real and per capita spending is considerable ($947 for Brazil and $2,097 for Portugal per capita). What makes the difference even greater is In light of the above discussion, this study aims to examine the methods of public initiatives and policies to manage obesity in Brazil and Portugal over the past two decades. 1. INTRODUCTION The main analysis framework consists of the principles and guidelines of the Global Strategy on Diet, Physical Activity and Health [3], recommending that countries develop national strategies and action plans on Physical Fitness and Healthy Eating. These strategies and actions focus on the adult population and include educating consumers, agricultural policies to ensure availability of healthy foods, pan governmental policies to promote physical activity to promote, prevent, monitor, investigate and evaluate healthcare services. Academic literature advocating health promotion was also reviewed [15]. The Global Burden of Disease (GBD) Study 2017 [9] found that the unsuitable eating habits of the Portuguese were the third risk factor for the loss of years of a healthy life. In the same study, the unhealthy eating habits of Brazilians were found to be the fourth risk factor for such loss. 2.1. Selection of Documents The document database for this study was created through online searches of the Brazilian and the Portuguese Ministry of Health websites, for laws, regulations, resolutions, and guidance manuals via searches for documents and information on the Global database on the Implementation of Nutrition Actions (GINA) and on the Nutrition Landscape Information System (NLiS) from the World Health Organization (WHO) website. Global and regional initiatives led by the UN have stimulated discussion about nutrition, food security, and food system transformation as a strategy to reduce obesity and other forms of malnutrition. Methodologies were sought for not only the individual but also families, communities, and the environment [10]. This novel approach required significant transformation of the healthcare infrastructure as well as coordinated public policies to address obesity and other social issues, such as human rights, culture, and the economy, which are beyond the reach of the healthcare system. 2.2. Organization of Information Documents that addressed healthy eating, physical activity, pre, and severe obesity, and non-communicable chronic diseases were organized in chronological order of publication by two independent reviewers. Subsequently, the documents were read carefully by two reviewers to identify references to any action or initiative to promote health or prevent obesity in the laws and regulations of both countries. A third reviewer was brought in when there was no consensus about the classification of the actions identified in the documents. Similar actions detected in more than one document were grouped into categories. Brazil’s free Unified Health Care System (SUS) was introduced in 1988 aiming to make healthcare a right for all and a duty of the State. It guarantees full, universal, and equal access to health services [11]. In Portugal, the right to healthcare was recognized in 1971 [12], and its National Health System was created in 1979. Both countries have struggled to ensure that their populations have access to good and comprehensive healthcare services. Despite using different policies and initiatives, they have made progress but also suffer inequities that need to be overcome [13]. 2. METHODOLOGY The scope review methodological framework was used to conduct this study. The scope review is characterized by the most appropriate approach for a comprehensive synthesis of evidence from a given field of knowledge and aims to identify gaps and provide guidance for future research priorities [14]. 208 The Open Public Health Journal, 2021, Volume 14 da Graça da Costa et al. the fact that Brazil’s Unified Health Care System (SUS), responsible for treating almost 80% of the population, received less than half (46%) of 2014’s total healthcare spending while the Portuguese public system, looking after 81.5% of the citizens, was given 64.8% [16]. That means Brazilian autho- rities used less than half of the total healthcare spending (3.8% of GDP) to care for the great majority of the population. Differences like these have resulted in each country planning public policies differently in order to deal with similar health issues. has called for joint efforts by the first, second, and third economic sectors, and indeed, the whole society to stop morbific behaviors. In recent decades, Brazil has reduced instances of deficient malnutrition but also increased obesity [6, 17]. In 1999, the country introduced the National Food and Nutrition Policy (PNAN) in alignment with WHO guidelines, seeking to entrench policies to eliminate malnutrition via the Unified Health Care System (SUS) [18]. In 2011, these policies were revised to include principles of humanization in healthcare practices, respect for the culture and food diversity, individual rights to choose and also social determination, multi- disciplinary and intersectoral food and nutrition concepts, and food security with sovereignty (Table 1). In this study, the publication, such as The Global Strategy on Diet, Physical Activity and Health by the World Health Organization is seen as the turning point in the battle against lifestyle-related chronic diseases [2, 5]. The Global Strategy Table 1. Timeline laws and regulations for addressing obesity in Brazil and Portugal (1999-2019). 2. METHODOLOGY The Open Public Health Journal, 2021, Volume 14 209 A Critical Review of Obesity in Healthcare Systems BRAZIL 2016 Monitoring of Protection and Promotion of the Mediterranean Diet [52] Priority Program–Promotion of Healthy Eating [53] Regulation of food supply to vending machines on healthcare facilities – Beverages [54] Special consumption tax on sugar-sweetened beverages [55] 2017 Integrated Strategy for the Promotion of Healthy Eating [56] Regulation for bars and canteens on administrative facilities [57] Hospital Diets and Nutritional Care [58] Overweight (pre-obesity) and obesity 2005 National Program to Fight Obesity [59] 2015 Integrated Assistance Process for Pre-obesity in Adults [60] 2017 Optimization of the Therapeutic Approach to obesity [61] Severe obesity 2009 Regulation for the Program of Surgical Treatment of Obesity [62] 2012 Best Practices for the Approach to Obese Patients eligible for Bariatric Surgery [63] 2018 Program of Surgical Treatment of Obesity [64] () Care line also includes severe obesity – Source: own elaboration 1 () Care line also includes severe obesity – Source: own elaboration autonomy, and self-care [15]. The RAS is based on social determinants, intersectoral and social participation. The launch of the Strategic Plan of Action for reducing chronic non- communicable diseases in 2011 was followed by the introduction of the Health Care Network for People with Chronic Diseases (2013), later revised to include strategies to address overweight and obesity care. Thus, the process of care for overweight and obesity was inserted in the RAS of people with chronic illness. It emerged as a way of mobilizing resources and expanding healthcare practices by organizing the flows of care at different levels of service, starting at primary healthcare [15]. Coincidentally, with the launch of The Global Strategy on Diet, Physical Activity and Health by WHO [2], the Healthy Brazil Action was also introduced in the same year in Brazil. It aimed to promote physical outdoor activities and healthier eating habits described in the Food Guide for the Brazilian Population (Table 1). It was concomitantly launched and was a milestone in Brazil’s approach to making nutritional recommendations. It revitalized and strengthened the food identity of the local population. In 2014 The Guide was revised, and this new version used a multidimensional approach that featured cultural and qualitative aspects. 2. METHODOLOGY In 2006 the Organic Law on Food and Nutritional Security (LOSAN) established the National System of Food and Nutritional Security (SISAN) [19], through which the National Food and Nutritional Security Policy (PNSAN) [20] was implemented (2009). SISAN was designed with the aims of reducing inequality of opportunity, promoting sustainable and healthy food systems, and ensuring the right to adequate food through the creation of intersectoral bureaus across all levels of government. It also included representation from civil society and the Councils for Food and Nutritional Security. Recently, The National Council for Food and Nutritional Security (CONSEA) exerted pressure on policy-makers, resulting in the framing of laws directed at regulating food advertising and the expansion of the National Pact for Healthy Eating (Table 1). Also, the General Coordination Office for Food and Nutrition Policy of Brazil’s Ministry of Health, in charge of implementing actions in line with the National Food and Nutrition Policy (PNAN), adopted creative strategies such as the Healthy Gym Program [21] and the Healthy Weight Program (intersectoral action directed at workers) amongst others [22]. It was only later, in 2013, that specific policies to promote integrated care for overweight and obesity were introduced. That took place even after the surgical treatment of obesity was made available through the public healthcare system in 2007 (Table 1). Challenges to the implementation of these actions revolve around the suppressed demand for specialist care and the transformation of primary healthcare practices to provide integrated primary and specialist care and other services across the country (Tables 2 and 4). However, there have been some setbacks as the current Brazilian administration (2019-2022) has withdrawn subsidies to municipalities and restricted access to basic services; for instance, the operation of overweight and obesity care services is no longer mandated [23]. There seems to be a contradiction in the approach of the Brazilian Health Authority to the treatment of obesity. In 2007, it introduced a highly complex procedure covered by the public health system (2007) [24], but only in 2013 was bariatric surgery included in the integrated care model for treatment of people with Chronic Diseases (RAS) [25]. Unlike Brazil, whose civil society has greatly contributed to the policymaking process through participation in conferences and councils, Portugal introduced policies on overweight based on academic research and following guidelines by the European Community. 2. METHODOLOGY BRAZIL Year Laws and Regulations Promotion of healthy eating and physical activity 1999 National Food and Nutrition Policy (revised in 2011) [18] 2005 Healthy Brazil Action–outdoor gyms [29] Food Guide for the Brazilian Population (revised in 2014) [30] 2006 National Health Promotion Policy (revised in 2014) [31] Organic Law on Food and Nutritional Security [19] 2009 National Food and Nutritional Security Policy [20] Actions Matrix for Food and Nutrition in Primary Health Care [32] 2010 Regulation of Food Advertising [33] 2011 Health Gym Program [21] Strategy for salt intake reduction (revised in 2013) [34] 2012 Reference Framework of Food and Nutrition Education for Public Policies [35] 2013 Healthy Weight Program (workers) [22] 2014 National Pact for Healthy Eating [36] Overweight and Obesity 2013 Care Line for Overweight and Obesity(*) [37] 2014 Strategies for the Care of People with Chronic Diseases– Obesity [38] Regional Organization of the Care Line for Overweight and Obesity [25] Intersectoral Strategy for Prevention and Control of Obesity [39] Severe obesity 2007 Treatment of severe obesity in the Unified Health Care System (SUS) [24] 2013 Technical regulation for High Complexity Care Services for Individuals with Obesity [40] Chronic non-communicable diseases (CNCD) 2006 Basic care notebook nº 12 - comprehensive and humanized approach to overweight patients [41] 2011 Plan for Coping with CNCD in Brazil - 2011 to 2022 [42] 2013 Health Care Network for People with Chronic Diseases [43, 44] 2014 Strategies for the care of a person with chronic disease [45] Perspectives and challenges in the healthcare for people with obesity in the Brazilian Health System [46] 2017 Redefinition of lines of care for overweight and obesity [47] PORTUGAL Promotion of healthy eating and physical activity 2005 National Plan for an Integrated Action on the Health Factors Related to Lifestyles [48] 2012 Program - Promotion of Healthy Eating [49] (revised in 2019) [50] 2015 Strategy for salt intake reduction [51] Table 1. Timeline laws and regulations for addressing obesity in Brazil and Portugal (1999-2019). Table 1. Timeline laws and regulations for addressing obesity in Brazil and Portugal (1999-2019). 2. METHODOLOGY In 2005, Portugal created the National Platform an Integrated Action on the Health Factors Related to Lifestyles, including a specific action plan to tackle obesity. As for the promotion of healthy eating, there have been some one-off actions [26]. Initiatives that stood out within the same period of time were the National Program to Fight Obesity within the National Health Plan (2004-2010), at the time still sectoral in nature and characterized by basic Healthcare Networks (RAS) represent the Brazilian strategy to promote changes in the care model, adopting the principles of integrality, humanization, multi-professional care, professional/user co-responsibility, relationship building, 210 The Open Public Health Journal, 2021, Volume 14 da Graça da Costa et al. assistance, and the Platform against Obesity (2008) through which actions of the National Program were delivered. proposed and detailed in the current official documents from both countries (in effect in 2019). Table 2 shows individual- level actions and programs for health promotion, disease prevention, and control of obesity. Unlike the Brazilian system, where primary care services focus on procedures, the Portuguese system offers an individualized approach for obesity care (Table 2). Specialized care is not always available in Brazilian public hospitals, but it is accessible in every Portuguese hospital. Also, unlike Portugal, Brazil has failed to deliver some of the needed specialized outpatient care services which are mandated in its policies. As a consequence, Brazil has not been able to satisfy the demand for bariatric needs. In fact, the lack of these services is a major bottleneck within the Unified Health Care System (SUS), which has long struggled to ensure universal healthcare access.Thus , Brazil has a long way to go before it affects implementation of its policies and commitments [65]. Such obstacles might be the reason why the Brazilian National Health Plan’s approach to care for over- weight and obesity is generic rather than specific; however, this needs to be better understood. Brazil faces numerous health challenges and has focused on primary health care, inter- sectoral actions, and involvement of the community in collaborative health-promoting activities. However, it was only later, after the revision of the National Health Plan (2012-2016; extended to 2020) [27], that the fight against obesity was given priority. In 2012 the National Strategy for the Promotion of Healthy Eating (PNPAS) was launched, bringing Portugal’s policies into alignment with the objectives of the European Commission and WHO. 2. METHODOLOGY It is one of the eight priority programs coordinated by the Directorate-General of Health (DGS). Moreover, one of the high-priority goals of the National Health Plan was to control the incidence rates and prevalence of overweight and obesity in school-aged children and slow down the rise of obesity by 2020 [27]. The current Portuguese Health Plan includes intersectoral, health-promoting actions on reducing the health risks of smoking, obesity, sedentarism, alcohol [27], and more recently, healthy eating programs. These actions have been directed at the younger population (Table 2). The initiatives for the prevention and control of obesity have been stimulated by the European Commission, especially those aimed at promoting Mediterranean food practices [28]. As for high complexity care for obesity, the Regulation for the Program of Surgical Treatment of Obesity, introduced by Portugal in 2009, and the 2012 and 2018 actions show their growing concern about the issue (Table 1). In Brazil, treatment of severe obesity was instituted in the Unified Health Care System (SUS) in 2007. At the community level, Brazilian policies created environments that encourage healthy and collective physical activity and eating habits. Communities participate in sectors such as agriculture, sports and leisure, culture, environment, and urban planning to serve their health needs (Table 3). The Journal Community Health Worker is an important com- munication tool in this pursuit because it reports on all these sectors [66]. Both countries’ public systems address obesity with systematic but different processes. The Portuguese system adopts three levels of care: primary, secondary, and tertiary. Individualized assistance ranging from overweight to obesity without complications is provided in primary care. Patients with class-2 obesity suffering from comorbidities and class-3 obesity are assisted in hospitals offering bariatric surgery. In addition to multidisciplinary assistance, patients are entitled to cosmetic surgery after weight loss following bariatric surgery [61]. Portugal uses a multi-pronged approach to manage obesity. This includes programs at schools and workplaces, training the primary food producers, creating regulations, and communicating with social marketing [61]. Another important feature of the Portuguese method is the prevention of iatrogenic harm from prescription drugs and the use of inappropriate diets [61]. Brazil’s National Health Promotion Policy, introduced in 2006 and revised in 2014 [31], is based on state autonomy and distributed responsibility and encourage, among other things, the importance of healthy eating, regular physical activity. It also stimulates interactions between health and well-being and social and environmental factors. Tables 2, 3, and 4 were created to list the initiatives 2. METHODOLOGY This strategy was built on the cooperation of 7 Ministries, Finance, Internal Administration, Education, Health, Economy; Agriculture, Forestry and Rural Development and Sea. celebrated a significant reduction in childhood obesity from 37.9% in 2008 to 29.6% in 2019, showing signs that it is close to reaching its established goals [75]. References to goal indicators found in the official docu- ments suggest that there are key elements in assessing the progress of policies aimed at combating overweight and promoting healthy eating (Table 5). The Brazilian Strategic Plan of Action for tackling chronic non-communicable diseases [42] and National Plan for Food and Nutritional Security [76] make specific reference to these indicators. They are also briefly described in the Portuguese National Health Plan (2012-2016; extended to 2020) and detailed in some of their specific programs [50, 60, 61]; a great number of indicators were assessed to measure the burden of obesity on the Portuguese people and their healthcare system [77]. However, these specific indicators have not been used when assessing the performance of the primary healthcare functional units [78] nor the accrediting healthcare services [79] in Portugal. This indicates that these policies are still being implemented but, unlike in Brazil, their primary healthcare units do not centralize these actions. In a study on food and nutrition policies (2011) [72], Brazil and Portugal were found to have the widest income inequality gaps on their respective continents. In Brazil, intersectoral actions and social participation were identified as strategies, and in Portugal, the Ministry of Agriculture tackled poverty and poor-quality food. Still, there are many societal issues with a contact in many policy areas that remain challenges for addressing chronic diseases [73]. Although many countries are developing multisectoral policies aimed at the prevention and control of chronic di- seases, mainly obesity, advancements in the assessment of their effectiveness and impact are still needed [74]. Portugal recently When comparing actions developed by Brazil and Portugal, a number of convergences can be observed. Each country has enhanced one aspect of the approach or another. Table 2. Individual-level actions and initiatives for the prevention and care of obesity listed in Brazilian and Portuguese official documents (in effect in 2019). 2. METHODOLOGY Population-level actions in Brazil revolve around com- munication, information, regulation, food inspection, nutritional labeling, and increasing the supply of good quality food (Table 4). The Portuguese policies show more control by the State, focusing their attention on specific groups. These groups include children, youth, needy families, hospitalized patients, and pregnant women. The goal of their policies is to in still better food choices and food availability. In Portugal, the State has strong regulating power over the food industry. Brazil, on the other hand, has been heavily lobbied to prevent advances in regulations and transparency [70]. Brazil, however, adopted a different approach based on risk classification. Although the patient can receive individual assistance from the Matrix Support Teams of Family Health Support Centers (NASFs [38], the Brazilian method is heavily focused on categorized assistance. In Brazil, SUS at the primary care level, is responsible for therapeutic modalities and care planning. This is based on patient risk assessment, available resources, social determinants of health, and partnership amongst primary care centers (particularly Family Health Support Centers) and the community. SUS engaged communities in regular physical activity, dancing, sports competitions, games, and workshops [29, 38]. The Portuguese Strategy for the Promotion of Healthy Eating (PNPAS) involves numerous population-targeted actions, while in Brazil, similar programs are community- oriented. In both nations, the programs aim to ensure the physical and economic availability of food for the people while at the same time encouraging an appreciation and consumption of healthy foods. They both also focus on epidemiological monitoring, reduction of risk factors, improvements in the healthcare system, promotion of healthy lifestyles, and policies on the adequacy of food and food systems [26]. The Tables 2, 3, and 4 were created to list the initiatives The Open Public Health Journal, 2021, Volume 14 211 The Open Public Health Journal, 2021, Volume 14 211 A Critical Review of Obesity in Healthcare Systems Portuguese Platform against Obesity (2008) was the first intersectoral policy of its kind in the country, with the purpose of promoting healthy eating and laid the foundations for the PNPAS (2012) [26]. Portugal has gradually set legal mechanisms to facilitate local interventions for the prevention and control of obesity through intersectoral actions [71]. The Integrated Strategy for the Promotion of Healthy Eating (2017) [56] was a major step in consolidating Portuguese policy to address overweight and obesity. 2. METHODOLOGY Brazil Portugal Primary Health Care: Support to Self-care for healthy weight [24, 36] Diagnosis of obesity [24, 36] Assessment of dietary intake [36] Weight monitoring [25] Diagnosis of arterial Hypertension and Diabetes Mellitus [24, 36] Individualized guidance [25, 36] Physical Activity prescription [25] Urgent, priority transport in specially-equipped vehicles for obese patients [40] Specialized care: Treatment by Diet therapy [25, 36] Psychotherapy for overweight and obese patients [25] Physical activity prescription [25] Specialized, outpatient care for bariatric surgery patients [24] Preoperative and follow-up tests [21, 36] Medicine Assistance Program for pre-bariatric surgery patients [24] Multidisciplinary assistance for post-bariatric surgery patients [24, 25, 40] Pharmacotherapy for post-bariatric surgery patients [25, 36] High-complexity, specialized care: Surgical treatment [36, 40] Plastic surgery [25, 40] Across all levels of care: Embracement of obese individual sat health care units [24] Therapeutic evaluation for users with overweight and obesity according to risk strata [24] Primary Health care: Early detection of overweight [61] Individualized care for obesity [60] Obesity appointments for class-1 and class-2 obesity patients [61] Medical follow-up/ Nurse/ Nutritionist/Psychologist [60, 61] Hospital care: A multidisciplinary approach to class-2 obesity with comorbidities and class-3 obesity [63] Bariatric surgery [62] Preoperative and postoperative therapeutic evaluation [61, 63] Across all levels of care: Used-centred care [61] Surveillance of Healthy individuals [61] Table 2. Individual-level actions and initiatives for the prevention and care of obesity listed in Brazilian and Portuguese official documents (in effect in 2019). tions and initiatives for the prevention and care of obesity listed in Brazilian and Portuguese 212 The Open Public Health Journal, 2021, Volume 14 da Graça da Costa et al. Table 3. Community-level actions and initiatives for the prevention and care of obesity are listed in Brazilian and Portuguese official documents (in effect in 2019). Table 3. Community-level actions and initiatives for the prevention and care of obesity are listed in Brazilian and Portuguese official documents (in effect in 2019). 2. METHODOLOGY Brazil Portugal Primary Health Care: Support group for hypertensive and diabetic individuals [24, 36] Support groups for weight control [38] Local participatory planning [36] Intersectoral actions: School Health Program Community fitness equipment [25, 36, 42] Cycle paths, parks, squares, walking paths [35, 42] Promotion of corporal practices for the young and the elderly [42] Promotion of healthy eating habits and lifestyle among the beneficiaries of the Family Grant Program [42] Enhancement of local food culture [36, 42] Strengthening of fresh food production and supply preferably from agroecological farms [36, 42] Increase in healthy food supply – food banks [42] Promotion of healthy eating habits and physical activity [24] Food and nutritional education in social facilities [36] Monitoring of workers’ weight [36] Culinary practices [36] Strengthening of community leadership and social participation [36] Intersectoral actions among schools, residents’ associations, and other public bodies [25, 36] Multisectoral Actions: Regulation of food and drinks sold at schools, health facilities, and scientific events [61] Incentives to local food consumption [49] Mediterranean Diet Food Guide– Adherence of the Ministry of Education to the Mediterranean Diet Food Guide [52] Transversal actions with other society sectors (agriculture, sports, environment, education, independent bodies, and social security) to promote the adoption of the Mediterranean diet [49, 50, 52] Promotion of the reduction of salt and trans fats content in bread [51] Tools for planning affordable, healthy menus [49] Adoption of new food standards for vending machines in healthcare facilities [54] Definition of the foods included in the food baskets given to people/families in need [67] Local strategies for the promotion of healthy eating habits [68] Local and regional projects [68] Greater availability of water, fruit, and vegetables [49, 61] nity-level actions and initiatives for the prevention and care of obesity are listed in Brazilian and Portuguese ts (in effect in 2019). Table 4. Population-level actions and initiatives for the prevention and care of obesity are listed in Brazilian and Portuguese official documents (in effect in 2019). 2. METHODOLOGY Brazil Portugal Ministry of Health: Campaigns for the promotion of physical activity and healthy eating habits [42] Regulation and agreements for the reduction of the content of fats, salt, and sugar in food products [42] Regulation of food advertising and food labeling [36, 42] Social marketing strategies – advertising [42] Food and Nutritional Surveillance [42] Inspection of food labeling and advertising [36] Strategy for salt intake reduction [34] Ministry of Health: Pre-obesity and Obesity Observatory [59] Control of sales of food products with a high content of salt, sugar, and fats [49, 61] Good practices for food product labeling, advertising, and marketing [49] Consumer information [49] Promotion of the consumption of overlooked food products [56] Campaigns for the promotion of information [61] Promotion of healthy eating in pregnancy [69] Systematic collection of indicators of nutritional status and food consumption [49] Partnerships with the food industry and restaurants to increase the production of healthy food products [49] Primary sector policies in alignment with health, sustainable goals [61] ion-level actions and initiatives for the prevention and care of obesity are listed in Brazilian and Portuguese ts (in effect in 2019). Table 4. Population-level actions and initiatives for the prevention and care of obesity are listed in Brazilian official documents (in effect in 2019). For instance, while multi-actor proposals in Brazil are more comprehensive and better characterized as intersectoral, in Portugal, there are more actions aimed at individuals within the healthcare system better equipped to meet the needs of obese individuals. Other government departments in Brazil have occasionally spearheaded initiatives for combating obesity; Brazil’s Ministry of Social Development coordinated the implementation of the Intersectoral Strategy for the Prevention and control of Obesity, involving 20 ministries (Inter- ministerial Food and Nutritional Security Chamber (CAISAN) [39]. The global economic crisis of 2008 brought about economic deregulation, privatization, and commercialization of healthcare in search of process efficiency. However, given the universal welfare and social protection policies of Brazil, these economic measures have not been able to meet the current challenges facing healthcare as the population grows older and non-communicable chronic diseases increase. Also, as a result of the New World Order in response to the assorted shortcomings of capitalism, labour relations in the health sector have deteriorated, and resources are scarce, directly impacting the universality of healthcare [80]. 2. METHODOLOGY The Open Public Health Journal, 2021, Volume 14 213 A Critical Review of Obesity in Healthcare Systems Table 5. Goals and indicators of public policies for addressing overweight in the Brazilian and Portuguese Health Care systems (in effect in 2019). Goals: Launched in 2015, the 2030 Agenda for Sustainable Development offered a strong counterpoint to the economic crisis in an effort to promote healthy living and well-being across all ages and reduce premature deaths due to non- communicable chronic diseases and has been widely discussed internationally since then [81]. Different geopolitical blocs have supported and encouraged global initiatives by WHO and the Food and Agriculture Organization (FAO) through joint declarations, action plans, and cooperation agreements, including the White Paper on a Strategy for Europe on Nutrition, Overweight and Obesity-related health issues by the Commission of the European Communities (2007) [82], the Plan of Action for the Prevention of Obesity in Children and Adolescents by the Pan American Health Organization (PAHO), and the establishment of multi-actors: working groups and networks involving government sectors, academia, civil society, private sector, and politicians. These global and regional initiatives have influenced the formulation of health policies in both Brazil and Portugal; the latter has also been under additional pressure from the European Commission to take measures for the protection and promotion of health but more than that, to rationalize its public finances. In this respect, both countries are faced with a dilemma as labour relations in the health sector deteriorate [80]. Against this background, numerous actions to prevent and control obesity have been introduced to ease the pressure on healthcare systems. Although some studies have indicated their effectiveness, a significant number of controlled studies are needed to corroborate the findings [15]. 2. METHODOLOGY BRAZIL Goals: Halt the rise in obesity in adults by 2025 [42] Increase the prevalence of physical activity during leisure time [42] Increase the intake of fruit and vegetables [42] Reduce the average intake of salt [42] Reduce the intake of fatty meat [42] Perform transversal assessment of body weight in the population [39, 76] Monitor nutritional status in primary care [42, 76] Assessment indicators: Prevalence of overweight and obesity by age group (transversal data) [42] Family food consumption (transversal data) [42] Coverage of anthropometric and food consumption data on the Food and Nutrition Surveillance System (SISVAN) [42] PORTUGAL Goals: Reduce the average salt content in main foods by 10% before 2020 [50] Reduce the average sugar content in main foods by 10% before 2020 [50] Reduce the fatty acid content in main foods by 2% before 2020 [50] Increase the number of people who consume fruit and vegetables by 5% before 2020 [50] Increase the number of people who know the Mediterranean diet by 20% before 2020 [50] Control the incidence and prevalence of overweight and obesity in school-aged children – slow down their rise by 2020 [27, 50] Assessment indicators: Mortality related to obesity: number of deaths, death rate, standardized death rate, standardized death rate in people under 65, standardized death rate in people aged 65 and over, standardized death rate in people under 70, standardized death rate in people aged 70 and over [77] Hospital care: users, discharge, length of hospital stay, day case, no day case, outpatient care cases, and deaths; related to localized adiposity: users, discharge, length of hospital stay, day case, no day case, outpatient care cases, and deaths; related to overweight and obesity-associated malignant neoplasm of colon/rectum, rectosigmoid junction and anus; related to overweight and obesity-associated malignant neoplasm of the breast; related to overweight and obesity-associated malignant neoplasm of the prostate; related to overweight and obesity-associated diabetes [77] Prevalence of overweight between 19 and 64 years old [60, 77] Prevalence of obesity between 19 and 64 years old [60, 77] Prevalence of individuals with BMI (Body Mass Index) between 25-30 kg/m2 [60, 77] Prevalence of individuals with BMI (Body Mass Index) of30 kg/m2 and above [60, 77] Prevalence of Systemic Arterial Hypertension [60, 77] Prevalence of consumption of breakfast, fruit, vegetables, sweets, soft drinks, coffee [77] Regular physical activity [60] Food insecurity [49, 77] Salt and trans fats intake [77] Users’ satisfaction [60] Cardiovascular risk [60] Prevalence of weight loss by 5 to 10% [61] AUTHORS’ CONTRIBUTIONS [7] Luciane da Graça da Costa, Thabata Koester Weber and Maria Rita Marques de Oliveira contributed to conception and design, Luciane da Graça da Costa contributed to data acquisition. Luciane da Graça da Costa, Thabata Koester Weber and Maria Rita Marques de Oliveira contributed to data analysis and interpretation, Luciane da Graça da Costa, Adriana Aparecida de Oliveira Barbosa e Maria Rita Marques de Oliveira helped in drafting of this paper. Luciane da Graça da Costa, Thabata Koester Weber, Isabel Monteiro, Flora Correia and Maria Rita Marques de Oliveira contributed to critical revision of the intellectual content, Flora Correia and Maria Rita Marques de Oliveira supervised the study. Brazil. Ministry of Health. Department of Situation Analysis Vigitel [7] Brazil 2011: Protective and Risk Factors for Chronic Diseases by Telephone Survey Brasília 2019.http://bvsms.saude.gov.br/bvs/publicacoes/vigitel_brasil_2011_f atores_risco_doencas_cronicas.pdf [8] Lopes C, Torres D, Oliveira A, et al. National Food and Physical [8] Activity Survey, IAN-AF 2015-2016: Methodological report University of Porto 2017.https://ian-af.up.pt/sites/default/files/IAN-AF%20Relatorio%20 Metodol%C3%B3gico.pdf Lopes C, Torres D, Oliveira A, et al. National Food and Physical 2017.https://ian-af.up.pt/sites/default/files/IAN-AF%20Relatorio%20 [9] Stanaway J, Murray CJL, Afshin A, et al. Global, regional, and [9] nacional comparative risk assessment of 84 behavioural, environmental and occupational, and metabolic risks or clusters of risks for 195 countries and territories, 1990-2017: A systematic analysis for the Global Burden of Disease Study 2018; 392(10159): 1923-94. Haddad L, Hawkes C, Webb P, et al. A new global research agenda [10] for food. Nature 2016; 540(7631): 30-2. [http://dx.doi.org/10.1038/540030a] [PMID: 27905456] FUNDING This study was financed in part by the Coordination for the Improvement of Higher Education Personnel - Brazil (CAPES) - Finance Code 001 Monteiro BR, Pisco AMSA, Candoso F, Bastos S, Reis M. Primary [12] healthcare in Portugal: 10 years of contractualization of health services in the region of Lisbon. Ciênc saúde coletiva 2017; 22(3): 725-36. Gomes PS. Access to contemporary health in Brazil and Portugal as a [13] social right 2014.http://site.ufvjm.edu.br/revistamultidisciplinar/files/2014/10/Ace sso-%C3%A0-sa%C3%BAde-contempor%C3%A2neo-no-Brasil-e- em-Portugal-como-um-direito-social.pdf Porto/Porto/Portugal; c) Regional Health Administration North (ARS - Norte-IP/ Porto/Portugal. REFERENCES World Health Organization. Obesity: Preventing and managing the [1] global epidemic: Report of a WHO consultation on obesity. Geneva, Switzerland: World Health Organization 1998. World Health Organization. Obesity: Preventing and managing the [1] global epidemic: Report of a WHO consultation on obesity. Geneva, Switzerland: World Health Organization 1998. Brazil has focused on primary health care and intersectoral strategies with community engagement. However, the chief advantage of this has been more than offset by unequal healthcare (notably specialized care) and modest government engagement in regulating food and food advertising. Portugal takes a more traditional approach; primary care units provide a range of individualized healthcare services, from nutritional monitoring to care for overweight and class 2-obesity. Por- tuguese hospitals include outpatient care for class 2-obesity with comorbidities and class-3 obesity and bariatric surgery if recommended. Comprehensive actions occur concertedly, and in spite of being encouraged by the Directorate-General of Health (DGS), there is almost no national program parti- cipation because priorities and healthcare spending are determined more granularly at the regional and municipal levels. Though Portugal’s government takes a more active role in regulating food products than Brazil, it takes a lesser role in the delivery of healthcare services. World Health Organization. Global strategy on diet, physical activity [2] and health Fifty-seventh world health assembly World Health Organization 2004.www.who.int/gb/ebwha/pdf files/WHA57/A57 R17-en.pdf World Health Organization. Global strategy on diet, physical activity [2] and health Fifty-seventh world health assembly World Health Organization 2004.www.who.int/gb/ebwha/pdf_files/WHA57/A57_R17-en.pdf 2004.www.who.int/gb/ebwha/pdf_files/WHA57/A57_R17-en.pdf World Health Organization. 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[http://dx.doi.org/10.1016/S0140-6736(16)31679-8] [PMID: 27733284] Coutinho JG, Gentil PC, Toral N. [Malnutrition and obesity in Brazil: [6] Dealing with the problem through a unified nutritional agenda]. Cad Saude Publica 2008; 24(Suppl. 2): S332-40. [http://dx.doi.org/10.1590/S0102-311X2008001400018] [PMID: 18670713] CONFLICT OF INTEREST The authors declare no conflict of interest, financial or otherwise. Aromataris E, Munn Z, Eds. JBI Manual for Evidence Synthesis. JBI [14] 2020. [cited: 05 th Feb 2021] [http://dx.doi.org/10.46658/JBIMES-20-01] CONSENT FOR PUBLICATION Brazil Law nº 8080, September 19, 1990 Provides information about [11] conditions for the promotion, protection and recovery of health, the organization and functioning of the corresponding services and other provisions Official Brazilian Diary of the Union 1990.https://www2.camara.leg.br/legin/fed/lei/1990/lei-8080-19-setem bro-1990-365093-publicacaooriginal-1-pl.html Not applicable. CONCLUSION Obesity is a global phenomenon that has economically burdened healthcare systems and strained their capacity to respond to other healthcare needs. Specialized care is required and costly in terms of structure, equipment, and labour deployment, and training. The key to tackling obesity is implementing lifestyle changes. It is worth stressing that this study was intended to solely 214 The Open Public Health Journal, 2021, Volume 14 214 The Open Public Health Journal, 2021, Volume 14 da Graça da Costa et al. report health actions aimed at combating obesity identified in the official documents of Brazil and Portugal rather than critically evaluate their implementation or results. Both count- ries have sought to meet global guidelines in their policies, developing guidelines and standards to induce and guide local dynamics. Porto/Porto/Portugal; ACKNOWLEDGEMENTS Reduction of sodium in foods: an analysis of voluntary [34] agreements in Brazil Brazilian Institute for Consumer Protection 2014.https://www.idec.org.br/uploads/publicacoes/publicacoes/cadern o-idec-sodio-alimentos.pdf [35] Brazil Ministry of Social Development and Fight Against Hunger [35] Landmark of food and nutrition education for public policies - Brasília, DF: MDS; National Secretariat for Food and Nutritional Security 2012.https://www.nestle.com.br/nestlenutrisaude/Conteudo/diretriz/M arco_Referencia_de_Educacao_Nutricional_Alimentar.pdf [20] Brazil Ministry of Health Decree nº 7272, August 25, 2010 Regulates [20] Law nº 11,346, September 15, 2006, which creates the National System of Food and Nutritional Security - SISAN with a view to guaranteeing the human right to adequate food, institutes the Policy Food and Nutritional Security - PNSAN, establishes the parameters for the preparation of the National Food and Nutritional Security Plan and and other provisions Official Brazilian Diary of the Union 2010.http://www.planalto.gov.br/ccivil_03/_ato2007-2010/2010/decret o/d7272.htm [36] Brazil Decree nº 8553, November 3, 2015 National Pact for Healthy [36] Eating Official Brazilian Diary of the Union, Brasilia 2015.http://www.planalto.gov.br/ccivil_03/_Ato2015-2018/2015/Decr eto/D8553.htm [21] [37] Brazil. Ministry of Health. Ordinance GM/MS nº 424, March 19, [37] 2013. Redefines the guidelines for the organization of prevention and treatment of overweight and obesity as a priority line of care for the Health Care Network for People with Chronic Diseases. Official Brazilian Diary of the Union, Brasilia 2013.bvsms.saude.gov.br/bvs/saudelegis/gm/2013/prt0424_19_03_201 3.html Brazil Ministry of Health Ordinance GM/MS nº 2681, November 7, [21] 2013 Redefines the Health Academy Program within the scope of the Unified Health System (SUS) Official Brazilian Diary of the Union 2013.http://atencaobasica.saude.rs.gov.br/upload/arquivos/201510/011 14701-20141103160921ms-prt2681.pdf Brazil Ministry of Health Manual for Implementing the Healthy [22] Weight Program Brasília, DF 2013.http://189.28.128.100/dab/docs/portaldab/publicacoes/manual_pe so_saudavel.pdf [38] Brazil Ministry of Health Strategies for the care of the person with [38] chronic disease: Obesity Official Brazilian Diary of the Union, Brasilia 2014.http://189.28.128.100/dab/docs/portaldab/publicacoes/caderno_3 8.pdf Jaime PC, Delmuè DCC, Campello T, Silva DOE, Santos LMP. A [23] look at the food and nutrition agenda over thirty years of the Unified Health System. 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Revista Nutricias 2013; 18: 26-9.https://sigarra.up.pt/ffup/pt/pub_geral.show_file?pi_doc_id=4947 [41] Brazil Ministry of Health Obesity: Basic Attention Notebook nº 12 [41] Brasília: Ministry of Health 2006.http://189.28.128.100/dab/docs/publicacoes/cadernos_ab/abcad1 2.pdf Portugal Ministry of Health Directorate-General for Health National [27] Health Plan Review and Extension to 2020 Lisbon: Ministry of Health; 2015.http://pns.dgs.pt/files/2015/06/Plano-Nacional-de-Saude-Revisao -e-Extensao-a-2020.pdf.pdf Brazil Ministry of Health Strategic Action Plan to Tackle [42] Noncommunicable Diseases (NCD) in Brazil 2011; 2011-22.https://portaldeboaspraticas.iff.fiocruz.br/wp-content/uploads/ 2020/09/plano_acoes_enfrent_dcnt_2011.pdf Portuguese Association of Nutritionists. ACKNOWLEDGEMENTS Burlandy L, Teixeira MRM, Castro LMC, et al. [Models of care for [15] individuals with obesity in primary healthcare in the state of Rio de Janeiro, Brazil]. 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https://openalex.org/W3048709074	https://bmcecolevol.biomedcentral.com/track/pdf/10.1186/s12862-020-01667-8	English	null	Distant hybrids of Heliocidaris crassispina (♀) and Strongylocentrotus intermedius (♂): identification and mtDNA heteroplasmy analysis	BMC evolutionary biology	2,020	cc-by	9,818	© 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. Distant hybrids of Heliocidaris crassispina (♀) and Strongylocentrotus intermedius (♂): identification and mtDNA heteroplasmy analysis Yaoyao Zhan†, Jingxian Sun†, Yingying Li, Dongyao Cui, Weijie Zhang, Limeng Yang and Yaqin Yaoyao Zhan†, Jingxian Sun†, Yingying Li, Dongyao Cui, Weijie Zhang, Limeng Yang and Yaqing Chang* Zhan et al. BMC Evolutionary Biology (2020) 20:101 https://doi.org/10.1186/s12862-020-01667-8 Zhan et al. BMC Evolutionary Biology (2020) 20:101 https://doi.org/10.1186/s12862-020-01667-8 Open Access Abstract Background: Distant hybridization between the sea urchin Heliocidaris crassispina (♀) and the sea urchin Strongylocentrotus intermedius (♂) was successfully performed under laboratory conditions. A new variety of hybrid sea urchin (HS hybrid) was obtained. However, the early-development success rates for the HS hybrids were significantly lower than those of purebred H. crassispina or S. intermedius offspring. In addition, it was difficult to distinguish the HS-hybrid adults from the pure H. crassispina adults, which might lead to confusion in subsequent breeding attempts. In this study, we attempted to develop a method to quickly and effectively identify HS hybrids, and to preliminarily investigate the molecular mechanisms underlying the poor early-development success rates in the HS hybrids. Results: The hybrid sea urchins (HS hybrids) were identified both morphologically and molecularly. There were no significant differences in the test height to test diameter ratios between the HS hybrids and the parents. The number and arrangement of ambulacral pore pairs in the HS hybrids differed from those of the parental lines, which might serve as a useful morphological character for the identification of the HS hybrids. A primer pair that identified the HS hybrids was screened by comparing the mitochondrial genomes of the parental lines. Moreover, paternal leakage induced mitochondrial DNA heteroplasmy in the HS hybrids, which might explain the low rates of early development success in these hybrids. Conclusions: The distant-hybrid sea urchins were accurately identified using comparative morphological and molecular genetic methods. The first evidence of mtDNA heteroplasmy after the distant hybridization of an echinoderm was also provided. Keywords: Heliocidaris crassispina, Strongylocentrotus intermedius, Distant hybridization, Hybrids identification, MtDNA heteroplasmy Background widely used as model organisms in several biological areas, including embryonic development [1], the origins of innate immunity [2], species evolution [3], and marine ecology [4]. Because the human-edible portion of the sea urchin (the gonad) is tasty and nutritious, some sea ur- chin species are major fishery resources in Asian, Medi- terranean, and Western countries [5]. Sea urchins are typical inhabitants of shallow coastal areas. Over the past century, sea urchins have been * Correspondence: yqkeylab@hotmail.com †Yaoyao Zhan and Jingxian Sun contributed equally to this work. Key Laboratory of Mariculture & Stock Enhancement in North China’s Sea, Ministry of Agriculture and Rural Affairs, Dalian Ocean University, Dalian, Liaoning 116023, People’s Republic of China Page 2 of 14 Page 2 of 14 Zhan et al. BMC Evolutionary Biology (2020) 20:101 Zhan et al. BMC Evolutionary Biology (2020) 20:101 tolerance, we previously performed a successful distant hybridization between H. crassispina (♀) and S. interme- dius (♂) in our laboratory using a temperature- controlled method [9]. In this method, we spawned and collected gametes at 26 °C (H. crassispina ♀) and 21 °C (S. intermedius ♂); performed inseminations at 26 °C; and allowed the fertilized eggs to grow and develop at 24 °C [9]. As expected, the larvae produced by the H. crassispina (♀) and S. intermedius (♂) hybrids (HS hy- brids) exhibited increased tolerance of high tempera- tures, as compared to S. intermedius [9]. However, the early-development success rates for the HS hybrids were significantly lower than those of purebred H. crassispina or S. intermedius offspring (Fig. 1), which might increase the costs of artificial reproduction. Moreover, we found that it was difficult to distinguish the HS-hybrid adults from the pure species H. crassispina adults (Fig. 2); this might lead to confusion in subsequent breeding at- tempts. Thus, effective methods for HS hybrid identifica- tion, as well as investigations of the molecular mechanisms underlying the low early development suc- cess rates, are urgently needed to support and promote the cultivation of HS hybrids. Among the edible sea urchins, the temperate urchin Strongylocentrotus intermedius is considered to have the best quality gonads [6]. This species is naturally distrib- uted along the intertidal and subtidal rocky shores of Hokkaido (Japan), the Korean Peninsula, and far eastern Russia [5, 7]. In 1989, S. intermedius was introduced to northern China from Japan, and artificial breeding was begun [7]. Currently, S. intermedius is the predominant commercially-valuable sea urchin species cultivated along the coast of the north Yellow Sea in China [7]. However, because the thermal tolerance of the species ranges only from −1 °C to 23 °C [7], the cultivation and promotion of S. intermedius in south coastal areas of China have been seriously restricted. Hybridization has been well documented as a way to create new germplasms and enrich breeding materials [8, 9]. Indeed, crossbreeding has been widely employed to improve growth, survival, stress resistance, and other traits associated with commercial quality in fish [10], shellfish [11], crustaceans [12], and echinoderms [13, 14]. The sea urchin Heliocidaris crassispina is naturally distributed along the southeastern coast of China [15]. For example, the delayed elimin- ation of paternal mtDNA might result in a high malformation rate in hybrid yellow catfish during early development [36]. As early as 2000, Steel et al. [40] re- ported heteroplasmy in the brittlestar Astrobrach con- strictum and showed that heteroplasmy formed due to mtDNA paternal leakage. However, to our knowledge, this remains the only report of mtDNA paternal leakage and heteroplasmy in echinoderms. As mtDNA hetero- plasmy may affect biological processes, species or popu- lation diversity, and even evolution, we therefore consider it valuable to broadly assess mtDNA hetero- plasmy in as many species as possible. Fig. 2 Morphological comparisons among living individuals. HC_F: Heliocidaris crassispina Fujian population; SI_C: Strongylocentrotus intermedius cultured population; HS hybrid: H. crassispina (♀) × S. intermedius (♂) hybrids In the present study, we attempted to develop a method to quickly and effectively identify HS hybrids, and to preliminarily investigate the molecular mecha- nisms underlying the poor early-development success rates in the HS hybrids. First, external morphological features were analyzed and compared among H. crassis- pina, S. intermedius, and the HS hybrids. The complete mitochondrial genomes for H. crassispina and S. inter- medius were then investigated: nucleotide composition and the codon usage profiles of the protein-coding genes (PCGs) were analyzed, and the secondary structure of each identified tRNA gene was described. A molecular primer pair, which can be used to identify living HS hy- brids, was designed and validated based on a comparison of the mitochondrial genomes of H. crassispina and S. intermedius. Lastly, mtDNA heteroplasmy analysis was performed to investigate the molecular mechanisms underlying the low early-development success rates in the HS hybrids. width to test length and the ratio of test height to test length were shown to be strongly associated with a geo- graphic location in the sea urchin Hemicentrotus pul- cherrimus [20]. The number of ambulacral pore pairs is another key character used for morphological classifica- tion in sea urchins [21]: Margit [21] divided the family Strongylocentrotidae into an oligopore group and a poly- pore group based on the number of ambulacral pore pairs. It has also been suggested that the developmental trajectories of the arrangement of ambulacral pore pairs were a key character supporting heterochronic evolution in the late Cretaceous echinoid genus Gauthieria [22]. This species can withstand seawater temperatures as high as 30 °C [16]. However, the market recognition and price of H. crassispina is relatively low due to the poor quality of the gonads of this species [7]. External morphology is the traditional basis for species identification [17, 18]. In sea urchins, test height, test width, and the ratio of test height to test width have been used to discriminate groups of wild H. crassispina in the South China sea [19]. In addition, the ratio of test In an attempt to cultivate a new variety of sea urchin with both high gonad quality and high temperature Fig. 1 Rate of success at each developmental stage in Heliocidaris crassispina purebred offspring, Strongylocentrotus intermedius purebred offspring, and H. crassispina (♀) × S. intermedius (♂) hybrids. HC_F: Purebred offspring of the H. crassispina Fujian population; SI_C: Purebred offspring of the S. intermedius cultured population; HS: H. crassispina (♀) × S. intermedius (♂) hybrids. “” indicates an extremely significant difference (P < 0.01) between the two groups connected by the line Fig. 1 Rate of success at each developmental stage in Heliocidaris crassispina purebred offspring, Strongylocentrotus intermedius purebred offspring, and H. crassispina (♀) × S. intermedius (♂) hybrids. HC_F: Purebred offspring of the H. crassispina Fujian population; SI_C: Purebred offspring of the S. intermedius cultured population; HS: H. crassispina (♀) × S. intermedius (♂) hybrids. “” indicates an extremely significant difference (P < 0.01) between the two groups connected by the line Zhan et al. BMC Evolutionary Biology (2020) 20:101 Page 3 of 14 Page 3 of 14 Fig. 2 Morphological comparisons among living individuals. HC_F: Heliocidaris crassispina Fujian population; SI_C: Strongylocentrotus intermedius cultured population; HS hybrid: H. crassispina (♀) × S. intermedius (♂) hybrids animals [28]. However, in recent years, a growing body of evidence has indicated the existence of parental mtDNA transmission and mtDNA heteroplasmy in ani- mals [29]. MtDNA heteroplasmy refers to the existence of more than one mtDNA populations within an individ- ual [29]. In different conditions, mtDNA heteroplasmy has different biological significances. For example, mtDNA heteroplasmy is correlated closely with various disease and aging phenotypes in mice and human beings [30–32], while in amphibians, birds, and arthropods, mtDNA heteroplasmy is considered one of the main fac- tors generating biodiversity [28, 33–35]. In addition, mtDNA heteroplasmy has been identified in several spe- cies of fish [36–39]. Mitochondrial DNA (mtDNA) sequences, which com- prise a small portion of the total DNA in a eukaryotic cell, are double-stranded and circular; in most species, mtDNA is inherited solely from the mother [23]. mtDNA sequences are typically 15–20 kb in length, en- coding two ribosomal RNA (rRNA) genes, 22 transfer RNA (tRNA) genes, and 13 protein-coding genes [24]. Compared with nuclear DNA, mtDNA has very low levels of recombination and is likely conserved, but mtDNA sequences evolve rapidly [25]. These features render mtDNA sequences useful tools for species identi- fication and the resolution of taxonomic controversies [26, 27]. It has been universally accepted that the mtDNA follows strict maternal inheritance in most Morphological identification of the HS hybrids Morphological identification of the HS hybrids The heights and diameters of the tests of living Helioci- daris crassispina (Fujian population) (HC_F), Strongylo- centrotus intermedius (cultured population) (SI_C), and HS hybrid individuals were measured to calculate the height-diameter ratio for each line. The results indicated that the height-diameter ratio of the HS hybrid was Zhan et al. BMC Evolutionary Biology (2020) 20:101 Page 4 of 14 smaller than height-diameter ratios of the parental lines (Table 1), but this difference was not statistically signifi- cant (P > 0.05). Fig. 3 Ambulacral pore pair arrangements and numbers. a: Aboral side of test, Heliocidaris crassispina Fujian population; b: Aboral side of test, Strongylocentrotus intermedius cultured population; c: Aboral side of test, H. crassispina (♀) × S. intermedius (♂) hybrid. a: Ambulacral pore pairs, H. crassispina Fujian population; b: Ambulacral pore pairs, S. intermedius cultured population; c: Ambulacral pore pairs, H. crassispina (♀) × S. intermedius (♂) hybrids The tests of sacrificed individuals were observed under a stereomicroscope. Microscope observations revealed six pairs of ambulacral pores in each row on the oral side of HC_F, and seven to nine (mostly eight) pairs of ambulacral pores in each row on the aboral side, which were arranged on the ambulacral plate in an arc shape (Fig. 3). There were five ambulacral pore pairs in each row on both the oral and the aboral side of SI_C, ar- ranged in an oblique arc on the ambulacral plate (Fig. 3). There were four to five ambulacral pore pairs in each row on the oral side of the HS hybrids. These rows were arranged in order but gradually increased at the equator. In each row on the aboral side, we primarily observed seven ambulacral pore pairs. In contrast to the neat ar- rangement of ambulacral pore pairs in both parents, the ambulacral pore pairs on the aboral side of the HS hy- brid were scattered, distorted, and irregular. The num- bers and arrangements of the pore pairs were obviously different from those of HC_F (Fig. 3). Mitochondrial genomes of HC_F and SI_C Because mitochondrial genomes may vary among sea ur- chin species and populations, we designed nine primer pairs based on four reference sequences from the NCBI database (reference sequence accession nos: KC479025.1, KC490912.1, NC_023774.1, and NC_ 023772.1). From these reference sequences, we devel- oped six primer pairs (I, IV, and VI–IX; Table 2) to amplify the HC_F mitochondrial genome, and five pri- mer pairs (I–V; Table 2) to amplify the SI_C mitochon- drial genome. The complete mitochondrial genomes of HC_F and SI_C were cloned and sequenced using poly- merase chain reactions (PCRs). Fig. 3 Ambulacral pore pair arrangements and numbers. a: Aboral side of test, Heliocidaris crassispina Fujian population; b: Aboral side of test, Strongylocentrotus intermedius cultured population; c: Aboral side of test, H. crassispina (♀) × S. intermedius (♂) hybrid. a: Ambulacral pore pairs, H. crassispina Fujian population; b: Ambulacral pore pairs, S. intermedius cultured population; c: Ambulacral pore pairs, H. crassispina (♀) × S. intermedius (♂) hybrids The mitochondrial genome of HC_F (GenBank acces- sion no. MH899145) was a double-stranded circular DNA with a total length of 15,708 bp and an A + T con- tent of 58.9% (Fig. 4a; Table S1; Table S2). The complete genome contained 37 genes: 13 PCGs, two rRNA genes, and 22 tRNA genes. The largest non-coding region (127 bp) was located between tRNAThr and tRNAPr° and was presumably the control region. Together, the protein- coding regions encoded 3822 amino acids (excluding termination codons). The proportion of A + T across the protein-coding regions was 58.2%, and the third codon showed the highest A/U base preference (61.6%). The length of the mitochondrial tRNA was between 68 and 73 bp. With the exception of tRNAser (AGN), which Table 1 Morphological characters of the Heliocidaris crassispina Fujian population, the Strongylocentrotus intermedius cultured population, and the H. crassispina (♀) × S. intermedius (♂) hybrids Sea urchin Test Height (mm) Test Diameter (mm) Test Height / Test Diameter HC_F 16.11(±1.28) 32.11(±2.14) 0.50 SI_C 14.26(±1.11) 27.89(±1.18) 0.51 HS hybrid 15.30(±1.69) 31.55(±1.24) 0.48 HC_F H. crassispina Fujian population, SI_C S. intermedius cultured population, HS H. crassispina (♀) × S. intermedius (♂) hybrids Zhan et al. BMC Evolutionary Biology (2020) 20:101 Page 5 of 14 Table 2 Primers and reaction conditions for mitochondrial DNA fragment amplification No. Mitochondrial genomes of HC_F and SI_C Primer Sequence (5′-3′) HC_F SI_C Annealing temperature Extension time Position (bp) Identity (%) Position (bp) Identity (%) I HSM1F TAACGGATTAAAGCACAGCACTGAA 9879-9903 96.00 9877-9901 100.00 61 °C 3 min 30 s HSM1R CGCATAGAGCTTGAAGGGAATTTAA 13,094-13,118 96.00 13,089-13,113 100.00 II HSM2F TCTTGTTTTCTTGTTTTTGTGAGTT 2812-2836 68.00 12,874-12,898 100.00 54 °C 3 min HSM2R CTCGTGTATCAACATCCATTCC 3541-3562 63.64 886–907 95.45 III HSM3F TGCCATGATTGCAATAGGAGT 825–845 85.71 825–845 100.00 57 °C 3 min 30 s HSM3R ATCTACAAAGTGTCAGTATCAGGCA 4251-4275 92.00 4250-4274 100.00 IV HSM4F CCACTTCTCAACCCATCACCACTTT 4212-4236 92.00 4211-4235 96.00 53 °C 3 min 30 s HSM4R CTATTCCTTGGGGGCCTATTTCTTC 8060-8084 80.00 8058-8082 100.00 V HSM5F TTTTATCTCCTCCCTTTTTATYTCT 8008-8032 76.00 8006-8030 96.00 55 °C 2 min HSM5R CCTCWAAAGTAGTTAAGATTGGGAC 9955-9979 76.00 9953-9977 96.00 VI HcM1F ATCCTGCCTTCCGTTATTTTA 9879-9903 96.00 9877–9901 100.00 53 °C 2 min HcM1R CAACAGTGGTTTGGTCCTTCT 13,094-13,118 96.00 13,089-13,113 100.00 VII RandomF CCGCAAGGGAAAGATGAAATAC 14,289-14,310 100.00 14,285-14,306 100.00 52 °C 4 min RandomR GGGGTGTTATTCTTCTAAGTATTGA 2600-2624 84.00 2598-2622 100.00 VIII HcM2F GAAGGACAAGAACTGGAGAC 2096-2116 100.00 1698-1717 75.00 53 °C 3 min HcM2R CTTTGCGAGATAGATTTAGC 4851–4870 100.00 10,672-10,691 33.33 IX HcM3F ACTTTTGTTTTTCAATAAATCCCTCCA 7680-7706 100.00 8631-8657 70.37 55 °C 2 min 30 s HcM3R TTTCTTTCTAACCACCCTTTTCACC 10,229-10,253 100.00 10,230-10,254 88.00 X RDF TATCATTTAGTAGACCAAAGCCCAT 3559-3583 100.00 3557-3582 100.00 52 °C 40 s RDR CCTGTAGCGACAAAGAAGGTAGAAC 4118-4142 80.00 4117-4141 100.00 lacks a dihydrouracil arm, the other secondary structures were typical cloverleaves. deletions and no single nucleotide polymorphism except for the two Gs in the polyguanine portion of the control region. The mitochondrial genome of SI_C (GenBank acces- sion no. MH899146) had a total length of 15,704 bp and an A + T content of 58.9% (Fig. 4b; Table S1; Table S3). Gene number, gene composition, and the location of the putative control region were consistent with the results of the HC_F analysis. The putative control region of SI_ C was 125 bp. In total, 3820 amino acids (excluding the stop codon) were encoded by the protein-coding regions, which had an A + T ratio of 58.3%. The third codon also had a strong preference for A/U bases (60.8%). The pre- dicted secondary structure of tRNAser (AGN) also lacked a dihydrouracil arm. In addition to the populations of H. crassispina and S. intermedius discussed above, we also selected two widely-distributed and well-studied sea urchin species [S. droebachiensis (SD) and S. purpuratus (SP)], as well as three common edible sea urchin species from northern China [H. pulcherrimus (HP), Mesocentrotus nudus (MN) and Glyptocidaris crenularis (GC)], for further analysis. Mitochondrial genomes of HC_F and SI_C The mitochondrial genomes of these nine sea urchin species/populations were compared and analyzed (Table S1). The average genetic dis- tances (K2Ps) among the 13 PCGs in the mitochon- drial genomes of HC_F, SI_C, and other common sea urchin species were calculated using MEGA 7 (Fig. 5a). The results indicated that the distance be- tween HC_F and HC_K was 0.01. Compared with the genomes of Strongylocentrotus purpuratus and Crypto- cidaris crenularis, the K2P distances for HC_F and HC_K were identical. When compared to the ge- nomes of other sea urchins, HC_F K2P distances were slightly greater than those of HC_K (0.01). The K2P distance between SI_C and SI_K was 0.02. Compared with H. pulcherrimus, the SI_C K2P distances were slightly lower than those of SI_K (0.01). Compared Mitochondrial genome comparisons among nine sea urchins Electrophoresis gels, showing the results of PCR amplification using primer pairs that were used to amplify mtDNA fragments from the cultured population of S. intermedius. The fragments in the inner ring are more similar to the female parent (H. crassispina), while the fragments in the outer ring are more similar to the male parent (S. intermedius). The PCR products amplified by primer pair V, which was selected for identification, are boxed in red Fig. 4 Amplification of mtDNA in Heliocidaris crassispina (♀) and Strongylocentrotus intermedius (♂) hybrids. a: Mitochondrial genome map for the H. crassispina Fujian population. b: Mitochondrial genome map for the S. intermedius cultured population. c: mtDNA fragment amplification diagram for the H. crassispina (♀) × S. intermedius (♂) hybrids. Primer pair V was selected for detection and identification; the product amplified by primer pair V is boxed in red. HC_F: H. crassispina Fujian population; SI_C: S. intermedius cultured population; HS hybrid: H. crassispina (♀) × S. intermedius (♂) hybrid. d. Electrophoresis gels, showing the results of PCR amplification using primer pairs that were used to amplify mtDNA fragments from the cultured population of S. intermedius. The fragments in the inner ring are more similar to the female parent (H. crassispina), while the fragments in the outer ring are more similar to the male parent (S. intermedius). The PCR products amplified by primer pair V, which was selected for identification, are boxed in red differentiation in this gene between the two groups was greater than for all other gene pairs (0–0.30). with other sea urchin genomes, the K2P distances for SI_C and SI_K were identical. The genetic distances between HC_F and HC_K and between SI_C and SI_ K for the 13 PCGs were calculated using MEGA 7 software (Fig. 5b). The distance between the PCGs of HC_F and HC_K was average, and the differentiation level was low (no more than 0.01). However, COI dif- fered noticeably between SI_C and SI_K (0.10); the Mitochondrial genome comparisons among nine sea urchins The HC_F mitochondrial genome was 6 bp longer than that of H. crassispina (Korean population) (HC_K), as was the HC_F control region. HC_F had single nucleo- tide polymorphisms (SNPs) at bases 120 and 121 in the control region, in addition to six Gs in the polyguanine portion of the control region. Compared with S. interme- dius (Korean population) (SI_K), the mitochondrial gen- ome of SI_C was 4 bp longer, while the control region of SI_C was 2 bp longer. SI_C has no base insertions or Zhan et al. BMC Evolutionary Biology (2020) 20:101 Page 6 of 14 with other sea urchin genomes, the K2P distances for SI_C and SI_K were identical. The genetic distances between HC F and HC K and between SI C and SI differentiation in this gene between the two groups was greater than for all other gene pairs (0–0.30). Fig. 4 Amplification of mtDNA in Heliocidaris crassispina (♀) and Strongylocentrotus intermedius (♂) hybrids. a: Mitochondrial genome map for the H. crassispina Fujian population. b: Mitochondrial genome map for the S. intermedius cultured population. c: mtDNA fragment amplification diagram for the H. crassispina (♀) × S. intermedius (♂) hybrids. Primer pair V was selected for detection and identification; the product amplified by primer pair V is boxed in red. HC_F: H. crassispina Fujian population; SI_C: S. intermedius cultured population; HS hybrid: H. crassispina (♀) × S. intermedius (♂) hybrid. d. Electrophoresis gels, showing the results of PCR amplification using primer pairs that were used to amplify mtDNA fragments from the cultured population of S. intermedius. The fragments in the inner ring are more similar to the female parent (H. crassispina), while the fragments in the outer ring are more similar to the male parent (S. intermedius). The PCR products amplified by primer pair V, which was selected for identification, are boxed in red Fig. 4 Amplification of mtDNA in Heliocidaris crassispina (♀) and Strongylocentrotus intermedius (♂) hybrids. a: Mitochondrial genome map for the H. crassispina Fujian population. b: Mitochondrial genome map for the S. intermedius cultured population. c: mtDNA fragment amplification diagram for the H. crassispina (♀) × S. intermedius (♂) hybrids. Primer pair V was selected for detection and identification; the product amplified by primer pair V is boxed in red. HC_F: H. crassispina Fujian population; SI_C: S. intermedius cultured population; HS hybrid: H. crassispina (♀) × S. intermedius (♂) hybrid. d. Molecular identification of the HS hybrids Based on the assumption that mtDNA paternal leakage and heteroplasmy occurred in the HS hybrids, we attempted to identify molecular markers using these phenomena that would be suitable for the identification Zhan et al. BMC Evolutionary Biology (2020) 20:101 Page 7 of 14 Fig. 5 Genetic distance analysis. a: Mean distances among common sea urchins for 13 mitochondrial protein-coding genes; standard error is given in brackets. b: Genetic distance between the mitochondrial protein-coding genes of different pairs of populations. HC_F: Heliocidaris crassispina Fujian population; SI_C: Strongylocentrotus intermedius cultured population; HC_K: H. crassipina Korean population; SI_K: S. intermedius Korean population; SD: Strongylocentrotus droebachiensis; SP: strongylocentrotus purpuratus; HP: Hemicentrotus pulcherrimus; MN: Mesocentrotus nudus; GC: Glyptocidaris crenularis while target bands did appear for all 30 of the PCRs per- formed with HS-hybrid total DNA as the template. The latter bands were clear and bright, and the successful identification rate was 100% (Fig. 6). of the HS hybrids. Two sets of PCR amplifications were performed with the five primer pairs used for SI_C mito- chondrial genome amplification (primer pairs I–V; Table 2): one set with the total DNA of the HS hybrids as the template, and one set with the total DNA of the HC_F population as the template. As shown in the results, pri- mer pairs I, II, IV, and V were successfully amplified in the HS hybrids, while primer pairs II and V were not successfully amplified in HC_F (Fig. 4d). When these two primer pairs were used, the amplification results dif- fered noticeably. Because the amplification products of primer pair V were shorter than those of primer pair II, the use of primer pair V saves time. Therefore, primer pair V was used for subsequent detection and identifica- tion (Fig. 4d). MtDNA heteroplasmy in the HS hybrids Electrophoresis gel, showing the PCR products amplified using the identification primer pairs. b: 30 H. crassispina (♀) × S. intermedius (♂) hybrids. Electrophoresis gel, showing the PCR products amplified using the identification primer pair Fig. 6 Identification of the Heliocidaris crassispi pina (♀) × Strongylocentrotus intermedius (♂) hybrids a: 30 H crassispina from the Fujian population Fig. 6 Identification of the Heliocidaris crassispina (♀) × Strongylocentrotus intermedius (♂) hybrids. a: 30 H. crassispina from the Fujian population. Electrophoresis gel, showing the PCR products amplified using the identification primer pairs. b: 30 H. crassispina (♀) × S. intermedius (♂) hybrids. Electrophoresis gel, showing the PCR products amplified using the identification primer pair digested. However, in the PCR-amplified and digested HS hybrids, we observed not only digested fragments (i.e., bands at 428 and 156 bp) but also undigested frag- ments. These results were identical across life stages (embryo, larva, and adult; Fig. 7b). were performed using the primer pair X (Table 2) that amplifies the same fragment of mtDNA in both parents; comparisons of the PCR products identified differing di- gestion sites. For example, the EcoR I digestion of PCR- amplified SI_C mtDNA should produce two fragments, 428 and 156 bp in length. In contrast, PCR-amplified HC_F resists EcoR I and should not be digested (Fig. 7a). After PCR and enzyme digestion, only one HC_F band appeared. The length of the band was between 500 and 700 bp, indicating that the PCR products were not digested. However, SI_C generated two bands: one less than 200 bp, and one between 400 and 500 bp. This indi- cated that the PCR product had been completely MtDNA heteroplasmy in the HS hybrids As paternal mtDNA fragments were amplified from the HS hybrids, we therefore attempted to further verify the occurrence of mtDNA paternal leakage and hetero- plasmy in the HS hybrids. After sequencing and multiple sequence alignment, we identified four mtDNA frag- ments from the HS hybrids that were successfully ampli- fied using primer pairs I, II, IV, and V. We found that sequence I from the HS hybrids was more similar to ma- ternal HC_F (99.29%), while sequences II, IV, and V from the HS hybrids were more similar to paternal SI_C (94.06, 98.71, and 99.59%, respectively; Fig. 4c). There- fore, we preliminarily speculated that the mitochondrial genome of the HS hybrids contained mitochondrial frag- ments from both paternal lines. To further confirm the simultaneous presence of biparental mtDNA in mito- chondrial genome of the HS hybrids, we used restriction digestion to distinguish the biparental mtDNA. PCRs In order to verify the validity and stability of the primers, we performed PCR amplifications for a large number of samples. Simultaneously, 30 HC_F and HS hybrid individuals were identified. The PCR products were verified using electrophoresis and compared. The electrophoresis results showed that, when primer pair V was used, target bands did not appear in any of the 30 PCRs performed with HC_F total DNA as the template, Page 8 of 14 Zhan et al. BMC Evolutionary Biology (2020) 20:101 were performed using the primer pair X (Table 2) that lifi h f f DNA i b h digested. However, in the PCR-amplified and digested HS h b id b d l di d f Fig. 6 Identification of the Heliocidaris crassispina (♀) × Strongylocentrotus intermedius (♂) hybrids. a: 30 H. crassispina from the Fujian population. Electrophoresis gel, showing the PCR products amplified using the identification primer pairs. b: 30 H. crassispina (♀) × S. intermedius (♂) hybrids. Electrophoresis gel, showing the PCR products amplified using the identification primer pair Fig. 6 Identification of the Heliocidaris crassispina (♀) × Strongylocentrotus intermedius (♂) hybrids. a: 30 H. crassispina from the Fujian population. Electrophoresis gel, showing the PCR products amplified using the identification primer pairs. b: 30 H. crassispina (♀) × S. intermedius (♂) hybrids. Electrophoresis gel, showing the PCR products amplified using the identification primer pair Fig. 6 Identification of the Heliocidaris crassispina (♀) × Strongylocentrotus intermedius (♂) hybrids. a: 30 H. crassispina from the Fujian population. Discussion Lanes 3–5: the biparental mtDNA signal was detected at each stage. HC_F: H. crassispina Fujian population; SI_C: S. intermedius cultured population; HS hybrids: H. crassispina (♀) × S. intermedius (♂) hybrid Fig. 7 Enzyme digestion of fragments from the parents of the Heliocidaris crassispina (♀) × Strongylocentrotus intermedius (♂) hybrids and from the pure offspring at each developmental stage. a: A 584 bp region was PCR amplified using total DNA as a template. The amplified fragment from the male parent (S. intermedius) contains an EcoR I-sensitive site. Thus, digestion of this PCR product produces two fragments (428 and 156 bp). However, the amplified fragment from the female parent (H. crassispina) is EcoR I-resistant. b: The fragments amplified from paternal mtDNA can be selectively digested and thus distinguished from the fragments amplified from maternal mtDNA (H. crassispina). Lane 1: PCR product resulting from maternal mtDNA amplification. This product could not be digested by the EcoR I restriction enzyme and was thus 584 bp long. Lane 2: PCR product resulting from paternal mtDNA amplification was successfully digested by the EcoR I restriction enzyme to produce 428 and 156 bp fragments. Lanes 3–5: the biparental mtDNA signal was detected at each stage. HC_F: H. crassispina Fujian population; SI_C: S. intermedius cultured population; HS hybrids: H. crassispina (♀) × S. intermedius (♂) hybrid Fig. 7 Enzyme digestion of fragments from the parents of the Heliocidaris crassispina (♀) × Strongylocentrotus intermedius (♂) hybrids and from the pure offspring at each developmental stage. a: A 584 bp region was PCR amplified using total DNA as a template. The amplified fragment from the male parent (S. intermedius) contains an EcoR I-sensitive site. Thus, digestion of this PCR product produces two fragments (428 and 156 bp). However, the amplified fragment from the female parent (H. crassispina) is EcoR I-resistant. b: The fragments amplified from paternal mtDNA can be selectively digested and thus distinguished from the fragments amplified from maternal mtDNA (H. crassispina). Lane 1: PCR product resulting from maternal mtDNA amplification. This product could not be digested by the EcoR I restriction enzyme and was thus 584 bp long. Lane 2: PCR product resulting from paternal mtDNA amplification was successfully digested by the EcoR I restriction enzyme to produce 428 and 156 bp fragments. Lanes 3–5: the biparental mtDNA signal was detected at each stage. HC_F: H. crassispina Fujian population; SI_C: S. intermedius cultured population; HS hybrids: H. Discussion Morphological size comparisons have been successfully used to identify hybrids in several aquatic animal groups, including carp [41], catfish (Silurus meridionalis ♀and Silurus soldatovi ♂) [42], and flounder (Scophthalmus maximus♀and Platichthys stellatus♂) [43]. Although a previous study suggested that Hemicentrotus Zhan et al. BMC Evolutionary Biology (2020) 20:101 Page 9 of 14 Fig. 7 Enzyme digestion of fragments from the parents of the Heliocidaris crassispina (♀) × Strongylocentrotus intermedius (♂) hybrids and from the pure offspring at each developmental stage. a: A 584 bp region was PCR amplified using total DNA as a template. The amplified fragment from the male parent (S. intermedius) contains an EcoR I-sensitive site. Thus, digestion of this PCR product produces two fragments (428 and 156 bp). However, the amplified fragment from the female parent (H. crassispina) is EcoR I-resistant. b: The fragments amplified from paternal mtDNA can be selectively digested and thus distinguished from the fragments amplified from maternal mtDNA (H. crassispina). Lane 1: PCR product resulting from maternal mtDNA amplification. This product could not be digested by the EcoR I restriction enzyme and was thus 584 bp long. Lane 2: PCR product resulting from paternal mtDNA amplification was successfully digested by the EcoR I restriction enzyme to produce 428 and 156 bp fragments. Lanes 3–5: the biparental mtDNA signal was detected at each stage. HC_F: H. crassispina Fujian population; SI_C: S. intermedius cultured population; HS hybrids: H. crassispina (♀) × S. intermedius (♂) hybrid Fig. 7 Enzyme digestion of fragments from the parents of the Heliocidaris crassispina (♀) × Strongylocentrotus intermedius (♂) hybrids and from the pure offspring at each developmental stage. a: A 584 bp region was PCR amplified using total DNA as a template. The amplified fragment from the male parent (S. intermedius) contains an EcoR I-sensitive site. Thus, digestion of this PCR product produces two fragments (428 and 156 bp). However, the amplified fragment from the female parent (H. crassispina) is EcoR I-resistant. b: The fragments amplified from paternal mtDNA can be selectively digested and thus distinguished from the fragments amplified from maternal mtDNA (H. crassispina). Lane 1: PCR product resulting from maternal mtDNA amplification. This product could not be digested by the EcoR I restriction enzyme and was thus 584 bp long. Lane 2: PCR product resulting from paternal mtDNA amplification was successfully digested by the EcoR I restriction enzyme to produce 428 and 156 bp fragments. Discussion crassispina (♀) × S. intermedius (♂) hybrid test size between the HS hybrids and the parental lines, although the tests of the HS hybrids appeared flatter than those of the parents. Thus, we excluded test size as pulcherrimus populations from different regions were distinguishable based on test size [20], we found no sta- tistically significant differences in traits associated with Zhan et al. BMC Evolutionary Biology (2020) 20:101 Page 10 of 14 Zhan et al. BMC Evolutionary Biology (2020) 20:101 Page 10 of 14 Page 10 of 14 hypothesize that complex mechanisms might control whether mtDNA differs consistently between the hybrid and the maternal parent. Consequently, additional stud- ies are necessary to determine whether mtDNA can be used as a reliable molecular marker for hybrid identification. a basis for HS hybrid identification. Consistent with a previous study [21], which stated that the number of ambulacral pore pairs was a key morphological character differentiating sea urchin species, we found an obvious difference in the number of ambulacral pore pairs be- tween the HS hybrids and the parental lines. This char- acter might thus prove useful for HS hybrid identification during artificial breeding. However, be- cause the sea urchin must be sacrificed and the test must undergo complex treatment before the number of ambulacral pore pairs can be counted, it is difficult to apply this method in practice. Furthermore, sequencing and analysis of mtDNA frag- ments indicated that some HS hybrid fragments had a higher homology with the paternal parent than with the maternal parent. mtDNA heteroplasmy analysis subse- quently suggested that paternal leakage had induced mtDNA heteroplasmy during hybridization. It has been shown that high rates of malformation and death might be closely correlated with the failure to eliminate pater- nal mtDNA during embryogenesis [36, 39, 49]. Here, mtDNA heteroplasmy was detected in both fertilized embryos and in blastulae. In addition, fertilization and blastula-hatching rates for the hybrids were extremely low. Thus, we speculated that mtDNA heteroplasmy due to paternal leakage might be a key factor affecting fertilization and hatching post-hybridization. Interest- ingly, mtDNA heteroplasmy was also detected in adult HS hybrids, but there was no significant difference in survival rates between HS hybrid adults and purebred adults. This suggested that the early developmental stages might be more sensitive to the harmful effects in- duced by mtDNA heteroplasmy than the late develop- mental stages. Discussion Some studies have indicated that paternal mtDNA might not be efficiently eliminated in interspe- cies hybrids [36, 39, 50]. Our results were consistent with these previous studies and also indicated that pater- nal mtDNA might not be eliminated in some distant hy- brids. In addition, our work raised questions about the dynamic processes comprising mitochondria uniparental inheritance (MUI). In addition, it remains unclear whether mtDNA intermolecular recombination occurred in the HS hybrids. Thus, future studies should aim to clarify not only the species-specific molecular mecha- nisms associated with the recognition and elimination of paternal mtDNA in hybrid species, but also the occur- rence of mtDNA intermolecular recombination in dis- tant hybrids. pp y p We also attempted to identify HS hybrids by searching for and screening genetic molecular markers. We first identified and characterized the mtDNA genomes of the parental lines (H. crassispina and S. intermedius). Se- quencing and bioinformatics analysis showed that dihy- drouracil arms were absent from the predicted secondary structures of tRNASer (AGN) in both H. cras- sispina and S. intermedius. Similar results were also re- ported in Mesocentrotus nudus, H. pulcherrimus, and Loxechinus albus [44–46]. We thus hypothesize that the lack of a dihydrouracil arm in the secondary structure of tRNASer (AGN) might be common in sea urchins. We also found that the genetic distance between the COI gene of the cultured population of S. intermedius (in this study) and the COI gene from the natural population of S. intermedius in Korea [47] was relatively large (0.100). This result might indicate that there is substantial gen- etic variation between cultured and natural populations of S. intermedius. In addition, this result suggested that the COI gene represents a potential molecular marker for the discrimination of cultured and natural popula- tions of S. intermedius. As the HS hybrids are very similar to the maternal parent in appearance, we initially predicted that the HS hybrids would strictly adhere to the maternal genetic in- heritance of mtDNA. However, when we amplified frag- ments of the HS hybrid and maternal parent genomes using the five primer pairs that were used to amplify the SI_C mtDNA fragments, the PCR products were not consistent. We noticed that primer pairs II and V, which were supposed to only amplify fragments from S. inter- medius, also amplified fragments from the HS hybrids. Discussion In particular, the use of primer pair V resulted in clear differences in PCR products between the HS hybrids and the maternal parent. Subsequent experiments con- firmed that the primer pair V accurately distinguished the HS hybrids from the maternal parent. This primer pair could thus potentially be used in aquaculture set- tings to distinguish HS hybrids from the maternal par- ent. Although Havelka et al. [48] suggested that mtDNA could not reliably distinguish hybrids from the maternal parent, our results indicated the opposite. Therefore, we Conclusions We accurately identified distant-hybrid sea urchins (HS hybrids) using comparative morphological and molecular genetic methods. Moreover, we provided the first evi- dence of mtDNA heteroplasmy after the distant hybridization of an echinoderm. Sea urchins Adult H. crassispina (representing one parental line) were collected from the coastal areas of Zhangzhou City, Page 11 of 14 Zhan et al. BMC Evolutionary Biology (2020) 20:101 Zhan et al. BMC Evolutionary Biology (2020) 20:101 Fujian Province, China (117°28′36″E, 23°42′51″N), and transported to the Key Laboratory of Mariculture & Stock Enhancement in the Ministry of Agriculture of the North China Sea at Dalian Ocean University, Dalian, China. The S. intermedius parents were obtained from a broodstock cultured at the Key Laboratory of Maricul- ture & Stock Enhancement. All specimens were kept in 1000 L recirculating seawater tanks (30 specimens per tank) at room temperature (5 °C–26 °C) under natural light. Specimens were fed kelp (Saccharina japonica). Korea (GenBank accession no. KC490911.1); and H. crassispina (HC_K) from Meamul-do, on the South Sea of South Korea (GenBank accession no. KC479025.1) were downloaded from the NCBI database (https://www. ncbi.nlm.nih.gov/). Similar fragments in these sequences were identified using multiple sequence alignments in DNAMAN (Lynnon Biosoft, USA). Based on these simi- larities, nine pairs of primers were designed to amplify the complete mitochondrial genomes of HC_F and SI_C (Table 2, I–IX). Primer pairs I and IV are universal primers, and were used to amplify mtDNA fragments from both HC_F and SI_C. Primer pairs II, III, and V were specific to SI_C. Primer pairs VI–IX were used to amplify mtDNA fragments in HC_F only. The fragments that were amplified by these primers partially overlapped. Using methods previously described [9], three lines were developed in September 2016 for the purposes of this research: H. crassispina ♀× H. crassispina ♂(HC_ F); S. intermedius ♀× S. intermedius ♂(SI_C); and the distant hybrid H. crassispina ♀× S. intermedius ♂(HS hybrid) (Table 3). All individuals were cultured under identical conditions for 1 year. pp PCRs were performed using TAKARA LA Taq (TaKaRa Bio, Japan) in a 50 μl volume, containing 5 μl of DNA template (~ 100 ng/μl), 2 μl of each primer (10 μM each), 5 μl of 10 × LA PCR buffer II (Mg2+ plus), 4 μl of dNTP mixture (2.5 mM), 1 μl of LA Taq polymerase (5 U/μl), and 31 μl of DEPC-Treated water. We used an Eppendorf Mastercycler (Eppendorf, Germany) to run the amplification program. Morphological analyses Living HC_F, SI_C, and HS hybrid individuals were ob- served, and morphological measurements (i.e., test height and test diameter) were taken following Luo et al. [19]. Using these measurements, the test height- diameter ratios were calculated. Individuals from all three sea urchin lines were sacrificed, and the spines and test skin were removed using a bristle brush. The num- bers and arrangements of the ambulacral pore pairs were observed under a stereomicroscope (LEICA M205 FA, Germany). Sea urchins The cycling conditions were as follows: pre-denaturation at 94 °C for 5 min; 35 cycles of denaturation at 94 °C for 30 s, annealing at various temperatures for 30 s, and extension at 72 °C for various durations; and a final termination at 72 °C for 5 min (the specific annealing temperatures and extension times used are shown in Table 2). The amplified products were detected using 1% agarose gel electrophoresis, and target bands were cut from the gel. Target bands were purified and fragments were recovered using SanPrep Column DNA Gel Extraction Kits (Sangon Biotech, China). The recovered fragments were cloned using pEASY-T1 Cloning Kits and Trans5α Chemically Com- petent Cells (Transgen Biotech, China). DNA fragments were cloned and plasmid DNA was sequenced by San- gon Biotech Co., Ltd. (Shanghai, China). Fragments from the parents of the HS hybrids were assembled using DNAMAN to obtain circular DNA sequences. Sequencing and mitochondrial genome assembly As mitochondrial genomes might vary among popula- tions, the complete mitochondrial DNA genomes of S. intermedius (SI_K), from Jumunjin, on the East Sea of South Korea (GenBank accession no. KC490912); H. pul- cherrimus from Tongyeong, on the South Sea of South DNA sample preparation Tube-foot tissues were sampled using sterilized ophthal- mic scissors and ground thoroughly in liquid nitrogen for DNA extraction. We used TIANamp Marine DNA Kits (Tiangen Biotech, China) to extract total DNA from sea urchins representing each of the three lines. DNA samples were quality controlled using 1% agarose gel electrophoresis and a SimpliNano (BioChrom, UK) ultra-micro-spectrophotometer. Authors’ contributions YYZ d YQC i d YYZ and YQC conceived and designed the experiments. JXS, DYC, YYL, and LMY performed the experiments. YYZ, JXS, WJZ, and YQC analyzed the data. YYZ and JXS wrote the paper. All authors read and approved the manuscript. YYZ and YQC conceived and designed the experiments. JXS, DYC, YYL, and LMY performed the experiments. YYZ, JXS, WJZ, and YQC analyzed the data. YYZ and JXS wrote the paper. All authors read and approved the manuscript. Ethics approval and consent to participate The animals used in this study are unregulated invertebrates. Animal welfare and experimental procedures were carried out in accordance with the Laboratory animals-General requirements for animal experimentation (Na- tional Standards of P. R. China, GB/T 35823—2018). Funding PCRs were performed using the total DNA from HC_F, SI_C, and the HS hybrid as templates, and five primer pairs that were used to amplify SI_C mtDNA fragments (Table 2, I–V). The electrophoresis results of the result- ing amplicons were compared. A pair of primers (Table 2, primer pair V) that effectively identified the HS hy- brids was selected. This primer pair was verified against 30 HC_F individuals and 30 HS-hybrid individuals to as- sess identification accuracy. This work was funded by the National Natural Science Foundation of China (No. 31672652). The funder provides experimental funds and supervision. Mitochondrial genome analysis We located 13 protein-coding genes (PCGs), two rRNA genes, and one control region using the DOGMA service (http://dogma.ccbb.utexas.edu/). The locations of most of the tRNA genes were identified using tRNAscan-SE Search Sever (http://lowelab.ucsc.edu/tRNAscan-SE/) in default search mode with the echinoderm mitochondrial genetic code. Some of the tRNA gene locations could not be identified by tRNAscan-SE; these genes were lo- cated using multiple sequence alignments based on the annotations of existing sequences in the NCBI database. Gene maps of the mitochondrial genomes of HC_F and Table 3 Conditions for the distant hybridization Sea urchin Gamete type Temperature (°C) Gamete collection Fertilization Incubation HC_F egg 26 (± 0.5) 26 (± 0.5) 24 (± 0.5) SI_C sperm 21 (± 0.5) HC_F Heliocidaris crassispina Fujian population, SI_C Strongylocentrotus intermedius cultured population Page 12 of 14 Page 12 of 14 Page 12 of 14 Zhan et al. BMC Evolutionary Biology (2020) 20:101 Zhan et al. BMC Evolutionary Biology (2020) 20:101 SI_C were generated using CG view Server (http:// stothard.afns.ualberta.ca/cgview_server/index.html). Competing interests The authors declare that they have no competing financial interest. Received: 19 February 2020 Accepted: 30 July 2020 References 1. Hwang J, Suh SS, Park M, Park SY, Lee S, Lee TK. Differential gene expression patterns during embryonic development of sea urchin exposed to triclosan. Environ Toxicol 2017;32:426–433. https://doi.org/https://doi.org/10.1002/tox. 22246. 1. Hwang J, Suh SS, Park M, Park SY, Lee S, Lee TK. Differential gene expression patterns during embryonic development of sea urchin exposed to triclosan. Environ Toxicol 2017;32:426–433. https://doi.org/https://doi.org/10.1002/tox. 22246. 2. Smith LC, Clow LA, Terwilliger DP. The ancestral complement system in sea urchins. Immunol Rev. 2001;180:16–34. 3. Cary GA, Hinman VF. Echinoderm development and evolution in the post- genomic era. Dev Biol, 2017;427:203–211. https://doi.org/https://doi.org/10. 1016/j.ydbio.2017.02.003. Availability of data and materials The datasets used and analysed during the current study available from the corresponding author on reasonable request. Data analysis All data were expressed as mean ± SD. SPSS 22 (IBM, USA) was used for all statistical analyses. Statistical sig- nificance was determined using the Tukey test following one-way ANOVAs. We considered P < 0.05 significant, and P < 0.01 extremely significant. 4. 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Abbreviations mtDNA: Mitochondrial DNA; rRNA: Ribosomal RNA; tRNA: Transfer RNA; PCR: Polymerase Chain Reaction; PCG: Protein-coding gene; MUI: Mitochondria uniparental inheritance; HS hybrids: The Heliocidris crassispina (♀) and Strongylocentrotus intermedius (♂) hybrids; HC_F: Heliocidaris crassispina (Fujian population); HC_K: Heliocidaris crassispina (Korea population); SI_C: Strongylocentrotus intermedius (cultured population); SI_K: Strongylocentrotus intermedius (Korea population); SD: Strongylocentrotus droebachiensis; SP: Strongylocentrotus purpuratus; HP: Hemicentrotus pulcherrimus; MN: Mesocentrotus nudus; GC: Glyptocidaris crenularis tRNAscan-SE and the ARWEN online service (130.235.46.10/ARWEN/) were used to predict the pos- sible secondary structures of the tRNAs. These second- ary structures were visualized using the Forna online service (http://rna.tbi.univie.ac.at/forna/). The mitochon- drial genomes of the eight other echinoderm species and populations in the NCBI database (Table S1) were com- pared with HC_F and SI_C using MEGA 7 [51], and the evolutionary distances among the PCGs were calculated (K2P). Consent for publication Not applicable. To detect differential restriction sites, multiple sequence alignments and restriction analyses were performed using the complete mtDNA sequences of the parental lines in DNAMAN. We designed a pair of primers that amplified the same mtDNA fragment from both the ma- ternal and the paternal mtDNA sequences, but carried different restriction sites (Table 2, primer pair X). Re- striction digestion was carried out on the PCR fragment, which allowed the amplified mtDNA fragments from SI_ C (but not HC_F) to be cut by the EcoR I restriction enzyme. Competing interests Competing interests The authors declare that they have no competing financial interest. Supplementary information pp y Supplementary information accompanies this paper at https://doi.org/10. 1186/s12862-020-01667-8. Additional file 1: Table S1. Abbreviations and mitochondrial DNA sequence accession numbers for the sea urchin species used in this study. Table S2. Characteristics of the mitochondrial genome of the Heliocidaris crassispina Fujian population. Table S3. Characteristics of the mitochondrial genome of the Stongylocrntrorus intermedius cultured population. pp y Supplementary information accompanies this paper at https://doi.org/10. 1186/s12862-020-01667-8. y Supplementary information accom Supplementary information accompanies this paper at https://doi.org/10. 1186/s12862-020-01667-8. 7. Chang Y. Biological research and culture of sea cucumber and sea urchin. Beijing: China Ocean Press; 2004. Additional file 1: Table S1. Abbreviations and mitochondrial DNA sequence accession numbers for the sea urchin species used in this study. Table S2. 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https://openalex.org/W4211169774	https://www.researchsquare.com/article/rs-1327887/latest.pdf	English	null	Zoonotic Trematode Prevalence In Galba Pervia (Lymnaeidae) And Experimental Infection Of Three Isolated Trematodes In The Intestine Of Duck	Research Square (Research Square)	2,022	cc-by	6,256	Zoonotic Trematode Prevalence In Galba Pervia (Lymnaeidae) And Experimental Infection Of Three Isolated Trematodes In The Intestine Of Duck Isolated Trematodes In Jian Li Guangxi Traditional Chinese Medical Universit Yijing Ren Guangxi University Lei Yang Guangxi University Jiani Guo Guangxi University Haiying Chen Inner Mongolia University Jiani Liu Inner Mongolia University Haoqiang Tian Inner Mongolia University Qingan Zhou Guangxi University Weiyi Huang Guangxi University Wei Hu Inner Mongolia University Xinyu Feng (  fengxinyu2013@163.com ) Inner Mongolia University https://orcid.org/0 Research Article License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Abstract Background: Food-borne diseases cause serious harm to public health and food safety. The snail species Galba pervia is an intermediate host for an array of parasitic trematodes. In this study, we performed a prevalence investigation on zoonotic trematode in G. pervia in Guangxi, China, and assessed the zoonotic potential of Trematode in the region. Methods: G. pervia was collected from 61 sites in 9 cities throughout Guangxi Provinces between 2012 to 2014. The larvae species were determined by combing morphological and molecular characteristics. Phylogenetic trees were constructed using neighbor-joining method with ITS2 sequences. The developmental cycles of the isolated trematodes were examined by experimental infection in ducks. The developmental characteristics of Echinostoma revolutum was recorded by dissecting infected ducklings from 1 dpi to 10 dpi. Methods: G. pervia was collected from 61 sites in 9 cities throughout Guangxi Provinces between 2012 to 2014. The larvae species were determined by combing morphological and molecular characteristics. Results: Species identification showed E. revolutum, Australapatemon sp., Hypoderaeum conoideum, Pharyngostomum cordatum and Echinostoma sp. parasitized in G. pervia. However, no Fasciola larvae had been detected. A Neighbor-Joining tree analysis of ITS2 sequences resulted in monophyletic clades comprised of all sequences from isolated larvae with high bootstrap support. The overall prevalence of Trematode larvae in G. pervia was 22.0% (1818/8258), while E. revolutum presented with the highest infection rate of 12.9% from 11 sampling sites. Ducklings exposed to Echinostoma sp., E. revolutum, and H. conoideum larvae were successfully infected. E. revolutum larvae matured at 10 dpi in the intestine of the duck, and the developmental characteristics of E. revolutum were characterized by the maturation of the reproductive and digestive organs around 6~8 dpi. Conclusions: The present investigation revealed the high prevalence of trematodes in the G. pervia in Guangxi, China. With existing trematode infection human cases together with wide geographical distribution of G. pervia, more insight into the risks of human health and its link to human infections are needed. Research Article License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 2/17 Background Food-borne trematode is a parasitic disease that seriously harms humans and animals [1, 2, 3]. Common food-borne fluke disease are fasciolosis, paragonimosis, schistosomatosis, gastrodiscosis, etc. Corresponding pathogens include fasciolidae, echinostomatidae, opisthorchiidae, heterophyidae, etc[3, 4, 5]. Food-borne trematodes can infect a wide range of mammals, including livestock and humans, causing severe veterinary and public health problems worldwide [6, 7]. Although the infection rates are low, several outbreaks have been reported recently [8, 9]. Galba pervia belongs to Mollusca, Gastropoda, Pulmonata, Basommatophora, Lymnaeidae, Galba [10]. It is an intermediate host for a variety of trematodes, some of which are zoonotic, such as F. gigantica, F. hepatica, Echinostoma revolutum, Echinochasmus perfoliatus and plays a vital role in the transmission and prevalence of these diseases [11]. The shell of G. pervia is thin and translucent with an ear-shaped Page 3/17 Page 3/17 aperture; the ratio apex/body is 10/8mm. Its natural habitats ranged from lakes, canals, ponds, and rice fields. Oviparous hermaphroditic G. pervia lives in large aggregation in suitable environments such as sewage sludge bottom or broken bricks and feeds on algae, hummus, and aquatic plants [12]. G. pervia is widely distributed in China and is the dominant host snail for transmitting Fasciola spp [13]. aperture; the ratio apex/body is 10/8mm. Its natural habitats ranged from lakes, canals, ponds, and rice fields. Oviparous hermaphroditic G. pervia lives in large aggregation in suitable environments such as sewage sludge bottom or broken bricks and feeds on algae, hummus, and aquatic plants [12]. G. pervia is widely distributed in China and is the dominant host snail for transmitting Fasciola spp [13]. Food-borne trematode is often infected by eating raw vegetables such as fish mint (Houttuynia cordata), lettuce (Lactuca sativa), parsley (Petroselinum crispum), and watercress (Nasturtium officinale) [14]. From 2011 to 2012, there was an outbreak of F. gigantica infection in Binchuan County, Dali Prefecture, Yunnan Province in China, and then the authors think that fish mint was most likely the source of diseases [9, 15]. Guangxi Zhuang Autonomous Region is contiguous to Yunnan and shares a similar climate, as well as lifestyles and dietary habits of the local people. Given that the Galba pervia is the important intermediate host of Fasciola in China [11, 16], it also has a wide distribution in Guangxi, representing a potential risk of parasitic zoonosis. Background Therefore, the main objective of this study was to investigate and identify presence of various trematode larvae in G. pervia in Guangxi, and assess the zoonotic potential of trematode for both animal and human in this area. Food-borne trematode is often infected by eating raw vegetables such as fish mint (Houttuynia cordata), lettuce (Lactuca sativa), parsley (Petroselinum crispum), and watercress (Nasturtium Identification of snails and isolation of trematodes larvae The snails were identified morphologically as G. pervia depending on systematic keys of the shell [12]. Then collected G. pervia were dissected under a stereomicroscope and carefully checked for trematodes larvae (rediae, cercariae, or metacercariae), and the larvae were separated from the tissue. We used MoticBA400 microscope to observe and record the body length and body width of each isolated trematode. The body length and body width of rediae, and the body length, body width, tail long and tail width of cercariae at each site were measured. The diameter and wall thickness of metacercaria were also measured. Study areas and snail collection To investigate the potential vector capacity of G. pervia in Guangxi Province, snails were collected from 54 sites in 9 cities, namely, Beihai, Fangchenggang, Guigang, Guilin, Liuzhou, Laibin, Nanning, Qinzhou, Wuzhou, and Yulin, from 2012 August to 2014 August (the number of snail samples per site was about 200). Two types of areas were included: Type 1 areas were rice cultivation areas (contains 51 sites, marked by circular shapes in Fig. 1G); Type 2 areas were the vegetation areas of crops which often used as the raw food (10 sites, marked by triangular shape in Fig. 1G). Details of each locality sampled are given in Table S1. In each sampling site, the snails were collected manually by the plastic scoop, transported to the laboratory, cleaned and rinsed five times in sterilized water, and then placed in plastic trays for subsequent experiments. Molecular examinations of the trematodes Next, a single larva with the identical morphology at each sampling site was selected and rinsed with sterilized distilled water three times before being used to extract parasite genomic DNA by a DNeasy Blood & Tissue kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The extracted DNA samples were stored at −20°C until PCR amplification. The PCR assay targeting the sequence of the internal transcribed spacer 2 (ITS2) gene was used to amplify trematode larvae. The universal primer pairs were designed as described by McManus et al. [17]. All the PCR products were directly sequenced after being purified. The obtained sequences were edited using DNASTAR software (www.dnastar.com/software/lasergene/) and aligned using ClustalX (http://www.clustal.org/clustal2/). The identity of individual specimens was ascertained by comparison with the sequences available in 'non-redundant' database in GenBank by BLAST (http://www.ncbi.nlm.nih.gov/blast/). The nucleotide sequences obtained in the present study have been deposited in the GenBank database under the accession numbers. Next, a single larva with the identical morphology at each sampling site was selected and rinsed with sterilized distilled water three times before being used to extract parasite genomic DNA by a DNeasy Blood & Tissue kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The extracted DNA samples were stored at −20°C until PCR amplification. The PCR assay targeting the sequence of the internal transcribed spacer 2 (ITS2) gene was used to amplify trematode larvae. The universal primer pairs were designed as described by McManus et al. [17]. All the PCR products were directly sequenced after being purified. The obtained sequences were edited using DNASTAR software (www.dnastar.com/software/lasergene/) and aligned using ClustalX (http://www.clustal.org/clustal2/). The identity of individual specimens was ascertained by comparison with the sequences available in 'non-redundant' database in GenBank by BLAST (http://www.ncbi.nlm.nih.gov/blast/). The nucleotide sequences obtained in the present study have been deposited in the GenBank database under the accession numbers. Phylogenetic tree construction with ITS2 Phylogenetic trees were constructed using the neighbor joining (NJ) method in MEGAX (26). The F. gigantica (MK321643), isolated from a cattle, was used as an out-group for the construction of the phylogenetic trees of Echinostoma sp. (KJ848453, KJ848454, and KJ848455), E. revolutum (AY168930, KM980474, KM980476 and KM980477), H. conoideum (AJ564385, KJ944311, KJ944312, and KJ944313), E. robustum (LC224084), E. friedi (AJ564383), E. miyagawai (MW199188), E. paraensei (AF336232), E. caproni (AJ564382), E. trivolvis (GQ463127), E. malayanum (JF412727), and Echinoparyphium recurvatum (AY168931 and KJ435270). For the construction of phylogenetic tree of Australapatemon sp. (KM980467, KM980468, KM980469, KM980470, and KM980471), Pharyngostomum cordatum (OL870492 and KJ137231), A. burti (KU950451), Austrodiplostomum ostrowskiae (KT72878), Alaria americana (MH521246), Diplostomum paracaudum (KJ889013) and Cyathocotyle prussica (MH521249), the Brachylaima sp. (JX010634) and Schistosoma japonicum (S72866) were used as out-groups. The phylogeny was tested with 1,000 bootstrap replicates, using the Kimura two-parameter model as a nucleotide substitution model and gamma distribution as rates among sites. Page 4/17 Page 4/17 Figure 2 Based on morphologically available keys, the species of isolated trematodes were identified by amplification of ITS2 region and verified through BLAST (blast.ncbi.nlm.nih.gov/Blast.cgi) with the highest identity after sequencing. Finally, we identified five different species of trematodes including Australapatemon sp., Echinostoma sp., E. revolutum, H. conoideum, and P. cordatum. The lengths of ITS2 were 292 bp, 429bp, 430bp, 432 bp and 294 bp, respectively. The nucleotide sequences obtained in the present study have been deposited in GenBank database under the accession numbers KM980466- KM980471 (Australapatemon sp.), KJ848453-KJ848455 (Echinostoma sp.), KM980474 and KM980476- KM980477 (E. revolutum), KJ944311-KJ944313 (H. conoideum) and OL870492 (P. cordatum). Experimental infections of isolated trematodes in the intestine of duck Five-day-old ducklings were fed with snails parasitized by isolated trematodes in the field. Each duckling was fed 20 G. pervia, and one duckling was dissected every day from the 1st to 10th day after ingestion. The trematodes were collected from the duck intestines using a complete helminthological dissection method [5], and high-resolution pictures of the collected trematodes were taken with the Motic BA400 microscope and additional accessories. The carmine staining of the press-and-fixed specimen was made according to the method provided by Kong Fanyao [6], and collar, spines, oral sucker, acetabulum, prepharynx, esophagus, testis, ovary was measured from digital images during daily observations. In addition, a single trematode was selected, and a small amount of tissue from the tail of the parasite was Page 5/17 cut out aseptically. After repeated rinsing with sterilized distilled water 2-3 times, DNA extraction was carried out according to the above method. ITS2 gene was amplified and sequenced using the same method, and the trematode species was verified. cut out aseptically. After repeated rinsing with sterilized distilled water 2-3 times, DNA extraction was carried out according to the above method. ITS2 gene was amplified and sequenced using the same method, and the trematode species was verified. Morphological characters and molecular identification of trematodes larvae Morphological characters and molecular identification of trematodes larvae The rediae of echinostomes were cylindrical, blunt at both ends, slightly pointed at the head and more pointed at the tail. The body was curved to the ventral surface with muscular feet, and the movement was slow. The tail of the cercariae is not forked. The head of H. conoideum cercariae shows prominent spines, as well as well-developed ventral suckers, pharynx, and intestines (Fig. 2D-F). The metacercaria were round and have two transparent walls (the outer wall was thicker than the inner wall). Abdominal suckers and refractive granules of larvae could be seen inside the cyst. Due to the movement of the larvae inside the sac, the small spines around its head were not easily observed. The rediae of Australapatemon sp. forms a distinct bulge at the head. The cercaria larvae had a forked tail, which was obviously longer than the body length. The cercaria of P. cordatum also had visible forked-tail, oral sucker and pharynx (Fig. 2J- L). forms a distinct bulge at the head. The cercaria larvae had a forked tail, which was obviously longer than the body length. The cercaria of P. cordatum also had visible forked-tail, oral sucker and pharynx (Fig. 2J- L). Overall information on the sampling and survey data G. pervia samples were collected from 54 sites (as shown in Fig. 1G) with about 38-214 snails in each site. Trematodes were found in 17 sites investigated following dissection, including Tianbao Reservoir and Hede village in Nanning city, Liushan Town, Liutang Village, Guangrong village, and Cha Village in Liuzhou City, and Maling Town in Guilin City. Various stages in the life history of this trematode (redia, cercaria and metacercariae) were found during dissection procedures. The prevalence of trematodes in G. pervia The overall trematodes infection rate was 22.0% (1818/8258). Echinostoma revolutum were detected in the snails from 11 sampling sites, with an infection rate of 12.9% (1069/8258); Hypoderaum conoideum infection was detected in the snails from two sampling sites, with an average infection rate of 3.8% Page 6/17 (315/8258). Infection of Australapatemon sp. was detected in the snails form 2 sampling sites, with an infection rate of 2.5% (206/8258); Infection of Pharyngostomum cordatum and Echinostoma sp. were detected at 1 sampling site with an infection rate of 0.4% (34/112) and 2.3% (194/8258), respectively. Phylogenetic analyses (315/8258). Infection of Australapatemon sp. was detected in the snails form 2 sampling sites, with an infection rate of 2.5% (206/8258); Infection of Pharyngostomum cordatum and Echinostoma sp. were detected at 1 sampling site with an infection rate of 0.4% (34/112) and 2.3% (194/8258), respectively. (315/8258). Infection of Australapatemon sp. was detected in the snails form 2 sampling sites, with an infection rate of 2.5% (206/8258); Infection of Pharyngostomum cordatum and Echinostoma sp. were detected at 1 sampling site with an infection rate of 0.4% (34/112) and 2.3% (194/8258), respectively. Ph l ti l (315/8258). Infection of Australapatemon sp. was detected in the snails form 2 sampling sites, with an infection rate of 2.5% (206/8258); Infection of Pharyngostomum cordatum and Echinostoma sp. were detected at 1 sampling site with an infection rate of 0.4% (34/112) and 2.3% (194/8258), respectively. Laboratory infection experiment with Echinostoma sp., E. revolutum, and H. conoideum Because there was no suitable second intermediate host for P. cordatum and definitive host for Australapatemon sp., we conducted an infection experiment for isolated three kinds of trematode to evaluate rates of parasite establishment in ducklings. Ducklings were individually exposed to Echinostoma sp., E. revolutum, and H. conoideum larvae and all were successfully infected. Subsequent observation on Ducklings (17 dpi) fed with Echinostoma sp. infected G. pervia, we detected eggs (195.8×143.8 µm) in the feces, and the morphological characteristics of adult Echinostoma sp. were presented as measures: body length 9.8 mm, width 1.2 mm, oral suker 638.9×399.2 µm, acetabulum 1591.2×1338.2 µm, pharynx 492.6×331.7 µm, anterior testis 1120.5×707.4 µm, posterior testis 1274.9×880.4 µm, ovary 818.9×527.9 µm. In contrast, we found H. conoideum eggs in ducklings fed with infected G. pervia from three sites from a median of 12 dpi (range: 9 dpi to 14 dpi). The morphological characteristics of adult H. conoideum were: body length 1.05 mm, width 1.5 mm, oral suker 424.2×293 µm, acetabulum 1610.2×1594.6 µm, pharynx 379.8×253.6 µm, anterior testis 1902.6×875.3 µm, posterior testis 2045.2×898.2 µm, ovary 751.7×553.9 µm, and also characterized by the possession of 50 spines. E. revolutum eggs (104.1×63.1 µm) were found on 10 dpi. The morphology of adult E. revolutum was characterized by: body length 8.5 mm, width 2.2 mm, oral suker 260×180.1 µm, acetabulum 741.6×598.3 µm, pharynx 193.1×150.6 µm, anterior testis 628×459.4 µm, posterior testis 725.5×557.9 µm, ovary 411.8×311.2 µm, and presence of a head collar with 37 spines. Phylogenetic analyses In total, 15 representative high-quality ITS2 sequence data was obtained. Figure 2 shows an NJ tree based on the submitted sequences and relevant GenBank sequences. The ITS2 sequences of Echinostoma sp. constituted a monophyletic clade (Fig. 3A shaded pink area), distinct from the clade formed by E. robustum, E. friedi and E. miyagawai. The sequences of E. revolutum and H. conoideum constituted a monophyletic group together with E. revolutum (AY168930) and H. conoideum (AJ564385) references (Fig. 3A shaded blue area). The ITS2 sequences of Australapatemon sp. formed a group with A. burti (KU950451) at 99% bootstrap value but formed a unique clade at 75% bootstrap value. Figure 3B showed that the ITS2 sequences of P. cordatum were identical to the reference sequences of P. cordatum (KJ137231). The developmental characteristics of E. revolutum in duckling host from juvenile to adult As there were not sufficient metacercariae of other trematodes, experiments were only designed to gain insight into how E. revolutum developed in duckling hosts. The developmental characteristics of E. Page 7/17 revolutum was recorded by dissecting infected ducklings from 1 dpi to 10 dpi (eggs in the feces were first detected). E. revolutum could be obtained in the small intestine from 1 to 7 dpi and then migrate and reside in the cecum and colon around 8-10 dpi. The body length developed from 490 µm to 8500.5 µm (a dramatic 17-fold increase). At 1 dpi, juveniles presented a circumoral collar bearing 37 spines in a double circle and characterized by clearly visible oral suckers, acetabulum, pharynx, esophagus, and cecum. At 1 dpi, the tiny structure of the testis appeared. By 4 dpi, the ovaries were beginning to organize and develop, and the seminal receptacle began to form. The tubular-shaped uterus loomed at 4 dpi, and maturation of the reproductive and digestive organs occurred around 6~8 dpi. The vitelline glands were the last to appear, and several eggs deposited in the uterus could be observed at 9 dpi. E. revolutum larvae matured at 10 dpi and excreted eggs (Fig. 4). The daily measurement of E. revolutum development was recorded in detail, as shown in Table S2. Figure 4 Discussion Numerous species of food-borne trematodes are endemic in developing nations and significantly impact public health [18, 19, 20]. Austropeplea, Galba, Lymnaea, Radix and Stagnicola etc. from the families Lymnaeidae act as intermediate hosts of trematodes with substantial implications for human health [10, 13]. The primary research focused on the capability of transmitting Fasciola sp., and at least 20 species of Lymnaeidae have been described as potential vectors of fascioliasis [21]. The results reported in the present paper demonstrate the presence of five trematode species in G. pervia. Morphological characteristics identified the larvae to species level by combining unequivocal molecular markers, which identified as E. revolutum; H. conoideum; Australapatemon sp. P. cordatum and Echinostoma sp., respectively. Different collection sites differed concerning the larvae species and intensity of snails present, which would link with meteorological parameters and habitat types. However, other trematode fauna, such as Fasciola, has not been detected, although Guangxi is one of the important regions of ruminant fascioliasis prevalence in the previous reports [22]. Our investigation indicated that E. revolutum was the most prevalent trematode species in Guangxi Province, with an infection rate of 12.9% among collected snails. In consideration of previous studies that Echinostomatidae have low intermediate host specificity [23]. In addition, Radix plicatula, R. swihoei, Gyraulus conrexiusculus etc. can also act as intermediate hosts [24], and all of above-mentioned snail species also have a wide distribution in Guangxi Province, so it implicates that the actual infection rate of Echinostomatidae trematodes may be much higher than the results found in this study. There are many species of echinostomes, which are tiny parasites that mainly parasitize the intestines of birds, mammals, and humans [21, 25, 26]. However, due to the high diversity of species and similar morphology, some species have not been fully morphologically described by the most used morphological traits, with a precise classification elusive. In addition, it is time-consuming to identify the adults by reintroducing the larvae to complete their life cycles, and the improper selection of the definitive Page 8/17 Page 8/17 experimental host will also lead to the failure of entering the next stage of the life cycle. Given these facts, Jonsson et al. proposed to apply gene markers or restriction fragment length polymorphism (RFLP) for molecular identification [17, 21, 27, 28]. ITS2 species-specific markers have been proven as suitable genetic markers for identifying and differentiating trematode species. Discussion Because the external morphology of trematode metacercariae from this study was quite similar, the ITS2 gene sequences of metacercariae were amplified to identify the metacercariae. The species identification results are consistent with morphological analyses, and the evolutionary relationships of trematode species were successfully elucidated and compared with reference sequences deposited in the public databases. As early as 1968, Lie [29] et al. proposed that the development of trematodes may be restricted by others due to the competition inside the snail when they take the same species of snail as the intermediate host. Subsequent studies revealed a similar competition relationship in the intermediate host of echinostomes [30] and schistosomes [31]. In our study, different trematode species have not been detected in one snail simultaneously. Meanwhile, although G. pervia snail can also serve as the intermediate host for Fasciola in Guangxi Province, we have not observed Fasciola infected G. pervia. This phenomenon may be caused by the cross-species competitive antagonisms of echinostomes with other trematodes, which led to a generally low or non-infection of Fasciola. Trematode is a parasite that can cause severe zoonotic diseases. Lie proposed that the transmission of the disease could be contaminated through competition among trematode larvae in intermediate hosts in 1973 [32]. Although theoretically, echinostomes could be used to reduce the economic losses caused by Fasciola, however, given its great harm to the livestock and poultry, echinostomes are not sound biological control agents for the control of Fasciola in practice. To unveil the trematode infection rate in larger areas in Guangxi Province, it is necessary to expand the sampling sites and select more species of snails for investigation. Further research is needed to determine the coexistence and coevolution of competitive species, especially two or more trematodes that reside in one snail host in natural communities. Echinostomes are a common intestinal parasitic trematode in poultry, which mainly affects the growth and development of the young while is less harmful to the adult. The developmental cycle of echinostomes in its terminal host is short and uncomplicated. Therefore, the animal developmental model of the echinostomes in its terminal host is suitable for studying the immune response between trematode and its host. The research results can also be used as a reference for other small intestinal flukes which induce the terminal host immune response, and related research has also been reported in recent years [3–4]. Discussion This study is mainly aimed at the observation of the growth and development of echinostomes from decapsulation of the cyst to the sexually mature adult stage in the intestinal tract of the terminal host. For the selection of experimental animals, mammals are not susceptible to echinostomes infection, so ducklings were used as the definitive host in our study. To provide a basis for subsequent related research, it needs to explore more animal models for echinostomes infection in the future. Conclusions Page 9/17 The present investigation revealed the prevalence of five trematodes species in the G. pervia in Guangxi Province, China. The results from our study not only provide a baseline information but also offered laboratory experimental models for assessing the potential zoonotic echinostomiasis from G. pervia. Further research is needed toward the understanding risk of human infection in combination with risk evaluation to ameliorate unwanted adverse effect during casual contact or exposure to infected G. pervia. Consent for publication All participants consented to have their data published. Authors' Contributors LJ and RYJ conceived and designed the study. LJ, RYJ, LY, GJN and CHY, LJN, THQ, ZQA and HWY collected and identified the snails, cercariae and metacercariae. RYJ and LJ analyzed the data and drafted the manuscript. LJ, FXY and HW helped in study design, study implementation and manuscript revision. LJ, FXY and HW critically revised the manuscript. All authors read and approved the final manuscript. Availability of data and materials The sequences data has already submitted to GenBank, and will be released to the public database until Dec 1, 2016. The GenBank accession numbers are KX781395 for the ITS2 of Australapatemon sp., KM980463~KM980465, KM980478~KM980479 for the Hypoderaeum conoideum, KM980474 and KM980476~KM980477 for the Echinostoma revolutum. Ethics approval and consent to participate Not applicable Funding This study was supported by the Guangxi Traditional Chinese Medical University Scientific Research Project (XP021059); National Parasitic Resources Center (NPRC-2019-194-30); Key Technology Project of Inner Mongolia Science and Technology Department (2021GG0171). Competing interests The authors declare that they have no competing interests. Acknowledgments Not applicable Page 10/17 References 1. Leonardo L, Hernandez L, Magturo TC, Palasi W, Rubite JM, de Cadiz A, et al. Current status of neglected tropical diseases (NTDs) in the Philippines. Acta Trop. 2020;203:105284. doi:10.1016/j.actatropica.2019.105284. 2. Li L, Liu X, Zhou B, Zhang S, Wang G, Ma G, et al. 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Res Rev Parasitol. 2005;66(1-4):69–74. 26. Anh NTL, Madsen H, Dalsgaard A, Phuong NT, Thanh DTH, Murrell KD. Poultry as reservoir hosts for fishborne zoonotic trematodes in Vietnamese fish farms. Vet Parasitol. 2010;169(3-4):391–4. 26. Anh NTL, Madsen H, Dalsgaard A, Phuong NT, Thanh DTH, Murrell KD. Poultry as reservoir hosts for fishborne zoonotic trematodes in Vietnamese fish farms. Vet Parasitol. 2010;169(3-4):391–4. 27. Wiroonpan P, Chontananarth T, Purivirojkul W. References Cercarial trematodes in freshwater snails from Bangkok, Thailand: prevalence, morphological and molecular studies and human parasite 27. Wiroonpan P, Chontananarth T, Purivirojkul W. Cercarial trematodes in freshwater snails from Bangkok, Thailand: prevalence, morphological and molecular studies and human parasite 27. Wiroonpan P, Chontananarth T, Purivirojkul W. Cercarial trematodes in freshwater snails from Bangkok, Thailand: prevalence, morphological and molecular studies and human parasite Page 12/17 Page 12/17 perspective. Parasitology 2021;148 3:366–83; doi:10.1017/S0031182020002073. https://www.ncbi.nlm.nih.gov/pubmed/33100233. perspective. Parasitology 2021;148 3:366–83; doi:10.1017/S0031182020002073. https://www.ncbi.nlm.nih.gov/pubmed/33100233. 28. Jousson O, Bartoli P, Pawlowski J. Molecular identification of developmental stages in Opecoelidae (Digenea). Int J Parasitol. 1999;29 11:1853–8. doi:10.1016/s0020-7519(99)00124-1. 28. Jousson O, Bartoli P, Pawlowski J. Molecular identification of developmental stages in Opecoelidae (Digenea). Int J Parasitol. 1999;29 11:1853–8. doi:10.1016/s0020-7519(99)00124-1. 29. Lie KJ, Basch PF, Heyneman D, Beck AJ, Audy JR. Implications for trematode control of interspecific larval antagonism within snail hosts. Trans R Soc Trop Med Hyg. 1968;62 3:299–319. doi:10.1016/0035-9203(68)90081-3. 29. Lie KJ, Basch PF, Heyneman D, Beck AJ, Audy JR. Implications for trematode control of interspecific larval antagonism within snail hosts. Trans R Soc Trop Med Hyg. 1968;62 3:299–319. doi:10.1016/0035-9203(68)90081-3. 30. Joe LK, Basch PF, Heyneman D, Fitzgerald F. Antagonism between two species of echinostomes (Paryphostomum segregatum and Echinostoma lindoense) in the snail Biomphalaria glabrata. Z Parasitenkd. 1968;30(2):117–25. doi:10.1007/bf00259720. 30. Joe LK, Basch PF, Heyneman D, Fitzgerald F. Antagonism between two species of echinostomes (Paryphostomum segregatum and Echinostoma lindoense) in the snail Biomphalaria glabrata. Z Parasitenkd. 1968;30(2):117–25. doi:10.1007/bf00259720. 31. Lie KJ: A possible biological control of schistosomiasis and other trematodes in snails. In: Southeast Asian Seminar on Parasitology & Tropical Medicine, Schistosomiasis & other Snail-transmitted Helminthiasis (4th), Manila, Feb 24-27 1969 Proceedings1969: 131-8. 31. Lie KJ: A possible biological control of schistosomiasis and other trematodes in snails. In: Southeast Asian Seminar on Parasitology & Tropical Medicine, Schistosomiasis & other Snail-transmitted Helminthiasis (4th), Manila, Feb 24-27 1969 Proceedings1969: 131-8. 32. Lie KJJEP. Larval trematode antagonism: principles and possible application as a control method. 1973;33 2:343–9. 32. Lie KJJEP. Larval trematode antagonism: principles and possible application as a control method. 1973;33 2:343–9. Figures Page 13/17 Figure 1 A, B: Type 1 areas were rice cultivation areas; C: G. pervia image in anterior view; D, E: Type 2 areas were the vegetation areas of agricultural crops which often used as the raw food; F: G. pervia image posterior view; G: 54 G. pervia snail collection sites in 9 cities in Guangxi Province. Page 14/17 Figure 2 Morphology of rediae, cercariae and metacercariae collected in G. pervia. A-C: The rediae, cercariae and metacercariae of Echinostoma sp.; D-F: The rediae, cercariae and metacercariae of Hypoderaeum conoideum; G-I: The rediae, cercariae and metacercariae of Echinostoma revolutum; J, K: The rediae an cercariae of Australapatemon sp.; L: The cercariae of Pharyngostomum cordatum. Figure 4 Development of E. revolutum in duckling host from 1 dpi to 10 dpi Development of E. revolutum in duckling host from 1 dpi to 10 dpi Figure 2 Morphology of rediae, cercariae and metacercariae collected in G. pervia. A-C: The rediae, cercariae and metacercariae of Echinostoma sp.; D-F: The rediae, cercariae and metacercariae of Hypoderaeum conoideum; G-I: The rediae, cercariae and metacercariae of Echinostoma revolutum; J, K: The rediae and cercariae of Australapatemon sp.; L: The cercariae of Pharyngostomum cordatum. Page 15/17 Page 15/17 Figure 3 Phylogenetic analyses of isolated trematodes based on the ITS2 seq sequences. A: Neighbor joining bootstrap consensus tree with 1000 echinostoms; B: Neighbor joining bootstrap consensus tree with 100 Figure 3 Phylogenetic analyses of isolated trematodes based on the ITS2 sequences and relevant GenBank sequences. A: Neighbor joining bootstrap consensus tree with 1000 bootstrap iterations for the rediae of echinostoms; B: Neighbor joining bootstrap consensus tree with 1000 bootstrap iterations for Australapatemon sp. and Pharyngostomum cordatum. Figure 3 Phylogenetic analyses of isolated trematodes based on the ITS2 sequences and relevant GenBank sequences. A: Neighbor joining bootstrap consensus tree with 1000 bootstrap iterations for the rediae of echinostoms; B: Neighbor joining bootstrap consensus tree with 1000 bootstrap iterations for Australapatemon sp. and Pharyngostomum cordatum. Phylogenetic analyses of isolated trematodes based on the ITS2 sequences and relevant GenBank sequences. A: Neighbor joining bootstrap consensus tree with 1000 bootstrap iterations for the rediae of echinostoms; B: Neighbor joining bootstrap consensus tree with 1000 bootstrap iterations for Australapatemon sp. and Pharyngostomum cordatum. Page 16/17 Page 16/17 Figure 4 Development of E. revolutum in duckling host from 1 dpi to 10 dpi Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. TableS1.docx TableS2.docx Figure 4 Development of E. revolutum in duckling host from 1 dpi to 10 dpi Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. Figure 4 Development of E. revolutum in duckling host from 1 dpi to 10 dpi Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. TableS1.docx TableS2.docx Page 17/17 Page 17/17
https://openalex.org/W4294677010	https://www.mdpi.com/2225-1154/10/9/132/pdf?version=1662548531	English	null	Coastal Flood Risks and the Business Community: Stakeholders’ Perception in Malta	Climate	2,022	cc-by	12,765	climate climate Citation: Spiteri, D.; Gauci, R. Coastal Flood Risks and the Business Community: Stakeholders’ Perception in Malta. Climate 2022, 10, 132. https://doi.org/10.3390/ cli10090132 Citation: Spiteri, D.; Gauci, R. Coastal Flood Risks and the Business Community: Stakeholders’ Perception in Malta. Climate 2022, 10, 132. https://doi.org/10.3390/ cli10090132 Community: Stakeholders’ Perception in Malta. Climate 2022, 10, 132. https://doi.org/10.3390/ cli10090132 Academic Editors: Maria Francesca Bruno and Matteo Gianluca Molfetta Received: 22 July 2022 Accepted: 29 August 2022 Published: 2 September 2022 Academic Editors: Maria Francesca Bruno and Matteo Gianluca Molfetta Received: 22 July 2022 Accepted: 29 August 2022 Published: 2 September 2022 Academic Editors: Maria Francesca Bruno and Matteo Gianluca Molfetta Keywords: coastal flooding; sea level rise; storm surges; business community; stakeholders’ perception; Malta 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. Article Daniel Spiteri * and Ritienne Gauci Department of Geography, Faculty of Arts, University of Malta, MSD 2080 Msida, Malta * Correspondence: daniel.spiteri.15@um.edu.mt Abstract: Resilience of coastal communities is increasingly required to adjust to the effects of climate change and its coast-related threats. Climate change is a major global threat to the environment, economy, and health of urban coastal lowlands. Flooding risks from both rising sea levels and increases in the frequency and severity of storm surges are considered to be amongst the most threatening consequences associated with climate change. The aim of this study was to assess the levels of socio-economic preparedness of low-lying urbanized towns in Malta for the impacts of coastal ﬂooding through the triangulation of stakeholders’ participation from three sectors: the business community, local councils, and specialized experts from the governmental and private sectors. The study also included ﬁeld collection of elevation data for each locality to capture the businesses’ distribution in relation to their height above sea level along the urban waterfront. One- way analysis of variance and NVivo were used to test and compare the business owners’ responses and the experts’ feedback, respectively. The main ﬁndings from the business community suggest that there are no long-term contingency plans or strategies in place to address potential ﬂooding impacts from rising sea levels and storm surges, and that the risks of driving owners out of business is high. From the feedback received by the local councils, it was observed that all of them signiﬁcantly lack the physical and ﬁnancial resources to effectively manage long-term coastal ﬂooding within their locality, forcing them to completely rely on central government for any future needs caused by the impact of coastal ﬂooding. From a central government perspective, it seems that all interviewed experts operate within a fragmented governance model, and mainly adhere to the set of responsibilities aligned with their respective roles within such a governance model. This evidence of governance disconnect requires more horizontal and vertical integration of cross-sectoral strategies to address coastal ﬂooding, within the broader framework of integrated coastal zone management as established by the Mediterranean ICZM protocol. Citation: Spiteri, D.; Gauci, R. Coastal Flood Risks and the Business Community: Stakeholders’ Perception in Malta. Climate 2022, 10, 132. https://doi.org/10.3390/ cli10090132 1. Introduction Climate change is considered to be one of the world’s greatest threats to the coast, the evidence of which is supported by decades of scientiﬁc studies published by the international community. Human activities have been found to be largely responsible for a spike in greenhouse gas emissions [1]. Global greenhouse gases are expected to peak between 2020 and 2025, in models that limit global warming to 1.5 ◦C and those that limit warming to 2 ◦C [2]. 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/). g It is estimated that by 2040–2050, the minimum sea level rise in the Mediterranean Basin is expected to be within the range of 9.8–25.6 cm [3]. Coastal ﬂooding and erosion are the main hazards affecting coastal areas, especially low-lying areas, which have the highest probability of experiencing damages from ﬂooding [4]. In the last century, global sea levels rose by about 20 cm; however, in the last two decades the rate of sea-level rise has nearly doubled, posing a signiﬁcant submersion threat to islands and low-lying areas across the https://www.mdpi.com/journal/climate Climate 2022, 10, 132. https://doi.org/10.3390/cli10090132 Climate 2022, 10, 132 2 of 24 globe [5]. Natural disruptive events such as coastal ﬂooding and storm surges are evidence of the extent to which weather and climate can affect our daily lives [6]. These coastal threats seriously undermine the physical and socio-economic assets of communities who have been surviving due to the functioning of these assets, but which are now becoming vulnerable by virtue of inadequate preparation and/or adaptation strategies [7,8]. Recent studies provide evidence of heavy impacts of ﬂooding on small and medium- sized business operations because of their lack of resources, making them susceptible to a wide range of indirect and direct effects [9–11]. Impacts ranged from indirect effects of event-based ﬂooding episodes to a wider range of long- and short-term direct effects, with only short-term impacts prioritized for immediate intervention [11]. In certain studies, around 40–60% of these small businesses ceased operations following a ﬂooding event due to most small business owners not having a disaster plan in place [10,12]. Cascading impacts also include sanitation and salinization problems that further aggravate the tourism industries present on the coast [13]. 1. Introduction Businesses were found to be more vulnerable when they operate in the context of other urban elements, such as road networks and industrial areas, the ﬂooding of which may cut off vital access to the area for transport users and workers [14,15]. The development of a business vulnerability index (BVI) against rising sea levels serves to assess how coastal zones naturally intersect with high-risk areas depending on the risk of other supporting land-use types being ﬂooded as well [14]. Post-ﬂooding costs, recovery and economic support to small coastal businesses are increasingly the subject of scientiﬁc attention in the literature. Financial impacts are being observed in post-ﬂooding insurance costs, with a considerable rise in insurance excesses and property insurance costs after an event [16]. Small enterprises were found to be more susceptible to ﬂooding impacts due to lower capacities of economic recovery. Insurance coverage is seen as an important instrument to promote resilient economies to address coastal ﬂooding and erosion, with some countries introducing variable levels of insurance coverage as a ﬁnancial protection against ﬂooding events or additional exclusion clauses. However, the likelihood of small and medium enterprises (SMEs) being under-insured remains relatively higher [11,16,17]. The effectiveness of governance for ﬂood risk management is critical for developing and maintaining national resilience, sustainable development, and well-being in the face of increasing climate change derived risks [18]. Flood risk mitigation therefore needs to be integrated with sustainable economic development, but questions remain as to how to engage with regional and local businesses within the governance process [19]. Studies show how business owners, even though they receive ﬂood risk information from national agencies, still have relatively little knowledge about the importance of managing ﬂood risks. Stakeholders’ engagement is therefore crucial to assess how business owners perceive ﬂood risks and impacts and to secure better participation within a governance process [20,21] to not only achieve sustainable economic development but also meet the Sustainable Development Goal (13) related to climate change [19]. 2. Study Area The city-island-state of Malta is the smallest EU nation, consisting of an archipelago of three main islands: Malta, Gozo and Comino situated in the central Mediterranean (Figure 1). Although it is traditionally considered to be a low-hazard country and safe from external physical wounding, recent studies of its public records show the presence of numerous natural hazards events through the ages [22]. In being a small highly urbanized archipelago, dependent on internal and external pressures (both socio-economic and envi- ronmental), Malta is faced with multiple internal and external pressures that increase its precariousness and vulnerability to such externalities [23]. Climate 2022, 10, 132 3 of 24 ration o ade (Fig Figure 1. Location of five selected sites of coastal towns: Gżira, Sliema and St Julian's (M Marsalforn and Xlendi (Gozo). Marsaskala, Marsaxlokk, Birzebbuġa and Msida were sele previous (2019) study by same authors. Figure 1. Location of ﬁve selected sites of coastal towns: G˙zira, Sliema and St Julian’s (Malta) and Marsalforn and Xlendi (Gozo). Marsaskala, Marsaxlokk, Birzebbu ˙ga and Msida were selected in a previous study (2019) by the same authors. Figure 1. Location of five selected sites of coastal towns: Gżira, Sliema and St Julian's (M Marsalforn and Xlendi (Gozo). Marsaskala, Marsaxlokk, Birzebbuġa and Msida were sele previous (2019) study by same authors. Figure 1. Location of ﬁve selected sites of coastal towns: G˙zira, Sliema and St Julian’s (Malta) and Marsalforn and Xlendi (Gozo). Marsaskala, Marsaxlokk, Birzebbu ˙ga and Msida were selected in a previous study (2019) by the same authors. The latest national report by the Seventh National Communication of Malta under the United Nations Framework Convention on Climate Change (UNFCC) identiﬁed several impacts related to climate change, including inundation, coastal erosion, loss of beaches and damages derived from high winds, storm surges and waves [24]. The previous UNFCC national reports identiﬁed vulnerabilities pertaining to rising sea levels which affect land-use such as ports, roads, coastal infrastructure and protected areas. The Strategic Plan for the Environment and Development (SPED, 2015) reported that extreme weather events are predicted to increase both in intensity and frequency, leading to increasing risks of ﬂooding [25]. Signiﬁcant land use pressures exist on the Maltese low-lying coasts ranging from tourism (29% of GDP), maritime activities, traditional services such as ﬁshing, swimming and artisanal salinas to important ecological and geoheritage services such as Natura2000 sites [26,27]. 2. Study Area In view of this, knowledge on stakeholders’ perception about the effects of climate change-related ﬂooding on the coastal business community is paramount since any change in sea level or coastal dynamics, such as waves, winds, or changes to its landscape due to erosion, would disrupt daily human operations like transport, housing, and work [28]. Malta ratiﬁed the Integrated Coastal Zone Management (ICZM) Protocol to the Barcelona Convention in 2019 (signed in 2008) and is still in process of adopting the Protocol principles into local legislation or policy. This study was conducted in ﬁve prime economic localities in the Maltese Islands i.e., G˙zira, Sliema, St Julian’s, Marsalforn, and Xlendi (Figure 1). Despite their economic importance, all ﬁve localities share the same low-lying coastal topography and a ribbon-type business distribution. Each coastal town has a high concentration of retail and recreational services spread along its waterfront and adjacent promenade (Figures 2–6). Climate 2022, 10, 132 limate 2022, 10, x Climate 2022, 10, x 4 of 24 Figure 2. A Gżira business establishment located close to the water’s edge. Figure 3. Businesses operating along the Sliema promenade. Figure 2. A G˙zira business establishment located close to the water’s edge. Figure 2. A Gżira business establishment located close to the water’s edge. Figure 3. Businesses operating along the Sliema promenade. Figure 3. Businesses operating along the Sliema promenade. Figure 2. A Gżira business establishment located close to the water’s edge. Figure 2. A G˙zira business establishment located close to the water’s edge. Figure 2. A Gżira business establishment located close to the water’s edge. Figure 2. A Gżira business establishment located close to the water’s Figure 2. A G˙zira business establishment located close to the water’s edge. Figure 2. A Gżira business establishment located close to the water’s Figure 3. Businesses operating along the Sliema promenade. Figure 3. Businesses operating along the Sliema promenade. Figure 3. Businesses operating along the Sliema promenade. Figure 3. Businesses operating along the Sliema promen Figure 3. Businesses operating along the Sliema prome Figure 3. Businesses operating along the Sliema promenade. Climate 2022, 10, 132 Climate 2022, 10, x limate 2022, 10, x 5 of 24 Figure 4. Business operating close to sea level at Spinola Bay, St Julian’s. Figure 4. Business operating close to sea level at Spinola Bay, St Julian’s. Figure 4. Business operating close to sea level at Spinola Bay, St Julian’s. Figure 4. 2. Study Area Business operating close to sea level at Spinola Bay, St Ju Figure 4. Business operating close to sea level at Spinola Bay, St Julian’s. Figure 4. Business operating close to sea level at Spinola Bay, St Ju Figure 5. Businesses located along the promenade of Xlendi Bay. Figure 5. Businesses located along the promenade of Xlendi Bay. Figure 5. Businesses located along the promenade of Xlendi Bay. Figure 5. Businesses located along the promenade of Xlend Figure 5. Businesses located along the promenade of Xlend Figure 5. Businesses located along the promenade of Xlendi Bay. Figure 5. Businesses located along the promenade of Xlend Figure 5. Businesses located along the promenade of Xlend Figure 5. Businesses located along the promenade of Xlendi Bay. Climate 2022, 10, 132 limate 2022, 10, x 6 of 24 Figure 6. Marsalforn businesses operating with outdoor seating close to the shore. Figure 6. Marsalforn businesses operating with outdoor seating close to the shore. Figure 6. Marsalforn businesses operating with outdoor seating close to the Figure 6. Marsalforn businesses operating with outdoor seating close to the shore. However, studies on the impacts of coastal flooding on the Maltese b community remain scarce. Spiteri (2019) assessed the impacts of rising sea lev storm surge flooding on coastal businesses through stakeholders’ consultation wi councils and the business communities in Marsaskala, Marsaxlokk, Birżebbu Msida [28]. None of the business owners or local councils ever had any plans to a such threats, even though the selected localities had experienced coastal flo particularly from episodic storm events. Such events have pushed local councils evaluating the vulnerability of their locality to coastal flooding, and to begin the However, studies on the impacts of coastal ﬂooding on the Maltese business commu- nity remain scarce. Spiteri (2019) assessed the impacts of rising sea levels and storm surge ﬂooding on coastal businesses through stakeholders’ consultation with local councils and the business communities in Marsaskala, Marsaxlokk, Bir˙zebbu ˙ga and Msida [28]. None of the business owners or local councils ever had any plans to address such threats, even though the selected localities had experienced coastal ﬂooding, particularly from episodic storm events. Such events have pushed local councils to start evaluating the vulnerability of their locality to coastal ﬂooding, and to begin the process of drawing up plans to protect their towns from future sea level rise and storm-derived ﬂooding [9]. 2. Study Area e a ua i g e u e a i i y o ei o a i y o oas a oo i g, a o egi e of drawing up plans to protect their towns from future sea level rise and storm-d flooding [9]. Other impact studies are mostly focused on the impacts of projected sea-level infrastructure and transport [29,30]. Rizzo (2019) assessed the reliance of businesses on an accessible and efficient coastal transport network and the vulne of the latter to flooding disruptions [15]. Through a multi-criteria analysis, a cas effect was observed during coastal flooding events in which flooding impacts road networks and severely restricts access to the business areas, including th Sliema and St Julian’s. Rapid urbanization on parts of the Maltese coast (suc g Other impact studies are mostly focused on the impacts of projected sea-level rise on infrastructure and transport [29,30]. Rizzo (2019) assessed the reliance of coastal businesses on an accessible and efﬁcient coastal transport network and the vulnerability of the latter to ﬂooding disruptions [15]. Through a multi-criteria analysis, a cascading effect was observed during coastal ﬂooding events in which ﬂooding impacts coastal road networks and severely restricts access to the business areas, including those in Sliema and St Julian’s. Rapid urbanization on parts of the Maltese coast (such as St Julian’s) since the Second World War has ampliﬁed the potential risks of damages derived from coastal ﬂooding events. However, such risks were perceived by the local community as seasonal and too infrequent to be of any national concern [31]. Julian’s) since the Seco derived from coastal flo 3. Materials and Methods In addition, Cloud Isle, the University of Malta’s open-source software hosting the latest Lidar data, was used to extract the elevation data and create a height proﬁle of the areas. B. Stakeholders’ participation: Three sets of mixed methods questionnaires/ interviews were conducted with the business community, local councils, and expert bodies. The local council and business questionnaires were structured to have mixed methods questions using “Likert scales” and “Yes/No” based questions combined with several open-ended questions. Experts were interviewed through open ended questions to encourage experts to share their insights, experiences, and professional opinions. The targeted business sample size was 50% of the total number of businesses; however, the number of interviewed businesses per locality was inﬂuenced by the uneven number of establishments in each locality. The total number of businesses across all ﬁve localities stood at 356. With a 50% sample size, the interviewed sample was calculated at 186 businesses with 95% conﬁdence level and a 5% margin of error [32]. The number of questionnaires distributed in each locality was calculated as follows: G˙zira: 40, Sliema: 50, St Julian’s: 78, Xlendi 7, and Marsalforn: 13. All 5 local councils were interviewed, along with 12 experts from the following private and governmental entities: i. The Malta Tourism Authority (hereinafter referred as MTA); ii. The Ministry for Transport, Infrastructure and Capital Projects (MTIP); e Ministry for Transport, Infrastructure and Capi iii. The Environmental Resource Authority (ERA); y iv. The Ministry for the Economy, Investment and Small Businesses (MEISB); y y v. The Malta Chamber of Commerce, Enterprise and Industry (MCCEI); The Malta Resource Authority (MRA); vii. The Malta Chamber of Small and Medium Enterprises (MCSME); viii. The Civil Protection Department (CPD); ix. The Ministry for the Environment, Sustainable Development and Climate Change (MESDCC); x. The Planning Authority (PA); xi. The Transport Malta (TM); and xii. The Transport Malta Ports and Yachting Directorate (PYD). xii. The Transport Malta Ports and Yachting Directorate (PYD). To analyse the experts’ feedback, NVivo was used to evaluate and interpret the responses on a thematic level and visually show any differences within the cluster analysis. The closed questions of the local council’s responses were analysed through the construction of ‘yes’ and ‘no’ matrix. The Likert scale responses in the business questionnaires were tested with analysis of variance (Kruskal Wallis H-Test) through Microsoft Excel (graphs) and IBM SPSS (statistics). Julian’s) since the Seco derived from coastal flo 3. Materials and Methods The scope of this statistical analysis was to test whether any differences or similarities exist between the replies of owners according to their business category and locality. Before carrying out each Kruskal Wallis H-Test, the Kolmogorov-Smirnov normality test conﬁrmed the use of a non-parametric analysis of variance. Julian’s) since the Seco derived from coastal flo 3. Materials and Methods derived from coastal flooding events. However, such risks were perceived by o u ity a ea o al a d too i f e ue t to be of a y atio al o e [31] The methods undertaken for this study were based on two types of investigation: community as seasonal and too infrequent to be of any national concern [31]. 3. Materials and Methods The methods undertaken for this study were based on two types of investiga A. Land-use mapping: This exercise aimed to plot the distribution, elevati types of businesses found along the coast of the five selected localities. The georefe coordinates were collected using an embedded GPS app available on iPhone simultaneously taking inventory of the number of businesses and classifying according to the type of business. The coordinates were then plotted into Q t a fo the oo di ate i to a et of a illu t ati the atial di t ibutio of y yp g A. Land-use mapping: This exercise aimed to plot the distribution, elevation and types of businesses found along the coast of the ﬁve selected localities. The georeferenced coordinates were collected using an embedded GPS app available on iPhone, while simulta- neously taking inventory of the number of businesses and classifying them according to the type of business. The coordinates were then plotted into QGIS to transform the coordinates into a set of maps illustrating the spatial distribution of coastal businesses in each locality. Other data could be extracted such as business clusters, sectors, and distribution in relation to the actual geographic distance from one another and the sea. For the creation of these maps, an Excel ﬁle was created for each business activity. As for the elevation data, the University of Malta’s own digital surface model (DSM) and digital terrain model (DTM) Climate 2022, 10, 132 7 of 24 7 of 24 for each locality were requested and imported into QGIS’s business distribution map to illustrate the business community’s elevation above sea level. In addition, Cloud Isle, the University of Malta’s open-source software hosting the latest Lidar data, was used to extract the elevation data and create a height proﬁle of the areas. for each locality were requested and imported into QGIS’s business distribution map to illustrate the business community’s elevation above sea level. 4.1. Businesses 4.1. Businesses 4.1.1. Mapping of Locations and Types of Coastal Businesses Figures 7–12 illustrate the coastal business distribution for each locality. The various coloured dots along the coastline represent the various sectors operating in each locality. In addition, the shaded areas superimposed on the business distribution, portrays the elevation of the area above sea level, with the lighter shade representing a low elevation while the darker shades exhibit a progressively higher elevation. Each map depicts various business sectors with catering, accommodation, and retail being the three most dominant sectors and primarily serving the tourism industry. The key ﬁnding from these maps is that, in each locality, a heavy concentration of businesses is situated in a low-lying area between 0–3 m above sea level, except for the backshore of St George’s Bay (Figure 10) where elevation increases rapidly due to an uphill topography. The land-use maps conﬁrm Climate 2022, 10, 132 8 of 24 d in a 8 of 24 d in a the high degree of concentration of a wide variety of businesses present along the lowest coastal elevations in the selected localities and how their exposure risks to coastal ﬂooding is related both to their close proximity to the shoreline, but also to their orientation to frequent north-westerly and north-easterly winter storms. Bay (Figure 10) where elevation increases rapidly due to an uphill topography. The land-use maps confirm the high degree of concentration of a wide variety of businesses present along the lowest coastal elevations in the selected localities and how their exposure risks to coastal flooding is related both to their close proximity to the shoreline, but also to their orientation to frequent north-westerly and north-easterly winter storms. Bay (Figure 10) where elevation increases rapidly due to an uphill topography. 4.1. Businesses The land-use maps confirm the high degree of concentration of a wide variety of businesses present along the lowest coastal elevations in the selected localities and how their exposure risks to coastal flooding is related both to their close proximity to the shoreline, b t l t th i i t ti t f t th t l d th t l i t t the high degree of concentration of a wide variety of businesses present along the lowest coastal elevations in the selected localities and how their exposure risks to coastal ﬂooding is related both to their close proximity to the shoreline, but also to their orientation to frequent north-westerly and north-easterly winter storms. Bay (Figure 10) where elevation increases rapidly due to an uphill topography. The land-use maps confirm the high degree of concentration of a wide variety of businesses present along the lowest coastal elevations in the selected localities and how their exposure risks to coastal flooding is related both to their close proximity to the shoreline, but also to their orientation to frequent north-westerly and north-easterly winter storms. Bay (Figure 10) where elevation increases rapidly due to an uphill topography. The land-use maps confirm the high degree of concentration of a wide variety of businesses present along the lowest coastal elevations in the selected localities and how their exposure risks to coastal flooding is related both to their close proximity to the shoreline, b t l t th i i t ti t f t th t l d th t l i t t q y y Figure 7. Gżira’s coastal businesses’ distribution. Figure 8. Sliema’s coastal businesses’ distribution. Figure 7. G˙zira’s coastal businesses’ distribution. but also to their orientation to frequent north-westerly and north-easterly winter stor Figure 7. Gżira’s coastal businesses’ distribution. Figure 8. Sliema’s coastal businesses’ distribution. Figure 8. Sliema’s coastal businesses’ distribution. Figure 7. Gżira’s coastal businesses’ distribution. Figure 7. G˙zira’s coastal businesses’ distribution. Figure 7. Gżira’s coastal businesses’ distribution. Figure 7. Gżira’s coastal businesses’ distribution. Figure 7. G˙zira’s coastal businesses’ distribution. Figure 7. Gżira’s coastal businesses’ distribution. Figure 7. Gżira’s coastal businesses’ distribution. Figure 7. G˙zira’s coastal businesses’ distribution. Figure 7. Gżira’s coastal businesses’ distribution. Figure 8. Sliema’s coastal businesses’ distribution. Figure 8. Sliema’s coastal businesses’ distribution. Figure 8. Sliema’s coastal businesses’ distribution. g Figure 8. Sliema’s coastal businesses’ distribution. Figure 8. Sliema’s coastal businesses’ distribution. 4.1. Businesses Climate 2022, 10, 132 Climate 2022, 10, x Climate 2022 10 x 9 of 24 9 of 25 9 of 25 Figure 9. Coastal businesses’ distribution in St Julian’s: Balluta to Spinola Bay (Part 1 of St Julian’s). Figure 9. Coastal businesses’ distribution in St Julian’s: Balluta to Spinola Bay (Part 1 of St Julian’s). . Figure 9. Coastal businesses’ distribution in St Julian’s: Balluta to Spinola Bay (Part 1 of St Julian’s). Figure 9. Coastal businesses’ distribution in St Julian’s: Balluta to Spinola Bay (Part 1 of St Julian’s). Figure 9. Coastal businesses’ distribution in St Julian’s: Balluta to Spinola Bay (Part 1 of St Julian’s). Figure 9. Coastal businesses’ distribution in St Julian’s: Balluta to Spinola Bay (Part 1 of St Julian’s). Figure 10. Coastal businesses’ distribution in St Julian’s: St George’s Bay (Part 2 of St Julian’s). Figure 10. Coastal businesses’ distribution in St Julian’s: St George’s Bay (Part 2 of St Julian’s). Figure 10. Coastal businesses’ distribution in St Julian’s: St George’s Bay (Part 2 of St Julian’s). g J g y ( J ) Figure 10. Coastal businesses’ distribution in St Julian’s: St George’s Bay (Part 2 of St Julian’s). Figure 10. Coastal businesses’ distribution in St Julian’s: St George’s Bay (Part 2 of St Julian’s). Climate 2022, 10, 132 Climate 2022, 10, x 10 of 24 10 of 25 Figure 11. Marsalforn’s coastal businesses’ distribution. Figure 12. Xlendi’s coastal businesses’ distribution. Figure 11. Marsalforn’s coastal businesses’ distribution. Climate 2022, 10, x 10 of 2 Figure 11. Marsalforn’s coastal businesses’ distribution. Figure 12. Xlendi’s coastal businesses’ distribution. Figure 12. Xlendi’s coastal businesses’ distribution. Figure 11. Marsalforn’s coastal businesses’ distribution. Figure 11. Marsalforn’s coastal businesses’ distribution. Figure 11 Marsalforn’s coastal businesses’ distribution Figure 11. Marsalforn’s coastal businesses’ distribution. Figure 11. Marsalforn’s coastal businesses’ distribution. Figure 11 Marsalforn’s coastal businesses’ distribution Figure 12. Xlendi’s coastal businesses’ distribution. Figure 12. Xlendi’s coastal businesses’ distribution. Figure 12. Xlendi’s coastal businesses’ distribution. g Figure 12. Xlendi’s coastal businesses’ distribution. Figure 12. Xlendi’s coastal businesses’ distribution. Climate 2022, 10, 132 11 of 24 11 of 24 4.1.2. Feedback from Business Owners Table 1 lists all the main responses provided by the business community. For some questions, similar responses were collected in each locality, while for others, there were some differences, mainly when comparing the Maltese against their Gozitan counterparts. Similar feedback was observed for matters related to relocation, contingency plans, coastal ﬂooding, and planning and management. In each locality, business owners do not have any contingency plans for the possibility of having to close down due to rising sea-levels or ﬂooding. This decision appears to be based on the seasonal experience of past ﬂooding events i.e., of suffering coastal ﬂooding in one-off events during winter, and how the owners always managed to recover and resume operations in the shortest possible time. Table 1. Feedback from interviews held with business communities of ﬁve selected towns. Key Findings Question Feedback G˙zira Sliema St Julian’s Marsalforn Xlendi Number of owners who were knowledgeable about coastal ﬂooding on the Likert Scale 57% 66% 56% 46% 42% Number of owners who were not aware of the risk of rising sea levels and storm surge ﬂooding before they established their business 53% 56% 60% 31% 14% Number of owners who did not envisage that rising sea levels and storm surge ﬂooding could pose a threat to their business 73% 70% 68% 31% 43% Number of owners who thought that rising sea levels and storm surge ﬂooding would affect their operations. 77% 82% 78% 92% 72% Number of owners who had contingency plans to avoid bankruptcy None None None None None Owners’ decision-making regarding location if they knew of the possible impacts of coastal ﬂooding before opening their establishment 92% 92% 84% 85% 72% Number of owners refusing relocation 88% 82% 86% 100% 71% Owners’ opinion on who they think should be addressing coastal ﬂooding The government by building and managing infrastructure. The government by building and managing infrastructure however, a few owners reported that it is impossible to solve. The government by building and managing infrastructure however, a few owners reported that it is impossible to solve. The government by building and managing infrastructure however, a few owners reported that it is impossible to solve. The government by building and managing infrastructure however, a few owners reported that it is impossible to solve. Table 1. Feedback from interviews held with business communities of ﬁve selected towns. 4.1.2. Feedback from Business Owners Climate 2022, 10, 132 12 of 24 12 of 24 As for relocation, most business owners believe that they will not relocate to inland locations, as they think that their type of business will only thrive and prosper in a coastal location which are the most visited areas by tourists. As shown in the land-use maps most of the catering, accommodation, and retail industries were found to be the dominant sectors on the selected waterfronts (Figures 7–12). In all localities, more than 50% of business communities were not aware of the risk of rising sea levels and storm surge ﬂooding before they established their business. They rarely or never envisaged that rising sea levels and storm surge ﬂooding could pose a threat to their operations. However, they agreed that rising sea levels and storm surge ﬂooding would affect their operations in future as they have already experienced operational difﬁculties from coastal ﬂooding in the past. y g When addressing the planning and management of coastal ﬂooding within their respective localities, similar mitigation and adaptation responses were noticed across the ﬁve localities. Most owners believe that to effectively address coastal ﬂooding, proper planning and management are fundamental, along with the required upgrades to the current infrastructure and, where necessary, new infrastructure to be developed along with regular maintenance. Some of the infrastructure proposed included breakwaters, reservoirs, road redesign, upgrading the drainage systems/gutters, early warning systems, and small dams (Xlendi). One major difference was observed between the Maltese and Gozitan business com- munities in terms of how knowledgeable they were on coastal ﬂooding in general and more speciﬁcally in their respective localities. When comparing the Gozitan communities (i.e., Marsalforn and Xlendi), the Gozitans were found to be more aware about coastal ﬂooding than their Maltese counterparts (Table 1). A series of business disruptions and damages caused by past ﬂooding events were mentioned by the owners during the interviews (Table 2). This inventory ranges from economic, infrastructural, and health and safety issues, to long-term maintenance and extremes, such as closing of business. Some of these damages and disruptions are manage- able and short term e.g., loss of sales, which was only temporary during a ﬂooding episode. However, some are more long term and of a more serious nature, which may require a temporary shutdown until ﬁxed, e.g., internal and external piping damages. Table 2. 4.1.2. Feedback from Business Owners An inventory of damages and disruptions sustained by the business communities during past coastal ﬂooding events. Table 2. An inventory of damages and disruptions sustained by the business communities during past coastal ﬂooding events. Table 2. An inventory of damages and disruptions sustained by the business communities during past coastal ﬂooding events. Damages Disruptions Broken glass Loss of sales Shorted appliances Loss of revenue Repainting Reduction in operational capacity Products discarded Accessibility Overﬂowing of internal and external drainage systems Closure Internal and external piping damages Foul smells derived from overﬂowing drainage Broken doors Overﬂowing of drains resulting in drainage release of bugs and insects Broken windows Maintenance Ruined furniture Cleaning Plumbing Loss of sales Loss of revenue Reduction in operational capacity Accessibility Closure Foul smells derived from overﬂowing drainage Overﬂowing of drains resulting in drainage release of bugs and insects Maintenance Cleaning Table 3 lists all the reasons provided by the owners for their answers to each qualitative question categorized by locality. In most questions, the responses were observed to be quite similar as these phenomena were found to affect each locality in a similar manner. Nevertheless, in certain questions, feedback was distinctly different, e.g., in question 7B the owners were asked how they think rising sea level will affect their business and to provide reasons for their answer. For this question, Xlendi business owners were the only ones who identiﬁed bankruptcy as an answer and how they would not be able to cope with the constant disruptions along with the daily overheads’ costs and limited revenue. 13 of 24 Climate 2022, 10, 132 Table 3. Reasons provided by the business owners in the qualitative section. Reasons Question Feedback G˙zira Sliema St Julian’s Marsalforn Xlendi Coastal ﬂooding experience Flooding, business disruptions, preventive measures Flooding, business disruptions, preventive measures Flooding, business disruptions, safety precautions Flooding, business disruptions Flooding, business disruptions Coastal ﬂooding disruptions Flooding, business disruptions Flooding, business disruptions Flooding, business disruptions, elevation. Flooding, business disruptions Flooding, bankruptcy Opting for coastal location Location, tourism, demand, challenges, relocation Location, tourism, seasonality, lack of knowledge, relocation, minor impacts Location, tourism, demand, ﬂooding, seasonality, risk Location, tourism, challenges Location, tourism, challenges Business recovery Preventive measures, insurance, conditional damages Insurance, preventive measures, conditional damages Insurance, recovery conﬁdence due to never/rarely ﬂooding, conditional damages, recovery period. Insurance, preparation, minor damages, recovery difﬁculties. Lack of knowledge, hope Lack of knowledge, seasonality, operational continuity, hope Lack of knowledge, seasonality, operational continuity, hope Lack of knowledge, hope Table 3. Cont. Table 3. Cont. Table 3. Cont. Table 3. Cont. Reasons Question Feedback G˙zira Sliema St Julian’s Marsalforn Xlendi Owners’ opinions on what the local and central government should do to mitigate coastal ﬂooding Management and planning, infrastructure maintenance, climate change mitigation and adaptation measures, government, lack of knowledge, ceasing coastal development Infrastructure, lack of knowledge, management, and planning, ceasing coastal development, environmental protection, mitigation, and support Infrastructure, lack of knowledge, management and planning, government, reducing and managing climate change, ceasing coastal development, scientiﬁc studies, ﬁnancial and scientiﬁc priority, local council Infrastructure, management and planning, local council, investment (educational and ﬁnancial), lack of knowledge, land reclamation, cease development Infrastructure, lack of knowledge, management, and planning, Relocation to those owners who said they might consider relocation Relocation within the same area Relocation within the same area but further inland Depends on trade, relocation within the same area but at higher elevation N/A Relocation within the same area but at higher elevation Relocation within the same area but at higher elevation In question 15, owners were asked whether they think about the possibility of business diversiﬁcation as a solution to future impacts of coastal ﬂooding, and what alternate business operations they would engage in. The catering businesses in St Julian’s claimed that, if possible, they would keep their current operations but switch their services as a delivery/take away restaurant. St Julian’s was once again the outlier as a few owners claimed that they would still opt for the same area but locate at a higher elevation above sea level. Others claimed that they would study the market forces at that time and would base their decision on relocation accordingly. g y With regards to physical preventive/adaptation measures (Table 4), business owners mentioned only short-term measures. Table 4. Inventory of preventive/adaptation measures. G˙zira Sliema St. Julian’s Xlendi Placing high enough slabs against the entrance Higher topographic elevation Higher topographic elevation Waterprooﬁng Entrance elevation Entrance elevation Entrance elevation Placing high enough slabs against the entrance Waterprooﬁng Waterprooﬁng Waterprooﬁng Placing high enough slabs against the entrance Work from home policy Table 4. Inventory of preventive/adaptation measures. Placing high enough slabs against the entrance 4.1.3. Analysis of Variance: Businesses’ Responses 4.1.2. Feedback from Business Owners Insurance, ﬁnance, damages Lack of contingency plans Preventive measures, lack of knowledge, operational continuity, retirement, minor effects, and rare ﬂooding events Preventive measures, seasonality, operational continuity, retirement, relocation, lack of knowledge, no major incidents Mitigation/adaptation measures, operational continuity, lack of knowledge, seasonality, business disruptions, no risk, never sustained any extensive or permanent damages Lack of knowledge, seasonality, operational continuity, hope Lack of knowledge, hope Authorities talks with the business communities Mitigation and adaptation measures Mitigation and adaptation measures Climate change and coastal ﬂooding mitigation and adaptation Climate change mitigation and adaptation measures, background information on climate change and, its consequences, and impacts Climate change impacts Owners’ opinions to mitigate coastal ﬂooding Management and planning, implementation of climate change mitigation and adaptation measures, infrastructure maintenance, ceasing coastal development, lack of knowledge Infrastructure, lack of knowledge, management, and planning, ceasing coastal development, environmental protection. Infrastructure, lack of knowledge, management and planning, government, reducing and managing climate change, ceasing coastal development, scientiﬁc studies, ﬁnancial and scientiﬁc priority Infrastructure, lack of knowledge, preventive measures Infrastructure, lack of knowledge, management, and planning, cease coastal development Table 3. Reasons provided by the business owners in the qualitative section. Climate change and coastal ﬂooding mitigation and adaptation Mitigation and adaptation measures Management and planning, implementation of climate change mitigation and adaptation measures, infrastructure maintenance, ceasing coastal development, lack of knowledge Owners’ opinions to mitigate coastal ﬂooding 14 of 24 Climate 2022, 10, 132 14 of 24 4.1.3. Analysis of Variance: Businesses’ Responses Most of the analysis of variance tests showed no difference in responses across the types of businesses in the ﬁve tested localities (Table 5). Out of 50 tests, only 5 results showed that there were differences. This means that the type of business category did not have any inﬂuence on the type of response received from the owners. Table 5 shows how St Julian’s is a strong outlier when compared with the other localities in having correlation between the various business categories and each tested question. This is key Climate 2022, 10, 132 15 of 24 15 of 24 to understanding how to better proﬁle the business community as stakeholders, and to determine what level and types of engagement should be exercised for each category of businesses. Table 5. Results from Kruskal Wallis H-Tests for selected businesses owners’ responses. Question G˙zira Sliema St. Julian’s Marsalforn Xlendi Question 1: How familiar are you with the issues of climate change and its implications? 0.430 0.765 0.005 0.520 0.269 Question 2: How informed are you about coastal ﬂooding (in your locality)? 0.665 0.464 0.013 0.559 0.346 Question 4: Were you aware about the risk of sea-level rise and possible coastal ﬂooding when you opened your current business? 0.716 0.687 0.015 0.643 1.000 Question 5: In recent years, have you experienced coastal ﬂooding? 0.463 0.520 0.694 0.875 0.189 Question 6: Have you ever envisaged that future sea-level rise and coastal ﬂooding could threaten your business? 0.752 0.665 0.035 0.668 0.978 Question 7: Do you think that sea-level rise and ﬂooding will affect your business? 0.199 0.607 0.505 0.801 0.978 Question 8: If you knew the risks that coastal ﬂooding (due to sea-level rise and storm surges) could have on your business, would you still have chosen a location along the coast? 0.273 0.162 0.943 0.052 0.153 Question 9: If such an event does happen in future, do you think that your business can recover from such a setback? 0.532 0.431 0.514 0.264 0.153 Question 15: Would it be possible to diversify your business operations, should current business operations not be possible anymore due to future impacts of coastal ﬂooding? 0.171 0.027 0.559 0.760 0.444 Question 16: How likely would you consider relocating your business? 0.789 0.916 0.214 0.616 0.053 Table 5. Results from Kruskal Wallis H-Tests for selected businesses owners’ responses. 4.2. Local Councils’ Feedback The ﬁve local councils provided mixed feedback on how coastal ﬂood risks are per- ceived. In terms of general sea level rise and storm surge ﬂooding risk assessments, all councils—except for Xlendi—identiﬁed sea level rise hotspots within their locality. Xlendi’s local council could not provide feedback on which areas are more susceptible to coastal ﬂooding. In addition, the local councils of Sliema, Marsalforn, and Xlendi’s do not think that the current local sea level rise projections of around 3.5 mm per year, are a threat to their town’s economy and local population. Conversely, the local councils of G˙zira and St Julian’s believe that coastal ﬂooding is a concern for their community. Climate 2022, 10, 132 16 of 24 With regard to the dissemination of information between the central and local govern- ments, residents, and the business community, all councils except Marsalforn reported that to date, they have not received any information. However, the councils of G˙zira, St Julian’s, and Xlendi reported that they had organized meetings with their business communities and residents to discuss the effects of coastal ﬂooding on their towns, while Sliema and Marsalforn reported that, to date, no such meetings had been held. It is rather incongruent to note how the only informed councils did not organize any meetings with the residents and business community, whereas three uniformed councils did reach out and organize such public meetings. 17 of 25 any meetings with the residents and business community, whereas three uniformed councils did reach out and organize such public meetings. Regarding infrastructure and planning, Gżira and St Julian’s reported that they do f p g For infrastructure and planning, G˙zira and St Julian’s reported that they do not have any infrastructure at risk to rising sea levels while the remaining councils reported that they do. This ﬁnding is contradicted by real life events - often reported in the media [33]—showing how coastal ﬂooding impacts the town’s main infrastructure (mainly roads). Regarding mitigation planning, only the local councils of G˙zira, Sliema, and Marsal- forn reported that in recent years the council did implement mitigation measures to reduce the effects of rising sea levels within their towns. In addition, G˙zira, St Julian’s, and Xlendi reported that they do have plans to protect their towns against rising sea levels. not have any infrastructure at risk to rising sea levels while the remaining councils reported that they do. 4.2. Local Councils’ Feedback This finding is contradicted by real life events - often reported in the media [33] showing how coastal flooding impacts the town’s main infrastructure (mainly roads). Regarding mitigation planning, only the local councils of Gżira, Sliema, and Marsalforn reported that in recent years the council did implement mitigation measures to reduce the effects of rising sea levels within their towns. In addition, Gżira, St Julian’s, and Xlendi reported that they do have plans to protect their towns against rising sea levels. 4.3. Government Experts’ Feedback 4.3 Government Experts’ Feedback Malta Tourism Authority (MTA) was relatively more aware of the risks, especially with regard to beaches and how the loss of sand and bathing areas will have a direct effect on tourism. In terms of business disruptions, the MCCEI predicts disruptions related to health and safety issues, reductions in business trafﬁc, cleanliness, and reductions in the prime value of real estate. The expected business disruptions mentioned included decline in walking trade, accessibility issues, and outside seating issues. In relation to government consultations with stakeholders, the MCCEI did organize discussions with potential stakeholders about the threats that may impact the coastal business communities through rising sea levels and storm surge ﬂooding. The MSCME has never organized any seminars/talks and to date, this is still not on the chamber’s agenda. g y g With regards to data on coastal ﬂooding risks, many of the interviewed institutions do not have longitudinal or national data. The MCCEI does not have any data showing the expected losses to revenue, jobs, labour force, and coastal business establishments, and stated that such data still needs to be modelled. The Ministry of the Economy, Investment, and Small Businesses (MEISB) has never collected economic data related to rising sea levels and storm surge ﬂooding. Both MTA and ERA have not conducted any studies on the risks of sea level rise and storm surge ﬂooding on coastal towns across the Maltese Islands as they claimed that this aspect is not within their remit. When new coastal infrastructural developments are proposed, Transport Malta (TM) is usually involved and assesses the likely impacts of rising sea levels and violent storms on these new developments. The MTA assesses every development proposal and submits rec- ommendations to the Planning Authority (PA), highlighting both the impacts and beneﬁts from such a project. However, with regard to management, planning, and regulations, the Ministry for Sustainable Development, Environment and Climate Change (MSDECC) does foresee future regulations pertaining to coastal development in low-lying areas that are highly susceptible to rising sea levels and storm surge ﬂooding. Currently, the MSDECC is in the process of updating the national Adaptation Strategy, which will inform policy makers and other authorities about measures and regulatory measures to better strengthen resilience to climate change. Coastal management actions as a response to coastal ﬂooding are also delivered speciﬁcally within institutional remits. 4.3. Government Experts’ Feedback 4.3 Government Experts’ Feedback The experts’ analysis is illustrated in Figure 13 which shows a dendrogram analysis of all of the main points mentioned by the experts grouped into codes and subcodes to illustrate the degree of similarity between them. Two main groups can be identiﬁed; the ﬁrst has codes between “Non SLR or CF to stakeholders’ discussion”, while the second group includes the rest (i.e., “Reasons for risk to SL and CF to Strategy Building”). The experts’ analysis is illustrated in Figure 13 which shows a dendrogram analysis of all of the main points mentioned by the experts grouped into codes and subcodes to illustrate the degree of similarity between them. Two main groups can be identified; the first has codes between “Non SLR or CF to stakeholders’ discussion”, while the second group includes the rest (i.e., “Reasons for risk to SL and CF to Strategy Building”). Figure 13. NVivo’s dendrogram illustrating similarity clustering provided based on experts’ feedback Figure 13. NVivo’s dendrogram illustrating similarity clustering based on experts’ feedback. Figure 13. NVivo’s dendrogram illustrating similarity clustering provided based on experts’ feedback Figure 13. NVivo’s dendrogram illustrating similarity clustering based on experts’ feedback. In terms of awareness to coastal flood risks, the responses from the key experts were varied. On the one hand, the Malta Chamber of Small and Medium Enterprises (MCSME) answered to not be sufficiently aware of the risks related to the effects of rising sea levels, and storm surge flooding on the coastal business communities. The Malta Resources Authority’s (MRA) only role pertaining to climate change is to serve as the GHG Inventory Agency; hence they shared no insights However other experts showed a In terms of awareness to coastal ﬂood risks, the responses from the key experts were varied. On one hand, the Malta Chamber of Small and Medium Enterprises (MCSME) answered to not be sufﬁciently aware of the risks related to the effects of rising sea levels, and storm surge ﬂooding on the coastal business communities. The Malta Resources Authority’s (MRA) only role pertaining to climate change is to serve as the GHG Inventory Agency; hence, they shared no insights. However, other experts showed a higher level of Climate 2022, 10, 132 17 of 24 17 of 24 awareness in their response. The Malta Chamber of Commerce, Enterprise, and Industry (MCCEI) classiﬁed rising sea levels and storm surge ﬂooding as a medium risk to Malta’s commercial activity. 4.3. Government Experts’ Feedback 4.3 Government Experts’ Feedback Presently, there is no nationwide assessment of the risks associated with coastal ﬂooding and its impact on coastal communities and businesses in Malta. The Ministry for Transport Infrastructure and Capital Projects (MTIP) addresses impacts on the basis of local community’s needs, with speciﬁc projects instigated mostly in the aftermath of storm events. Actions and works are carried out depending on the budget allocated on an annual basis. In addition, the PA does not foresee the introduction of future regulations pertaining to coastal development in low-lying areas that are highly susceptible to rising sea levels and storm surge ﬂooding, as the authority does not have any clear evidence on which to base such regulations. For the tourism sector, the MTA has not yet aligned its future policies and marketing strategies to reﬂect the impacts of climate change on coastal tourism, especially in relation to rising sea levels and storm surge ﬂooding. The reason provided is that marketing serves to attract high quality and environmental tourists in a positive manner, and not by highlighting issues whose risks and impacts are not yet assessed. TM does have yet plans to protect coastal infrastructure and other investments that are deemed to be at risk of being partially or fully submerged and mostly limits itself to repair works, such as repairing the original ﬂood valves at Sliema Ferries. p p g g Preventive measures are critically important to effectively manage coastal ﬂooding; however, more than one entity must be involved in order achieve maximum protection from these measures. The MSDECC reported that in the current Adaptation Strategy, measures are being proposed to protect the coastline, roads, and infrastructures from rising sea levels and storm surge ﬂooding, including ﬂash ﬂoods; however, the implementation of such measures does not fall under this ministry. The Civil Protection Department (CPD) Climate 2022, 10, 132 18 of 24 18 of 24 does receive calls for support during storm ﬂood events, but it does not have preventive measures to mitigate the onset of these requests for support during storm ﬂood events. does receive calls for support during storm ﬂood events, but it does not have preventive measures to mitigate the onset of these requests for support during storm ﬂood events. 5. Discussion In terms of the vulnerability of these businesses to coastal ﬂooding, the land use mapping task (Figures 7–12) shows how each establishment’s elevation (i.e., height above sea level) clearly indicates that most of the coastal businesses in the ﬁve selected localities are clustered around 0–3 m above sea level. This makes these businesses highly sensitive to coastal ﬂooding, and this was conﬁrmed by the interviewed business owners who reported that they have experienced coastal ﬂooding in the past. In fact, the Kruskal Wallis H-tests results for Question 5 (Table 5) established that there is no statistically signiﬁcant relationship between coastal ﬂooding and the type of business sector, in view of how the various business sectors operate adjacent to one another along the coast and therefore are equally exposed to the same level of risks from coastal ﬂooding (Figures 7–12). These ﬁndings build on those from Spiteri (2019) and others [10,14] who found that the coastal businesses operating in low lying areas are more susceptible to rising sea level and extreme storms. The land-use maps also conﬁrm that these coastal areas should be considered high risk areas not only by virtue of their elevation above sea level, but also because of the dense concentration of business services within a very small stretch of the coastal zones, which are also exposed to some of the most prevalent storms coming from the northwest and northeast sextants. This conﬁrms how the zones deemed to be at the highest risk of ﬂooding do not necessarily intersect with high-risk areas, as other factors apart from ﬂooding, such as land use, road networks and infrastructural quality increase the coastal area’s vulnerability [14]. y The feedback responses provide evidence that the Maltese business communities are not sufﬁciently informed by any government entity about the potential risks associated with rising sea levels and storm surge ﬂooding. Receiving information on ﬂoods and risk management may help to improve stakeholder involvement but the former should not be considered as the only strategy to increase knowledge about the importance of ﬂood risks management. In fact, studies have shown that business operators who had received ﬂood risk information had lesser knowledge on the importance of managing ﬂood risks then those who had not [19]. 4.3. Government Experts’ Feedback 4.3 Government Experts’ Feedback g q pp g Apart from the preventive measures, newly constructed and/or future planned infras- tructure must be designed in such a way that it can withstand rising sea levels and storm surge ﬂooding. TM reported that newly constructed and future planned roads are designed to cater for rising sea levels. Whenever possible, roads and waterfront infrastructure are constructed at higher levels; however most low-lying areas are densely built-up, with urban land-use closely situated to the water’s edge. TM also reported that during the recent coastal road upgrades, measures were implemented to protect these investments from rising sea levels and storm surge ﬂooding. To date, the Ports and Yachting Directorate (PYD) did not consider any mitigation measures to protect coastal infrastructure and towns from rising sea levels and storm surge ﬂooding as this is not the directorate’s area of responsibility. Their responsibility is to ensure the safe navigation and operation of vessels in Maltese territorial waters. Finally, in terms of transport, the ERA, MTIP, MSDECC, and TM identiﬁed road networks and public structures that are at risk from rising sea levels and storm surge ﬂooding. 5. Discussion Those owners and local councillors who reported having experienced coastal ﬂooding agreed that future ﬂooding caused by rising sea level and storm surges, may produce more deleterious business disruptions, particularly in terms of their operations. This feedback echoes that of Spiteri (2019) for other coastal localities in Malta. However, as with other studies, these disruptions were considered by the business owners to be seasonal and not substantially impactful during the high summer season [9]. The long-term notion of rising sea level impacts remains widely underestimated. Climate 2022, 10, 132 19 of 24 19 of 24 Other disruptions such as reduced accessibility and the reduction in business trafﬁc, were documented by the local councils and business owners as factors of major concern during ﬂooding events. A substantial number of business owners in this study complained about inaccessibility issues due to road ﬂooding since customers are not able to access the establishment when the roads are completely ﬂooded. These issues, which included lack of sales due to inaccessibility, were also reported by the experts from the MCCEI and MCSME and documented by Rizzo (2019). These ﬁndings continue to conﬁrm the views of how the vulnerability of the business community to coastal ﬂooding is compounded by other impacted land uses such as ﬂooded road networks [14,15]. p Another crucial issue was relocation prospects, given that most owners expressed a strong refusal to relocate in the likely event of coastal ﬂooding. In addition, owners working in the catering industry also reported that a coastal location is a must for their establishment to succeed for two main reasons: walk-in trade and the fact that people prefer coastal locations for leisure and dining. This signiﬁcantly elevates the establishments’ susceptibility to the risks of coastal ﬂooding. This refusal was tested by Kruskal Wallis H-Test for Q6 and Q18 of each locality (see Table 5) in which relocation refusal was voiced by the majority of the owners and did not vary according to the business category. NVivo coding of experts’ interviews also conﬁrmed that walk-in trade is an important source of revenue, which would therefore explain the refusal to relocate. 5. Discussion This issue was reported by Stafford and Renaud (2019), who investigated a high percentage of businesses that were at risk of relocation after episodic storm events, and how the communities nevertheless refused to relocate, due to the economic turnover generated by the coast as a touristic service area [10]. Amongst the business disruptions listed in Table 1, the one most experienced by local stakeholders was reduction in sales. However, during the analysis, three speciﬁc sales disruptions were identiﬁed. The ﬁrst was related to the outdoor seating areas (Figure 14) as the physical indoor dimensions of most establishments in the ﬁve studied localities were observed to be quite small and tended to have a larger outdoor seating capacity. Such an outdoor setup is the main factor responsible for sales reduction when impacted by road ﬂooding. Road ﬂooding signiﬁcantly reduces the businesses’ seating capacity and operations, and sales go down drastically as they cannot use the outside area. One study mentioned that ﬂooding or inundation of roads can cause devastation to coastal businesses and their sales [14]; likewise, the Maltese and Gozitan businesses (especially the catering industry) suffer similar decreases in sales. The second factor was that these ﬁve areas are well known for coastal ﬂooding events. Therefore, visitors avoid these areas during storms (regardless of whether they are ﬂooded or not) which in turn leads to a reduction in revenue. This is a very common pattern that was also observed by Craig et al. (2019) who found that businesses also experienced a drastic reduction in their daily sales during rainy days. The last factor, not mentioned in any of the literature, was the localized incidence of foul smell after a ﬂooding event. Several businesses in the catering industry reported that they suffer a reduction in sales, when powerful storms damage the sewage systems, resulting in the presence of a pungent smell while repairs are being carried out. These three factors in the reduction in sales were also noted by the MCCEI and MCSME experts. Despite both SME’s and the coast are considered as important economic engines, a research gap in business ﬂooding disruptions still exists, and which requires a multi- disciplinary approach to the assessment and implementation of mitigation measures. In addition, none of the interviewed businesses had any contingency plans in place in case coastal ﬂooding puts them out of business. This trend was also observed by Craig et al. 5. Discussion (2019) who found that most small business owners do not have a disaster plan in place and that around 40–60% ceased operations after they endured a natural disaster [12]. The reason given by Maltese and Gozitan business owners for not having any contingency plans was that all interviewees have managed to recover from episodic coastal ﬂooding. Climate 2022, 10, 132 20 of 24 ation to sinesses Figure 14. Coastal restaurants in Marsalforn, with their outdoor seating area densely occupying large parts of the coastal promenade. Figure 14. Coastal restaurants in Marsalforn, with their outdoor seating area densely occupying large parts of the coastal promenade. When all council members and experts were asked about their risk awareness in relation to rising sea levels and storm surges, all interviewed councillors agreed that they were extremely aware of these phenomena. Nevertheless, except for Xlendi, they reported that they rely on central government in terms of concrete actions and mitigation measures. This same concern was voiced by another four local councils in the study of Spiteri (2019) [9]. Good governance is central to any successful management of hazards and threats driven by climate change. However, given the multi-sectorial nature of ﬂooding phenomenon, challenges remain as to how to engage with other stakeholders in this effort. It was observed that such leading initiatives in mitigating ﬂood risk and informing the local business community about the risks, were sporadic and have not yet been concretely addressed [19]. Only one of the ﬁve councils reported that a government entity had informed them about the impacts imposed on their locality due to rising sea levels and storm surge ﬂooding. Nonetheless, it is important to mention that G˙zira, St Julian’s, and Xlendi reported that they have organized meetings with their business communities and residents to discuss these phenomena. When government experts were asked about concrete action in addressing these two phenomena, only those responsible for the environment gave detailed and speciﬁc answers while the rest all reported that they rely on other environmental ministries and departments. Due to limited resources and human capacity, both the MCSME and MCCEI are forced to prioritize the needs of the wider agenda of the business community and hence, have never addressed the speciﬁc nature of the risks posed by coastal ﬂooding to coastal businesses. 5. Discussion St Julian’s and G˙zira were the only two localities whose councillors reported that they do believe that rising sea levels are a threat to the town’s economy. When asked whether the councils had identiﬁed hotspots for sea level rise and storm surge ﬂooding, only Xlendi’s council responded in the negative. Similar questions were asked to some experts who reported that Sliema does have infrastructure and road networks that are susceptible to coastal ﬂooding; this was corroborated by Sliema’s local council. However, even though neighbouring localities (i.e., G˙zira and St Julian’s) share similar geographic and topographic characteristics, none of the experts mentioned them. The NVivo analysis identiﬁed a strategy building code within the Ministry for Transport, Infrastructure and Capital Projects which plays a key role in vulnerability/risk assessments. This code was also Climate 2022, 10, 132 21 of 24 21 of 24 identiﬁed in a UK study, where the authors identiﬁed that strategy building through spatial planning is key for effective governance for ﬂood and coastal erosion risk management [18]. identiﬁed in a UK study, where the authors identiﬁed that strategy building through spatial planning is key for effective governance for ﬂood and coastal erosion risk management [18]. Roads have a strong connection with the success of coastal businesses and the overall potential post-ﬂooding recovery of coastal territories. Although local councils and experts did not specify any high-risks roads, Sliema has one major road that is highly susceptible to coastal ﬂooding; this is Triq ix-Xatt which constitutes Sliema’s study area as all of the businesses interviewed operate along this road. Meanwhile in St Julian’s, there are multiple coastal roads, but like Sliema, the roads that are most susceptible to coastal ﬂooding are the roads located along the coast which host a high concentration of different businesses [15]. Prioritizing investment and planning climate change adaptation measures during the planning and construction of coastal roads remains paramount [28]. Another study conﬁrmed the dire need for such actions, as it was discovered that in both studies ﬁndings are corroborated by the author’s own study [26]. Coastal ﬂooding is often perceived as a seasonal phenomenon with minimal effects on the bay and urban environment, due to its seasonal (winter) frequency [31]. Although in this study, the business communities shared a similar perception, the local councillors, and experts considered it to be a more tangible, long-term phenomenon [28]. 5. Discussion Such a distinction in perception shows the need for better communication and wider consultation between the different stakeholders to create a more common public knowledge base about the science of coastal ﬂooding. g From an adaptation and mitigation measures perspective Busuttil (2011) recommended the implementation of soft and hard engineering methods to continuously safeguard the bay. The PA and ERA reported that they no longer advocate for the use of hard engineering methods but have instead transitioned to soft engineering methods coupled with green and blue infrastructure. However, both authorities reported that in certain scenarios, the implementation of green and blue infrastructure is not possible, and therefore other engineering methods may need to be considered. NVivo thematic analysis revealed how some responsibilities tend to overlap between authorities/entities (Figure 13). Similar to other studies, such as that carried out by UK’s Environment Agency [18], this ﬁnding suggests that there is some form of inter cooperation between the identiﬁed entities, irrespective of the distinct differences in their remits. 6. Conclusions The main key ﬁndings with respect to the business communities were as follows: (i) none of the interviewed businesses have any contingency plans to safeguard themselves against bankruptcy caused by coastal ﬂooding; (ii) most interviewed owners consider their business areas to be a prime locations, and conﬁrm that they would still opt for a coastal location for their business regardless of the risks linked to coastal ﬂooding; (iii) for the same reason expressed in (ii), they also refuse to consider relocation; (iv) owners explicitly stated that to resolve the recurring issues caused by coastal ﬂooding, the central government needs to heavily invest and plan in building adequate infrastructure along with the required maintenance of such infrastructure to help in mitigating the effects of coastal ﬂooding. The key ﬁndings with respect to the local councils were as follows: (i) G˙zira and St Julian’s claimed that they do not have any infrastructure at risk of coastal ﬂooding, while the other councils reported that they do; (ii) only Xlendi identiﬁed sea level rise hotspots within their locality; (iii) G˙zira, Sliema, and Marsalforn reported that they had implemented measures to mitigate the impacts of coastal ﬂooding while St Julian’s and Xlendi did not; (iv) ﬁnally, G˙zira, St Julian’s and Xlendi reported that they do possess plans to protect their towns from coastal ﬂooding, while Sliema and Marsalforn reported that to date, no such plans exist. The main ﬁnding from the experts’ interview was that all interviewed government and private organizations, entities, departments, and ministries operate within their legal framework and act within their own jurisdiction and there is limited horizontal and vertical cooperation in the risk management of coastal ﬂooding and its long-term hazard implications. Climate 2022, 10, 132 22 of 24 22 of 24 A long-term and strategic form of governance between the three different stakehold- ers for coastal protection from coastal ﬂooding is vital to maintain economic and social resilience, in the wake of increasing risks posed by climate change to city-island-states such as Malta [22,23,27,29,30]. Recommendations for coastal protection were expressed by the business communities such as the implementation of early warning systems as a highly effective mitigation tool, so that owners will have sufﬁcient time to protect their establishments from the upcoming events. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. 2. Shukla, P.R.; Skea, J.; Slade, R.; Al Khourdajie, A.; Vyas, P.; Luz, S.; Fradera, R.; Belkacemi, M.; Hasija, A.; Malley, J.; et al. Climate Change 2022 Mitigation of Climate Change Working Group III Contribution to the Sixth Assessment Report of the Intergovernmental Panel on Climate Change Summary for Policymakers; Cambridge University Press: Cambridge, UK; Cambridge University Press: New York, NY, USA, 2022. [CrossRef] j g 4. Aucelli, P.P.C.; Di Paola, G.; Rizzo, A.; Rosskopf, C.M. Present day and future scenarios of coastal erosio along the Italian Adriatic coast: The case of Molise region. Environ. Earth Sci. 2018, 77, 371. [CrossRef] 5. Shaftel, H.; Jackson, R.; Callery, S.; Bailey, D. Evidence\|Facts—Climate Change: Vital Signs of the P https://climate.nasa.gov/evidence/ (accessed on 1 February 2022). 3. Galassi, G.; Spada, G. Sea-level rise in the Mediterranean Sea by 2050: Roles of terrestrial ice melt, steric effects and glacial isostatic adjustment. Glob. Planet. Chang. 2014, 123, 55–66. [CrossRef] 6. Conclusions As key stakeholders and intermediaries between the central government and the citizens, local councils may have an important role to play in future risk assessments of coastal ﬂooding in their respective locality, especially to assess hotpots of vulnerability. The multi-faceted threats driven by climate change, require an equally multi-faceted and multi-sectoral management strategy to address them. Each threat, such as rising sea levels and storm surges, imposes cascading impacts that amplify the vulnerability and precariousness of low-lying coasts and their communities. Within this context, central government institutions need to ensure that there are no vacuums of climate change related policies and strategies in their governance structure. The process of collecting data about the economic costs of coastal ﬂooding (among other factors) is an important start to ﬁlls gaps in knowledge. A long-term assessment and vision of resource allocation and infrastructure modiﬁcations is also required to increase the level of coastal protection of these towns. In conclusion, more national research is required on the risks of coastal ﬂooding, not only to deﬁne and quantify the socio-economic risks, but also to collect more perception data from stakeholders, which will ultimately secure a stronger participatory role from the Maltese community. Author Contributions: Conceptualization: D.S.; methodology: D.S. and R.G.; software: D.S. and R.G.; validation: R.G.; formal analysis: D.S.; investigation: D.S.; resources: D.S. and R.G.; data collection: D.S.; writing original draft preparation: D.S.; review editing R.G.; visualization: R.G.; supervision: R.G.; project administration: R.G.; funding acquisition: R.G. All authors have read and agreed to the published version of the manuscript. Funding: The Article Processing Charges of this article were funded by University of Malta Research Seed Fund 2022 GEORP01-22. Institutional Review Board Statement: The research was reviewed and approved for ethics clearance by the University of Malta Research Ethics Board of the Faculty of Arts. Informed Consent Statement: Informed consent was obtained from all participants involved in the study. Informed Consent Statement: Informed consent was obtained from all participants involved in the study. Data Availability Statement: The full dataset that supports the ﬁndings of this study are available from the corresponding author, upon request. Data Availability Statement: The full dataset that supports the ﬁndings of this study are available from the corresponding author, upon request. References Song, J.; Peng, Z.-R.; Zhao, L.; Hsu, C.-H. 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https://openalex.org/W3164505414	http://ijcis.net/index.php/ijcis/article/download/28/32	English	null	Employee Admissions Information System Web-based at CV Galerindo Nusantara	International Journal of Computer and Information System	2,021	cc-by	2,876	Employee Admissions Information System Web-based at CV Galerindo Nusantara 1stEdy Susena, 2ndEdy Susanto, 3rdDwi Iskandar, 4thMuhamad Handi Stiawan 1,2,3,4Politeknik Indonusa Surakarta 1,2,3,4Jl.KH. Samanhudi 31 Mangkuyudan, Surakarta Surakarta, Indonesia 1edysusena@poltekindonusa.ac.id, 2edy_skp@poltekindonusa.ac.id, 3dwik@poltekindonusa.ac.id, 4muhamad.stiawan@poltekindonusa.ac.id Abstract—CV Galerindo Nusantara company still uses manual methods in the recruitment process, namely prospective employees must come to the company to apply for work and still use paper so that companies need a computerized system that can facilitate employee recruitment activities. Based on these problems the authors seek solutions to make it easier for companies to overcome these problems. In developing this system, the author uses the waterfall method, namely the work of a system that is carried out sequentially or linearly. The results of the web-based employee recruitment information system at CV Galerindo Nusantara which was created by the author are to produce a system that can make it easier for prospective employees and companies to carry out computerized employee recruitment activities as well as time efficiency and make it easier for prospective employees to access information systems. Keywords :System, Information, Employees, website 1. System A system is a collection of people who work together with systematic and structured regulatory provisions to form a single unit that carries out a function to achieve a goal. The system has several characteristics or characteristics consisting of system components, system boundaries, external environment, liaisonsystem, system input, system output, system processing and system objectives[2]. I. INTRODUCTION company can be developed with the help of system updates that can provide a new form of service so that the recruitment process is short without a longer process. The use of technology is now being used by various groups, one of which is a web-based information system. Almost all companies use a computerized system and take advantage of technology for the advancement of the company's business. The more sophisticated the technology used, the easier and faster the activities will be carried out in the business venture. The need for time and cost efficiency causes companies to feel the need to apply technology in their business. Human Resources (HR), namely as a driving force for an organization or company, if HR is not managed properly and is not properly managed to work together and synergize with each other, it is impossible for the organization or company to run properly and optimally as expected, it will but it must be understood and understood that humans are not like machines that work without feelings and are considered solely as a source of business energy to achieve an organizational or company goal[1] Galerindo Nusantara is one of the jobs in the field of food and beverage production. The establishment of Galerindo Nusantara was initially to meet the needs of food and beverage products in Indonesia. Galerindo Nusantara itself was first founded by Bintang Marsiwa Nusantara in 2018. Previously there was only a small restaurant on the side of the road called Ayam Bakar Bhesus. After several years finally established CV. Galerindo Nusantara which has two branches, namely Ayam Bakar Bhesus and GaleriPedas which is a restaurant in Surakarta. CV Galerindo Nusantara needs a system that can regulate and manage employee recruitment according to the company's needs. And in this era of increasingly sophisticated technological developments, the employee recruitment system at this company is still manual and there is no information system that makes it easy for prospective employees to apply for jobs where prospective workers do not need to manually write on paper to apply for jobs. Therefore, the authors want to provide a solution by creating a web-based information system to facilitate activities within the company. Based on developments, the existing service processes in the 3. Information Systems Information systems are organized systems for the collection, organization, storage and communication of information.This system is used by people and organizations to collect, filter, process, create and distribute data into information [9]. produce some information for the user. 2. Information Information is data that is processed to be more useful and meaningful for the recipient, as well as to reduce uncertainty in the decision-making process regarding a situation[2]. Based on the above opinion, the writer concludes that information is data that has been processed into a more useful form and can be used as a basis for making the right decision. Journal IJCIS homepage - https://ijcis.net/index.php/ijcis/index International Journal of Computer and Information System (IJCIS) Peer Reviewed – International Journal Vol :Vol. 02, Issue 02, May 2021 e-ISSN : 2745-9659 https://ijcis.net/index.php/ijcis/index International Journal of Computer and Information System (IJCIS) Peer Reviewed – International Journal Vol :Vol. 02, Issue 02, May 2021 e-ISSN : 2745-9659 https://ijcis.net/index.php/ijcis/index International Journal of Computer and Information System (IJCIS) Peer Reviewed – International Journal Vol :Vol. 02, Issue 02, May 2021 e-ISSN : 2745-9659 https://ijcis.net/index.php/ijcis/index 6. Website A website is a set of pages consisting of several pages containing information in the form of digital data, whether in the form of text, images, video, audio and other animations provided through an internet connection.More specifically, websites are pages that contain information that can be accessed by browsers and are able to provide useful information for those who access them[8]. 7. XAMPP XAMPP is free software, which supports many operating systems, which is a compilation of several programs. The function of XAMPP itself is as a stand-alone server (localhost), which consists of several programs, including: Apache HTTP Server, MySQL database, and language translators written in the PHP and Perl programming languages[5]. 1. Context Diagram a. Observation a. Observation The following is a context diagram of the Web-Based Employee Admissions Information System at CV Galerindo Nusantara. Observation is an observation of a person's behavior in certain situations. This observation aims to assess the problem. Assessment can be said to be professional if it is done by monitoring the behavior of others visually while recording information from the behavior obtained qualitatively or quantitatively[7]. In this observation method, the writer observes directly the object of research, namely by observing the staffing and managerial departments to obtain accurate data in CV Galerindo Nusantara Surakarta.. Figure 1. Context Diagram c. Literature Study Method Employee recruitment is guided by certain principles outlined by the internal and external companies, so that the implementation and results of the selection can be accounted for both legally and economically [6]. Literature study is a framework, concept or orientation for analyzing and classifying facts collected in the research conducted. Reference sources (books, journals, magazines) referred to should be relevant and up to date (state of art) and in accordance with those contained in Reference library[1]. In this literature study method, the authors collect data by reading books, literature, journals related to report writing. II. BASIC THEORY AND METHOD In this research method, there are 2 types of methods, namely data collection methods and systems development methods. 2.2 System Development Methods The system development method is a systematic or regular way that aims to analyze the development of a system so that the system can meet needs.In essence, the waterfall model system development method is the work of a system that is carried out sequentially or linearly.From the user side it is also more profitable because it can plan and prepare all the data and process requirements that will be needed. 3.1 Needs Analysis Databases are interconnected data that are grouped in a table or several tables and an application program that regulates how to access the data. Collections of this data are usually called databases, which contain real information, for example for a company[10]. At this stage of needs analysis, the writer made a system using the waterfall method.The analysis currently needed is a new system analysis because CV Galerindo Nusantara does not yet have a computerized employee recruitment system, namely the company only provides job vacancies and still uses paper for recruitment selection and applicants must come to the company with less effective procedures.So that the author wants to design and develop a new web-based information system that is effective using interconnected procedures. 2.1 Data Collection Methods The following is a system design at CV Galerindo N The following is a system design at CV Galerindo Nusantara The following is a system design at CV Galerindo N t The following are as follows: The data collection methods that the authors use are as follows: 1. Context Diagram 4. Employees Employees are the main wealth of every company, because their role determines the success or failure of the company to achieve its goals. Companies must always try to obtain and place qualified employees in every position and job so that the implementation of work is more efficient and effective [6]. Page 29 Journal IJCIS homepage - https://ijcis.net/index.php/ijcis/index International Journal of Computer and Information System (IJCIS) Peer Reviewed – International Journal Vol :Vol. 02, Issue 02, May 2021 e-ISSN : 2745-9659 https://ijcis.net/index.php/ijcis/index b. Interview Method The interview method is a process of conversation carried out by interviewers and interviewees with specific goals, with guidelines and can be face to face or through certain communication tools[3]. In this interview method, the author conducts questions and answers with the manager, namely Indah Pratiwi at CV Galerindo Nusantara which is related to the recruitment of employees to make references and flow in making information systems. Figure 1. Context Diagram Page 30 Journal IJCIS homepage - https://ijcis.net/index.php/ijcis/index International Journal of Computer and Information System (IJCIS) Peer Reviewed – International Journal Vol :Vol. 02, Issue 02, May 2021 e-ISSN : 2745-9659 https://ijcis.net/index.php/ijcis/index https://ijcis.net/index.php/ijcis/index 2. Tiered Diagram The following is a diagrammatic diagram of a Web- Based Employee Recruitment Information System at CV Galerindo Nusantara. Figure 4. DAD Level 1 Figure 4. DAD Level 1 Figure 4. DAD Level 1 Figure 4. DAD Level 1 3.3 System Implementation The following is an implementation of the web-based employee recruitment information system at CV Galerindo Nusantara. 3.4 System Testing This Web-based Employee Recruitment Information System, the authors conducted a test using the Black Box method, to achieve the goal that the system is feasible to use.Black Box testing focuses on the functional requirements of the software.Thus, this test makes it possible to obtain a set of input conditions that fully utilize the functional requirements for a program.The following is a table of system test results: Figure 9. Job Position View Table 1. System Testing Table No Process Design Expected results Result 1. Admin Login to the admin page Successfully entered the admin page was successful 2. Admin can Input Job Data Successfully Input Job data was successful 3. Admin can view user data Successfully seen user data was successful 4. User can login or register Successfully login or register to the system was successful 5. User can input data Successfully input data was successful Figure 9. Job Position View 6. Display Registration Implementation On this page, Applicants can create a new account to later log in to the job vacancies that have been provided. 6. Display Registration Implementation On this page, Applicants can create a new account to later log in to the job vacancies that have been provided. Figure 10. Registration Display Figure 10. Registration Display 1. Display Implementation Login 02, Issue 02, May 2021 e-ISSN : 2745-9659 https://ijcis net/index php/ijcis/index International Journal of Computer and Information System (IJCIS) Peer Reviewed – International Journal Vol :Vol. 02, Issue 02, May 2021 e-ISSN : 2745-9659 https://ijcis net/index php/ijcis/index Vol :Vol. 02, Issue 02, May 2021 e-ISSN : 2745-9659 https://ijcis.net/index.php/ijcis/index Figure 7. Applicant Data Display Figure 11. Applicant Dashboard View Figure 7. Applicant Data Display Figure 7. Applicant Data Display Figure 7. Applicant Data Display Figure 11. Applicant Dashboard View 4. Display Problem Test Implementation In this menu, the admin can input questions for the test to applicants, namely in the form of multiple choice or essays. 8. Display HR Dashboard Implementation The following is the HR dashboard display, on this page HRD can access the employee recruitment report. Figure 8. Display Test Questions Figure 12. FigureDashboard Hrd Figure 12. FigureDashboard Hrd Figure 8. Display Test Questions Figure 12. FigureDashboard Hrd 5. Job Vacancies Implementation Display On this page, the Admin can enter a Job Vacancy required by the company. 1. Display Implementation Login Figure2. Tiered Diagram This login implementation functions to enter the system, if the user name and password match, then they can enter the system, if the user name and password do not match then they cannot enter the system. 3. Data Flow Diagram (DAD) Level 0 The following is a picture of Level 0 Data Flow Diagram (DFD) Information System for Web-Based Employee Admissions at CV Galerindo Nusantara Figure 5. Login View Figure3. DAD Level 0 Figure 5. Login View Figure3. DAD Level 0 Figure3. DAD Level 0 2. Display Admin Dashboard Implementation 2. Display Admin Dashboard Implementation On this page the admin can select the menu and view the data that has been entered by the user so that the admin can edit, create, change, and delete data from this system. 4. Data Flow Diagram (DAD) Level 1 The following is a picture of Level 1 Data Flow Diagram (DFD) Information System for Web-Based Employee Admissions at CV Galerindo Nusantara. 4. Data Flow Diagram (DAD) Level 1 The following is a picture of Level 1 Data Flow Diagram (DFD) Information System for Web-Based Employee Admissions at CV Galerindo Nusantara. 2. Display Admin Dashboard Implementation On this page the admin can select the menu and view the data that has been entered by the user so that the admin can edit, create, change, and delete data from this system. Figure 6. Admin Dashboard Display Figure 6. Admin Dashboard Display Journal IJCIS homepage - https://ijcis.net/index.php/ijcis/index Page 31 Figure 6. Admin Dashboard Display 3. Display Applicant Data Implementation On this page, the admin can see the applicant's data input in the application details and the admin can change the applicant's status. Journal IJCIS homepage - https://ijcis.net/index.php/ijcis/in Figure 6. Admin Dashboard Display 3. Display Applicant Data Implementation On this page, the admin can see the applicant's data input in the application details and the admin can change the applicant's status. 3. Display Applicant Data Implementation p y pp p On this page, the admin can see the applicant's data input in the application details and the admin can change the applicant's status. p y pp p On this page, the admin can see the applicant's data input in the application details and the admin can change the applicant's status. Page 31 Journal IJCIS homepage - https://ijcis.net/index.php/ijcis/index International Journal of Computer and Information System (IJCIS) Peer Reviewed – International Journal Vol :Vol. IV. CONCLUSION Figure 10. Registration Display From the results and discussion that has been presented by the author with this web-based employee recruitment information system, the authors draw the conclusion that with the construction of this information system, the problem of employee recruitment becomes more effective. 7. Applicant Dashboard Implementation Display This page is the applicant's display when they have registered and logged into the system. 7. Applicant Dashboard Implementation Display This page is the applicant's display when they have registered and logged into the system. Journal IJCIS homepage - https://ijcis.net/index.php/ijcis/index Page 32 International Journal of Computer and Information System (IJCIS) Peer Reviewed – International Journal Vol :Vol. 02, Issue 02, May 2021 e-ISSN : 2745-9659 https://ijcis.net/index.php/ijcis/index https://ijcis.net/index.php/ijcis/index With this information system, applicants do not need to use paper to apply for jobs. And with this system, companies can inform more broadly about job vacancies. REFERENCES [1] Ameilia Zuliyanti Siregar dan Nurliana Harahap. (2019). Strategi Dan Teknik Penulisan Karya Tulis Ilmiah Dan Publikasi. Deepublish. [2] Anggraeni, Y. E., & Irviani, R. (2017). Pengantar Sistem Informasi (E. Risanto (ed.)). CV.ANDI OFFSET. [3] Edi, F. R. S. (2016). Teori Wawancara Psikodignostik. – yogyakarta : LeutikaPrio,2016. [4] Elbadiansyah. (2019). ManajemenSumberDayaManusia. CV IRDH. [5] Haqi, B. (2019). Aplikasi SPK Pemilihan DosenTerbaikMetode Simple Additive Weighting (SAW) Dengan Java (1st ed.). Deepublish [6] Marjuni, S. (2015). Manajemen Sumber Daya Manusia (P. D. H. St. Haerani, S.E., M.Si. (ed.)). CV.SAH MEDIA. [7] Ni’matuzahroh, & Prasetyaningrum, S. (2018). observasi: teori dan aplikasi dalam psikologi. Universitas Muhammadiyah Malang. [8] Sa’ad, M. I. (2020). Otodidak Web Programming: Membuat Website Edutainment. PT Gramedia Jakarta. [9] Suprihadi, E. (2020). Sistem Informasi Bisnis Dunia Versi 4.0. ANDI OFFSET. [10] Widodo, A. W., & Kurnianingtyas, D. (2017). Sistem Basis Data (1st ed.). UB Press. [11] Muryani, A. S., & Muqorobin, M. (2020). Decision Support System Using Cloud-Based Moka Pos Application To Easy In Input In Orange Carwash Blulukan Flash N0. 110 Colomadu. International Journal of Computer and Information System (IJCIS), 1(3), 66-69. [12] Rais, N. A. R., & Saputra, R. (2020). Online Sales System Analysis of PT. Nutrifood Indonesia through the distributor CV. Trio Sukses Mandiri Solo With Nutrimart Home Delivery (NHD) Application. International Journal of Computer and Information System (IJCIS), 1(2), 40-46. [13] Pujianto, H., & Rokhmah, S. (2021). Analysis of" E-Patient UNS" Application System for Online Registration of UNS Hospital Patients. International Journal of Computer and Information System (IJCIS), 2(1), 9-12. [14] Dwiyana, D., & Muqorobin, M. (2021). Analysis of Adi Soemarmo Solo Airport Parking Payment System. International Journal of Computer and Information System (IJCIS), 2(1), 1-3. [15] Efendi, T. F., & Wihartati, A. P. (2021). Decision Support System for Share Investment Using The Capital Assetpricing Method (CAPM). International Journal of Computer and Information System (IJCIS), 2(1), 18-22. Page 33 Journal IJCIS homepage - https://ijcis.net/index.php/ijcis/index
https://openalex.org/W4206517844	https://pure.knaw.nl/ws/files/496442303/7300_Lameris.pdf	English	null	Migratory vertebrates shift migration timing and distributions in a warming Arctic	Animal Migration	2,021	cc-by	18,666	Migratory vertebrates shift migration timing and distributions in a warming Arctic Lameris, Thomas; Hoekendijk, Jeroen; Aarts, Geert; Aarts, Aline; Allen, Andrew M.; Bienfait, Louise; Bijleveld, Allert I.; Bongers, Morten F.; Brasseur, Sophie; Chan, Ying-Chi; de Ferrante, Frits; de Gelder, Jesse; Derksen, Hilmar; Dijkgraaf, Lisa; Dijkhuis, Laurens R.; Dijkstra, Stanne; Elbertsen, Gert; Ernsten, Roosmarijn; Foxen, Tessa; Gaarenstroom, Jari; Gelhausen, Anna; van Gils, Jan A.; Grosscurt, Sebastiaan; Grundlehner, Anne; Hertlein, Marit L.; van Heumen, Anouk J.P.; Heurman, Moniek; Huffeldt, Nicholas Per; Hutter, Willemijn H.; Kamstra, Ynze; Keij, Femke; van Kempen, Susanne; Keurntjes, Gabi; Knap, Harmen; Loonstra, A.H. Jelle; Nolet, B.A.; Nuijten, Rascha; Mattijssen, Djan; Oosterhoff, Hanna; Paarlberg, Nienke; Parekh, Malou; Pattyn, Jef; Polak, Celeste; Quist, Yordi; Ras, Susan; Reneerkens, Jeroen; Ruth, Saskia; van der Schaar, Evelien; Schroen, Geert; Spikman, Fanny; van Velzen, Joyce; Voorn, Ezra; Vos, Janneke; Wang, Danyang; Westdijk, Wilson; Wind, Marco; Zhemchuzhnikov, Mikhail K.; van Langevelde, Frank published in Animal Migration 2021 document version Publisher's PDF, also known as Version of record document license CC BY Link to publication in KNAW Research Portal Link to publication in KNAW Research Portal citation for published version (APA) Lameris, T., Hoekendijk, J., Aarts, G., Aarts, A., Allen, A. M., Bienfait, L., Bijleveld, A. I., Bongers, M. F., Brasseur, S., Chan, Y.-C., de Ferrante, F., de Gelder, J., Derksen, H., Dijkgraaf, L., Dijkhuis, L. R., Dijkstra, S., Elbertsen, G., Ernsten, R., Foxen, T., ... van Langevelde, F. (2021). Migratory vertebrates shift migration timing and distributions in a warming Arctic. Animal Migration, 8(1), 110-131. 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 i h d ghts for the publications made accessible in the public portal are retained by the authors and/or other copyright owners 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 KNAW public portal for the purpose of private stu ch • Users may download and print one copy of any publication from the KNAW public portal for the purpose of private study or research. 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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. E-mail address: pure@knaw.nl Migratory vertebrates shift migration timing and distributions in a warming Arctic https://doi.org/10.1515/ami-2020-0112 received June 29, 2021; accepted October 10, 2021 Corresponding author: Thomas K. Lameris, Department of Coastal Systems, NIOZ Royal Netherlands Institute for Sea Research, Den Burg, Texel, The Netherlands Abstract: Climate warming in the Arctic has led to warmer and earlier springs, and as a result, many food resources for migratory animals become available earlier in the season, as well as become distributed further northwards. To optimally profit from these resources, migratory animals are expected to arrive earlier in the Arctic, as well as shift their own spatial distributions northwards. Here, we review literature to assess whether Arctic migra- tory birds and mammals already show shifts in migration timing or distribution in response to the warming climate. Distribution shifts were most prominent in marine mammals, as expected from observed northward shifts of their resources. At least for many bird species, the ability to shift distributions is likely constrained by available habitat further north. Shifts in timing have been shown in many species of terrestrial birds and ungulates, as well as for polar bears. Within species, we found strong variation in shifts in timing and distributions between populations. Ou r review thus shows that many migratory animals display shifts in migration timing and spatial distribution in reaction to a warming Arctic. Importantly, we identify large knowledge gaps especially concerning distribution shifts and timing of autumn migration, especially for marine mammals. Our understanding of how migratory animals respond to climate change appears to be mostly limited by the lack of long-term monitoring studies. Department of Animal Ecology, Netherlands Institute of Ecology (NIOO-KNAW), Wageningen, the Netherlands, Email: thomaslame- ris@gmail.com, Jeroen Hoekendijk, Geert Aarts, Allert I. Bijleveld, Sophie Brasseur, Ying-Chi Chan, Jan A. van Gils, Jeroen Reneerkens, Mikhail K. Zhem- chuzhnikov, Department of Coastal Systems, NIOZ Royal Netherlands Institute for Sea Research, Den Burg, Texel, The Netherlands Andrew M. Allen, Bart A. Nolet, Department of Animal Ecology, Netherlands Institute of Ecology (NIOO-KNAW), Wageningen, the Netherlands Jeroen Hoekendijk, Geert Aarts, Allert I. Bijleveld, Sophie Brasseur, Ying-Chi Chan, Jan A. van Gils, Jeroen Reneerkens, Mikhail K. Zhem- chuzhnikov, Department of Coastal Systems, NIOZ Royal Netherlands Institute for Sea Research, Den Burg, Texel, The Netherlands Andrew M. Allen, Bart A. Nolet, Department of Animal Ecology, Netherlands Institute of Ecology (NIOO-KNAW), Wageningen, the Netherlands Geert Aarts, Aline Aarts, Louise Bienfait, Morten F. Bongers, Frits de Ferrante, Jesse de Gelder, Hilmar Derksen, Lisa Dijkgraaf, Laurens R. E-mail address: pure@knaw.nl E-mail address: pure@knaw.nl Anim. Migr. 2021; 8:110–131 This work is licensed under the Creative Commons Attribution 4.0 License. Research Article Thomas K. Lameris, Jeroen Hoekendijk, Geert Aarts, Aline Aarts, Andrew M. Allen, Louise Bienfait, Allert I. Bijleveld, Morten F. Bongers, Sophie Brasseur, Ying-Chi Chan, Frits de Ferrante, Jesse de Gelder, Hilmar Derksen, Lisa Dijkgraaf, Laurens R. Dijkhuis, Sanne Dijkstra, Gert Elbertsen, Roosmarijn Ernsten, Tessa Foxen, Jari Gaarenstroom, Anna Gelhausen, Jan A. van Gils, Sebastiaan Grosscurt, Anne Grundlehner, Marit L. Hertlein, Anouk J.P. van Heumen, Moniek Heurman, Nicholas Per Huffeldt, Willemijn H. Hutter, Ynze J. J. Kamstra, Femke Keij, Susanne van Kempen, Gabi Keurntjes, Harmen Knap, A.H. Jelle Loonstra, Bart A. Nolet, Rascha J.M. Nuijten, Djan Mattijssen, Hanna Oosterhoff, Nienke Paarlberg, Malou Parekh, Jef Pattyn, Celeste Polak, Yordi Quist, Susan Ras, Jeroen Reneerkens, Saskia Ruth, Evelien van der Schaar, Geert Schroen, Fanny Spikman, Joyce van Velzen, Ezra Voorn, Janneke Vos, Danyang Wang, Wilson Westdijk, Marco Wind, Mikhail K. Zhemchuzhnikov, Frank van Langevelde Open Access. © 2021 Lameris et al., published by De Gruyter. This work 1 Introduction distribution northward to locations with later phenology. Animals making this northward shift may then experience the same timing of resource availability in spring without advancing migration timing (Figure 2), although it could also result in later arrival given longer migration distances. However, distributional shifts might be limited by topog- raphy or by increased travel costs for some Arctic animals. First, many terrestrial animals in the Arctic already find themselves at the most northern edge of the continent, resulting in shrinking habitat range or “polar squeeze” (Figure 2). Second, suitable habitats may not be present further north. For example, marine mammals are largely reliant on sea ice for feeding (and reproducing in case of pinnipeds), and may find themselves without any availa- ble habitat with the predicted disappearance of sea ice in summer [21]. Changes in timing of migration and repro- duction [22,23], and to a limited extent shifts in breed- ing distribution [24,25], have already been observed in Arctic migratory animals. Those species which have been unable to shift timing of reproduction sufficiently, often suffer from reductions in reproductive success and sur- vival [15,16]. As Arctic migratory animals are an essential part of local Arctic ecosystems [26,27], as well as provide important resources for local Indigenous Peoples [28,29], any changes in the migration timing and distribution of migratory animals will have far-reaching consequences. The Arctic region is characterised by strong seasonality. During winter, the Arctic forms an inhospitable environ- ment for most animals, with low temperatures, extensive snow and ice cover and long phases of darkness. The summer season is relatively short with long light phases and temperatures above freezing, associated with strong changes in environmental conditions, including melt of snow and break-up of sea ice. In both terrestrial and marine ecosystems, the Arctic summer is also a period of peak productivity, creating a short period of high resource abundance for many species. These strong sea- sonal changes in environmental conditions explain why many vertebrates occurring in the Arctic have a migratory lifestyle [1, Figure 1]. This allows them to profit from high resource abundance in the Arctic summer, while escaping harsh climatic conditions during winter. The Arctic climate is changing disproportionally fast [2], with temperature increases three times as fast as the rest of the globe [3], especially accelerating in recent decades [4]. 1 Introduction Increases in temperature throughout the year coincide with loss of Arctic sea ice, shorter seasonal duration of snow cover [5], and overall increased ‘green- ing’ of tundra regions [but with strong variation between sites, 6]. Such changes are predicted to further accelerate in the near future [7]. The warming climate has strong impacts on the availability of resources for Arctic migra- tory animals. Earlier disappearance of ice and snow in the season can result in shifts in timing and distribution of the main food resources for migrants, including vegetation [8,9] and arthropods on land [10–12], and phytoplank- ton abundance at sea [13,14]. If migratory animals do not change the timing of their migration and reproduction or their summer distribution in response, phenological mis- matches with their food resources may occur, potentially resulting in reduced fitness [15,16] which might affect pop- ulation dynamics. Here we review literature on Arctic migratory animals for evidence of shifts in timing of migration and shifts in distribution. We focus on groups of endothermic migra- tory vertebrates that perform seasonal migrations to, or within, the Arctic, including terrestrial and marine birds, ungulates, cetaceans, pinnipeds and polar bears (Figure 1). These animals differ strongly in their habitats (marine, coastal and terrestrial habitats) as well as in their diet (plankton, benthic invertebrates, fish, pinnipeds, plants, arthropods, rodents). We expect to find variation in both shifts in timing and distribution across taxonomic groups of Arctic migratory animals. Concerning migration timing, we predict that long-distance migrants (most birds and cetaceans, with one-way journeys on average exceeding 2000 kilometres) will show smaller shifts in migration timing in comparison to short-distance migrants (includ- ing some cetaceans, all ungulates, pinnipeds and polar bear), as long-distance migrants cannot predict condi- tions in the Arctic from their distant wintering grounds [30,31]. Concerning shifts in distribution, we expect terres- trial animals (including land-breeding marine birds), to be more constrained in making large shifts [32] compared to marine animals. For terrestrial animals, suitable alter- native habitat further northward may not be available due to lagging changes in suitable vegetation communities, or To adjust to changes in the timing of resource availa- bility in the Arctic, migratory animals could advance their timing of arrival on the breeding grounds. Migratory vertebrates shift migration timing and distributions in a warming Arctic Dijkhuis, Sanne Dijkstra, Gert Elbertsen, Roosmarijn Ernsten, Tessa Foxen, Jari Gaarenstroom, Anna Gelhausen, Sebastiaan Grosscurt, Anne Grundlehner, Marit L. Hertlein, Anouk J.P. van Heumen, Moniek Heurman, Willemijn H. Hutter, Ynze J. J. Kamstra, Femke Keij, Susan- ne van Kempen, Gabi Keurntjes, Harmen Knap, Rascha J.M. Nuijten, Djan Mattijssen, Hanna Oosterhoff, Nienke Paarlberg, Malou Pa- rekh, Jef Pattyn, Celeste Polak, Yordi Quist, Susan Ras, Saskia Ruth, Evelien van der Schaar, Geert Schroen, Fanny Spikman, Joyce van Velzen, Ezra Voorn, Janneke Vos, Danyang Wang, Wilson Westdijk, Marco Wind, Frank van Langevelde, Wildlife Ecology & Conservation Group, Wageningen University, Wageningen, The Netherlands Geert Aarts, Sophie Brasseur, Wageningen Marine Research, Wage- ningen University and Research, Den Helder, the Netherlands Ying-chi Chan, Jan van Gils, Conservation Ecology Group, Gronin- gen Institute for Evolutionary Life Sciences (GELIFES), University of Groningen, Groningen, the Netherlands Ou r review thus shows that many migratory animals display shifts in migration timing and spatial distribution in reaction to a warming Arctic. Importantly, we identify large knowledge gaps especially concerning distribution shifts and timing of autumn migration, especially for marine mammals. Our understanding of how migratory animals respond to climate change appears to be mostly limited by the lack of long-term monitoring studies. Nicholas Per Huffeldt, Greenland Institute of Natural Resources, Nuuk, Greenland & Arctic Ecosystem Ecology, Department of Biosci- ence, Aarhus University, Roskilde, Denmark Keywords: birds; mammals; marine mammals; phenologi- cal mismatch; range shift; migration phenology A.H. Jelle Loonstra, Altenburg & Wymenga, Feanwâlden, the Nether- lands Bart A. Nolet, Theoretical and Computational Ecology, Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, Amsterdam, the Netherlands Bart A. Nolet, Theoretical and Computational Ecology, Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, Amsterdam, the Netherlands Rascha J.M. Nuijten, Interdisciplinary Centre for Conservation Sci- ence, Department of Zoology, University of Oxford, Oxford, UK Rascha J.M. Nuijten, Interdisciplinary Centre for Conservation Sci- ence, Department of Zoology, University of Oxford, Oxford, UK This work is licensed under the Creative Commons Attribution 4.0 License. Open Access. © 2021 Lameris et al., published by De Gruyter. This work is licensed under the Creative C 111 Migratory vertebrates shift migration timing and distributions in a warming Arctic 1 Introduction Given the limited leeway to increase the speed of migration once underway [17,18] for birds which have relatively high travel speeds, this likely also necessitates advancements in migratory fuel deposition and departure from the wintering grounds [19]. Besides changes in spring migration in response to earlier resource availability, longer Arctic summer seasons associated with later freeze-up and snowfall [5] could also drive delays in the timing of autumn migration [20]. In addition to shifts in the timing of migration, animals may respond to a warming Arctic by shifting their summer 112 Lameris et al. Figure 1: Examples of warm-blooded vertebrates migrating to the Arctic, showing (top left), reindeer (Rangifer tarandus) crossing a frozen river on the way to their calving grounds, (top right), red knots (Calidris canutus) on a spring staging site in northern Norway, (bottom left), walrus (Odobenus rosmarus) resting on sea ice with their young, and (bottom right), a minke whale (Balaenoptera acutorostrata) in a fjord in Svalbard. Photographs by Paul Asman & Jill Lenoble (top left), GRID Arendal (top right), Alaska Region US Fish & Wildlife (bottom left), and Guillaeme Baviere (bottom right) Figure 1: Examples of warm-blooded vertebrates migrating to the Arctic, showing (top left), reindeer (Rangifer tarandus) crossing a frozen river on the way to their calving grounds, (top right), red knots (Calidris canutus) on a spring staging site in northern Norway, (bottom left), walrus (Odobenus rosmarus) resting on sea ice with their young, and (bottom right), a minke whale (Balaenoptera acutorostrata) in a fjord in Svalbard. Photographs by Paul Asman & Jill Lenoble (top left), GRID Arendal (top right), Alaska Region US Fish & Wildlife (bottom left), and Guillaeme Baviere (bottom right). Figure 2: (A-B) Maps showing examples of current (green) and hypothetical future summer distributions (orange) of purple sandpipers Calid- ris maritima (A) and harp seals Pagophilus groenlandicus (B) around Svalbard. While harp seals can shift their distribution northward with retreating sea ice, purple sandpipers are constrained by available land mass of Svalbard to shift their distributions far north. Median sea ice cover (during summer months 1981 - 2010) is shown as light grey area in the north, outlined by a solid black line, and hypothetical change in future ice cover is depicted by the white area outlined by dashed black line. Grey dashed arrows show migration directions of the sand- pipers and seals. Distributions and migration directions are based on [73,195,231]. 1 Introduction (C-D) With a warming climate, timing of food availability (blue lines) is expected to advance (red dashed lines), both in current distributions (D) as well as hypothetical future distribution ranges (C). To maintain a synchrony with these peaks in food availability, animals are expected to advance their own timing of migration, but may also be able to maintain a synchrony by shifting their distribution northward (towards distribution C) where the food becomes available later in the season. Figure 2: (A-B) Maps showing examples of current (green) and hypothetical future summer distributions (orange) of purple sandpipers Calid- ris maritima (A) and harp seals Pagophilus groenlandicus (B) around Svalbard. While harp seals can shift their distribution northward with retreating sea ice, purple sandpipers are constrained by available land mass of Svalbard to shift their distributions far north. Median sea ice cover (during summer months 1981 - 2010) is shown as light grey area in the north, outlined by a solid black line, and hypothetical change in future ice cover is depicted by the white area outlined by dashed black line. Grey dashed arrows show migration directions of the sand- pipers and seals. Distributions and migration directions are based on [73,195,231]. (C-D) With a warming climate, timing of food availability (blue lines) is expected to advance (red dashed lines), both in current distributions (D) as well as hypothetical future distribution ranges (C). To maintain a synchrony with these peaks in food availability, animals are expected to advance their own timing of migration, but may also be able to maintain a synchrony by shifting their distribution northward (towards distribution C) where the food becomes available later in the season. Figure 2: (A-B) Maps showing examples of current (green) and hypothetical future summer distributions (orange) of purple sandpipers Calid- ris maritima (A) and harp seals Pagophilus groenlandicus (B) around Svalbard. While harp seals can shift their distribution northward with retreating sea ice, purple sandpipers are constrained by available land mass of Svalbard to shift their distributions far north. Median sea ice cover (during summer months 1981 - 2010) is shown as light grey area in the north, outlined by a solid black line, and hypothetical change in future ice cover is depicted by the white area outlined by dashed black line. Grey dashed arrows show migration directions of the sand- pipers and seals. Distributions and migration directions are based on [73,195,231]. 2.2 Ungulate migration The Arctic is inhabited by a limited set of ungulate species, including reindeer (Rangifer tarandus), moose (Alces alces), muskox (Ovibos moschatus), Dall’s sheep (Ovis dalli) and snow sheep (Ovis nivicola). Movements of the three latter species are limited to short-distances (up to 100 km), which can be seasonal but often follow nomadic patterns [55] in search for suitable foraging grounds. On the other hand, moose [56,57] and reindeer are consid- ered partial migrants, and especially some populations of reindeer make large migratory movements up to 1300 km [58] from taiga wintering areas to calving grounds at coastal Arctic tundra zones. Other populations of rein- deer migrate shorter distances [59] or are resident [60]. By migrating, ungulates can winter in areas with more suit- able conditions for both adults and their offspring [56], travel northwards in spring along a wave of vegetation green-up [61], and match calving with local peaks in food quality [15,62]. Studies on the migrations of moose are largely limited to their southern ranges, and in this review we therefore focus on reindeer. 2.1 Bird migration The most abundant birds with Arctic distributions are seabirds, shorebirds, and waterfowl, with other less rep- resented species groups including passerines, grouse and birds of prey [1]. Most of these species are migratory [35] and spend the winter in more southern regions. The extent of these migrations varies enormously, with some seabird species wintering in Arctic waters [36,37], most waterfowl, passerines, and birds of prey wintering in tem- perate regions [38–40], and many shorebird and seabird species wintering in areas that range from temperate and tropical regions [41–43] down to Antarctic waters [44,45]. Differences in wintering areas, and therefore migration distance, likely relate strongly to availability of suitable wintering habitat with available resources. Fish-eating seabirds may be able to winter in Arctic waters as long as fish are available and accessible [46,47], while shorebirds, depending on benthic invertebrates, travel to temperate and tropical intertidal flats that do not freeze in winter [42]. Given long migration distances, many bird species require stopover sites to gain energy stores between leaps of migration [48]. During spring migration, at least some species of waterfowl appear to track peaks in food quality and availability [49–51] and the onset of ice break-up and snowmelt at staging sites [52,53]. This, however, does not appear to be the case for all species of waterfowl [51] nor for as it is limited by topographical barriers in the landscape such as the northern edge of the continent (Figure 2). In comparison, animals in marine environments that can freely navigate the oceans and enter the Arctic basin, may show stronger shifts in their distribution [32], for example by following the edge of pack ice, or the distribution shifts of their main prey [33,34]. shorebirds [54]. Birds of prey also track snowmelt during northward migration, possibly as areas with melting snow contain high availability of rodent prey [38]. All Arctic migratory birds reproduce during the Arctic summer, and many species (including waterfowl, shorebirds and marine birds) appear to attempt to synchronize their reproduction with prey availability for their offspring. In this review, we first introduce the focal taxonomic groups of endothermic vertebrates and their migrations. Thereafter we introduce the resources on which animals depend and discuss how the phenology and abundance of these resources are expected to change in a warming climate. Finally, following a systematic literature search, we review scientific literature on evidence for shifts in timing of migration and shifts in distribution of focal taxo- nomic groups, and quantify whether shifts in timing differ between taxonomic groups. 1 Introduction (C-D) With a warming climate, timing of food availability (blue lines) is expected to advance (red dashed lines), both in current distributions (D) as well as hypothetical future distribution ranges (C). To maintain a synchrony with these peaks in food availability, animals are expected to advance their own timing of migration, but may also be able to maintain a synchrony by shifting their distribution northward (towards distribution C) where the food becomes available later in the season. 113 Migratory vertebrates shift migration timing and distributions in a warming Arctic 2.4 Pinniped migration Several pinniped species are year-round residents in the Arctic regions, like harp seal (Pagophilus groenlandicus), ringed seal (Pusa hispida), hooded seal (Cystophora cris- tata), bearded seal (Erignathus barbatus), spotted seal (Phoca largha), ribbon seal (Histriophoca fasciata) and walrus (Odobenus rosmarus). In addition, the distribu- tions of other species like harbour seals (Phoca vitulina) and grey seals (Halichoerus grypus), Steller sea lion (Eume- topias jubatus) and northern fur seal (Callorhinus ursinus) extend into the polar regions. While pinnipeds feed at sea, they require land or ice to reproduce, moult and period- ically rest, which severely constrains their at-sea distri- bution. To avoid land predators, most Arctic pinnipeds spend the breeding and moulting season on land-fast ice or free-floating pack ice in late winter and spring [71], after which they disperse. While some individuals move long distances away from the ice (e.g. harp seals and walrus), most pinniped species remain associated with outer edges of the pack ice, where they feed on fish and invertebrates [72], while using the pack ice as resting platforms. Resident Arctic pinnipeds feed on prey that is present and accessi- ble in the Arctic regions year-round, which provides no strong incentive to leave the Arctic region altogether, as opposed to most birds and cetaceans. However, pinnipeds do show seasonal long-distance movements [73], but this seasonal migratory pattern mostly involves movement between foraging areas, breeding and moulting locations, largely driven by the extent of the pack ice [74]. 2.5 Polar bear migration tory pattern remains largely unknown. It has been sug- gested that whales migrate to reduce predation pressure on calves [64,65], or that the higher temperatures of low latitude waters bring thermoregulation benefits for calves (and/or adults) [66,67]. However, recent new evidence suggests that deferred skin moult could be the main driver of long-distance cetacean migration [67]. In colder waters, cetaceans reduce blood flow to their skin to conserve body heat. It appears that cetaceans migrate to warmer waters at lower latitudes to reduce heat loss during moult, a period during which they enlarge blood flow through the skin. Similarly, the pagophilic species that remain in the Arctic year-round, make seasonal migrations towards warmer waters in estuaries and shallows to moult [68–70]. Polar bears (Ursus maritimus) depend on sea ice platforms to hunt fatty, energy-dense pinniped prey [75], primarily ringed seals and bearded seals [76]. The bears consume around two-thirds of their annual food intake from Feb- ruary up to mid-April, when seals give birth on the sea ice [75,77]. When sea ice melts and becomes fractured in spring, the polar bears’ mobility and seal hunting tech- nique become inefficient [78], and bears either move with the receding pack ice, or migrate towards terrestrial hab- itats [75]. The summer is typically a period of fasting for polar bears during which they rely on endogenous energy reserves [79]. Most polar bears move back onto the pack ice after autumn, when pack ice extent increases, while pregnant female bears will enter terrestrial maternity dens along the coast where they give birth to their young [80]. 3 Changing resources under climate warming Temperatures in the Arctic are increasing year-round, with temperatures above freezing occurring earlier in spring [81], associated with earlier timing of snow melt, active layer melt and ice break-up [5]. These climatic changes can result in earlier availability of resources for migrants (although the relative importance of climatic factors may differ at regional scales, e.g. [82]). In addition, higher summer temperatures may lengthen the period of resource availability, and climatic changes may also cause a northward shift in the spatial distribution of resources, as well as of suitable habitats for migrants. We discuss these aspects in detail in the following sections. 2.3 Cetacean migration Cetaceans occurring in the Arctic are mostly represented by baleen whales (Mysticeti), including rorquals (Balae- nopteridae) and grey whales (Eschrichtius robustus), and fewer toothed whales (Odontoceti), including belugas (Delphinapterus leucas), narwhals (Monodon monoceros), sperm whales (Physeter macrocephalus) and northern beaked whales (Hyperoodon ampullatus). With the excep- tion of the pagophilic (i.e. sea-ice loving) beluga, narwhal and bowhead whale (Balaena mysticetus), which make seasonal migrations within the Arctic, cetaceans are sea- sonal visitors to the Arctic. In contrast to birds and ungu- lates that migrate to the Arctic for reproduction, migratory whales use the high latitude summer grounds exclusively for feeding, while reproduction occurs in low latitude winter grounds, where food availability is generally scarce or non-existent [63]. The mechanism driving this migra- 114 Lameris et al. 114 3.1 Earlier resource availability Other aspects such as light-dark cycles and topographical features (such as cliffs which seabirds require for nesting, shown in grey boxes) will not change, potenti- ally constraining distribution shifts of animals. Retreating ice cover Vegetation change Changes in thermal niches Light-dark cycle Topograhical features Retreating ice cover Vegetation change Changes in thermal niches Light-dark cycle Topograhical features Figure 3: Simplified food webs in marine Arctic habitats (left) and terrestrial habitats (right), with the focal groups of marine mig i l h bi i ( ) d i l i ( ) di l d i i l R ll d h Retreating ice cover Vegetation change Changes in thermal niches Light-dark cycle Topograhical features Changes in thermal niches Topograhical features Figure 3: Simplified food webs in marine Arctic habitats (left) and terrestrial habitats (right), with the focal groups of marine migrants (blue), terrestrial herbivore migrants (green) and terrestrial carnivores (orange) displayed in circles. Resources, as well as predators which do not display typical migratory behaviour, are displayed in black, smaller circles. Several marine fish species make semelparous migrations to streams and rivers to spawn (as indicated by the grey dashed arrow), and are thus available as resource to both marine and terrestrial animals. In marine habitats, migratory seabirds, cetaceans and pinnipeds are expected to adjust timing of migration and / or distribution with availability of zooplankton, benthic organisms such as bivalves, and fish, which in turn rely on phytoplankton (including ice algae). Polar bears rely on the presence of pack ice to prey on pinnipeds during winter months, but with a warming climate spend more time in terrestrial habitats where they have started to prey on bird’s eggs. In terrestrial habitats, herbivores feed on forage plants and time arrival with peaks in nutritional quality of plants. Shorebirds time migration in synchrony with availability of arthropods, and birds of prey rely on availability of rodents for successful reproduction. In turn, terrestrial predators such as Arctic foxes prey on bird’s nests, especially in years when cyclic rodent populations are depressed. Besides shifts in phenology of resources, climate warming may also impact habitat suitabi- lity via changes in thermal niches, retreating pack ice and vegetation community change (shown in orange boxes). Other aspects such as light-dark cycles and topographical features (such as cliffs which seabirds require for nesting, shown in grey boxes) will not change, potenti ally constraining distribution shifts of animals. 3.1 Earlier resource availability Lower trophic levels, including the food resources for many migratory animals (Figure 3), are known to rapidly adjust their phenology to a warming climate [83]. In ter- restrial habitats, earlier snowmelt and increasing temper- atures have led to an advancement in the growing season of many plants [84], and thereby an advancement in the moment of peak quality and availability of forage plants for herbivorous birds and ungulates [8,9,85]. Arthropods, which form the main prey for Arctic-breeding shorebirds and passerines [86], respond to earlier dates of snow and active layer melt and increasing temperatures by earlier emergence [10,22,87] and changes in their abundance Migratory vertebrates shift migration timing and distributions in a warming Arctic 115 115 Migratory vertebrates shift migration timing and distributions in a warming Arctic 115 Figure 3: Simplified food webs in marine Arctic habitats (left) and terrestrial habitats (right), with the focal groups of marine migrants (blue), terrestrial herbivore migrants (green) and terrestrial carnivores (orange) displayed in circles. Resources, as well as predators which do not display typical migratory behaviour, are displayed in black, smaller circles. Several marine fish species make semelparous migrations to streams and rivers to spawn (as indicated by the grey dashed arrow), and are thus available as resource to both marine and terrestrial animals. In marine habitats, migratory seabirds, cetaceans and pinnipeds are expected to adjust timing of migration and / or distribution with availability of zooplankton, benthic organisms such as bivalves, and fish, which in turn rely on phytoplankton (including ice algae). Polar bears rely on the presence of pack ice to prey on pinnipeds during winter months, but with a warming climate spend more time in terrestrial habitats where they have started to prey on bird’s eggs. In terrestrial habitats, herbivores feed on forage plants and time arrival with peaks in nutritional quality of plants. Shorebirds time migration in synchrony with availability of arthropods, and birds of prey rely on availability of rodents for successful reproduction. In turn, terrestrial predators such as Arctic foxes prey on bird’s nests, especially in years when cyclic rodent populations are depressed. Besides shifts in phenology of resources, climate warming may also impact habitat suitabi- lity via changes in thermal niches, retreating pack ice and vegetation community change (shown in orange boxes). 3.3 Northward shifts in suitable habitats and resources and a decreasing time-lag between ice algae and phyto- plankton blooms [95], which together form the main food resource for many species of bivalves and zooplankton [97]. Zooplankton may suffer from reductions in survival (measured as lower available zooplankton biomass) when they are not able to adjust their phenology in response to advanced phytoplankton blooms [97–99], as well as pos- sibly due to the segregation of ice algae and phytoplank- ton blooms. Nevertheless, other studies found stable zooplankton biomass following earlier phytoplankton blooms [100]. Zooplankton is the main food resource for fish species, and both zooplankton and fish are eaten by most baleen whales, pinnipeds and seabirds [101,102]. Both timing and abundance of zooplankton may affect the abundance and availability of fish species as prey for higher trophic levels. For seabirds and whales, impor- tant prey fish species may occur earlier in the season, or decrease in abundance due to phenological mismatches with algal and plankton blooms. A change in climate is expected to change the habitat suit- ability for migratory animals as well as for their resources, and both may show northward shifts of their distribu- tion in response (Figure 2). First of all, many organisms thrive within a specific ‘thermal niche’ or ‘thermal pref- erence’, and experience fitness reductions outside this niche [110,111]. With a warming climate, the location of this thermal niche and therefore the suitability of habitats is predicted to shift northwards [34,112]. This may lead to distribution shifts for endothermic vertebrates, as well as for their food resources, often ectothermic animals or vegetation. Although at high latitudes most endothermic animals live at temperatures well below their thermal maximum and thus have leeway under increasing temper- atures [113,114], habitat suitability of ectothermic organ- isms is more sensitive to changing temperatures [110], and these potentially make larger distribution shifts [115]. At the same time, vegetation communities and thereby dis- tribution of specific plant species appear to change at rel- atively slow rates [6,116]. Such shifts in the distribution of resources will also change habitat suitability for migrants. For example, in marine environments, many fish species are showing northward distribution shifts, changing local community composition of potential prey species for marine predators [117]. 3.3 Northward shifts in suitable habitats and resources At the same time, some fish species from lower latitudes may be unable to find refuge away from predators during the continuous light of polar summer, which may constrain their abundance under certain climate change scenarios [118]. 3.1 Earlier resource availability In marine environments, ice algae and phytoplankton form the most important primary producers and are the basis of the Arctic marine food web [93]. Ice algae grow under thinning ice edges, and their phenology is regu- lated by light [94]. While a warming climate would result in a more permeable ice layer and earlier ice algal blooms may be expected, short day-length at high latitudes limit phenological advancements of ice algae [94,95]. The other main primary producer, phytoplankton, blooms later in the season at ice edges, regulated by light and nutri- ent upwelling [95]. Earlier ice disappearance has caused an advancement in timing of phytoplankton blooms [96] [10]. The annual cycles of Arctic rodents, the main prey of Arctic raptors and skuas [88], seem to be little impacted by a warming climate [89] (but see reports on irregularity of these cycles [90]). At the same time, the accessibility of rodents as prey for birds is potentially dependent on snow cover, with high concentrations of accessible rodents around the time of snow melt [38]. Despite increasing temperatures, increasing precipitation in winter (another aspect of climate change in the Arctic, 91]) may also result in abundant snow and late melt thereof, which has the potential to largely disrupt reproduction of all terrestrial animals [92]. [10]. The annual cycles of Arctic rodents, the main prey of Arctic raptors and skuas [88], seem to be little impacted by a warming climate [89] (but see reports on irregularity of these cycles [90]). At the same time, the accessibility of rodents as prey for birds is potentially dependent on snow cover, with high concentrations of accessible rodents around the time of snow melt [38]. Despite increasing temperatures, increasing precipitation in winter (another aspect of climate change in the Arctic, 91]) may also result in abundant snow and late melt thereof, which has the potential to largely disrupt reproduction of all terrestrial animals [92]. 116 Lameris et al. Lameris et al. 116 4.2.1 Terrestrial and marine birds Most of the time series available on migration timing of terrestrial birds show no clear advancements in the timing of migration departure from wintering areas [16,22,122– 124]. However, in the last decades, some species of water- fowl and one shorebird have shown profound shifts in timing of departure [123,125–128], but these shifts appear mostly linked to changes in suitable stopover sites along their migratory route. At the same time, many terres- trial bird species have advanced timing of arrival in the Arctic, with studies showing species to adjust migration timing to annual variation in climatic conditions in the Arctic [16,22,129–131] and some studies also showing clear trends of advanced arrival over time [22,132–134]. While one study shows stronger shifts in advancement of arrival in short-distance migrants compared to long-distance migrants [133], other studies find no clear differences between short- and long-distance migrants [129]. Along with advancements in migration timing, some terrestrial bird species show advancements in the timing of repro- duction [22,135–139], but this is not found for all species [11,140,141], and advancements in reproduction timing can lag behind advancements in arrival [16]. Trends in autumn migration are mixed, with some species showing delayed arrival in wintering grounds, associated with higher temperatures at northern summering and staging sites [126,142]. Several other species show earlier arrivals of adult birds [142,143], possibly explained by disrupted breeding seasons. For every paper, we recorded (1) the study species and taxonomic group (bird, ungulate, cetacean, pinniped, polar bear), (2) the region where the study was conducted, and (3) whether evidence was reported for shifts in timing of spring migration, reproduction and autumn migration and shifts in summering distribution. For shifts in timing, we considered evidence to be significant shifts in timing over years (considering study periods of at least 5 years) or with changing climatic variables. For shifts in distribu- tion, we considered evidence to include both increases in the number of sightings (but only when evident that this was unrelated to population increase) as well as latitu- dinal change in observations [121]. We noted the rate of change in timing of spring and autumn migration (in days per year) when this was reported in studies. In contrast to many terrestrial bird species, less is known about changes in timing of migration in Arctic sea- birds. 3.2 Longer period of resource availability While a warming climate will advance the timing when resources become available, it can also impact the abun- dance of resources [10], as well as result in a longer period of availability of resources. Although their nutritional value declines over the season, forage plants are availa- ble for herbivorous birds until covered by first snowfall in autumn, which is occurring later in the season in recent years [5]. This is also beneficial for many ungulates, which, despite their ability to find forage plants under shallow layers of snow, cannot deal with deeper layers of snow [103] or ice crust formation [104]. Terrestrial arthropods may remain active as long as temperatures are above freezing, but their availability to predators could be limited due to a restricted number of generations emerg- ing in one season [105] but see [106], and Arctic shorebirds are thus unlikely to profit from a longer breeding season through re-nesting [107]. With increasing temperatures, warming Arctic waters may facilitate longer resource availability, by driving the emergence of a second phy- toplankton bloom [100,108] and a second generation of copepods [109] during autumn. Some factors of habitat suitability are closely linked to climatic conditions, including the thermal niche and resource distribution as explained above, as well as spe- cific environmental aspects of habitats, such as cover of sea ice as resting platforms for pinnipeds and hunting platforms for polar bears. While climate warming will directly change these aspects of habitat suitability, other aspects are geographically fixed, for example topographi- cal features (e.g. cliffs making up suitable nesting habitat for seabirds) and the duration of the light-dark cycle (which is fixed by latitude and date). Advances in the timing of resource availability and rapid-changing aspects of habitat suitability as a result of climate warming may drive northward distribution shifts of animals, but slow-changing or fixed aspects of habitat suitability may at the same time form constraints for distribution shifts [118,119]. Moreover, such differences in the rate of north- ward shifts could cause reductions in suitable habitat altogether. 117 Migratory vertebrates shift migration timing and distributions in a warming Arctic 4 Shifts in migration timing and distributions which shifts in migration and distribution were recorded. We quantified whether shifts in migration timing dif- fered between taxonomic groups by comparing slopes of reported shifts in spring and autumn migration. In addi- tion, comparing the number of species and study regions for which we found relevant studies allowed us to quan- tify how knowledge gaps differed between groups and regions. 4.1 Methods to quantify differences in responses between taxonomic groups To review whether vertebrates display shifts in migration timing and distributions in response to a warming climate, we searched for relevant papers using the Web of Science database. We used the search term Arctic AND (range shift OR migration timing) AND (bird OR avian OR ungulate OR herbivore OR cetacean OR whale OR beluga OR narwal OR pinniped OR seal OR walrus OR polar bear). This query resulted in 486 papers, from which we only included papers that (1) dealt with Arctic migratory species, (2) reporting on changes in either timing of spring or autumn migration, timing of reproduction or changes in distribu- tion, (3) either as trends over time (as measured over a period of at least 5 years) or (4) in relation to climatic and environmental conditions in the Arctic (as measured over a period of at least 3 years). To determine which species are considered ‘Arctic species’, we used species lists as provided by the Arctic Biodiversity Assessment [35], with the exception that we only included bird species for which the majority or complete population breeds in the Arctic. We excluded papers that did not report species-specific results. Eventually this selection resulted in 32 papers. In addition to these papers, we added 35 relevant papers (matching the criteria mentioned above) that we found within reference lists of the 32 selected papers, as well as relevant papers found within the reference lists of two review studies on marine mammals [74,120]. 4.2.3 Cetaceans An increasing asynchrony between the arrival of migra- tory cetacean species and local abundance of prey (due to spatial and temporal shifts) has been predicted [71], but long-term data on the timing of migration of Arctic ceta- cean species is rare, and the few available studies paint a mixed picture [120]. Spring migration phenology shows either no change (beluga whales) or has been delayed (bowhead and grey whale), which could suggest that these species might not keep up with advancing phenol- ogy of their prey in the Arctic. In autumn, delays in depar- ture from northern waters have been found for beluga whales, which appears to be a response to later ice for- mation [159]. However, further south along the migration route, migrating baleen whales are observed on autumn migration earlier in recent years [160]. 4.2.2 Ungulates A variety of trends on reindeer migration timing emerge from the literature. A delay in spring migration over time was found for reindeer populations on Newfoundland up to the year 2000 [59], earlier spring departures but not earlier arrivals were shown for populations in Northern Quebec between 2000 and 2011 [151] and no trends in departure dates but earlier arrival were found for popula- tions in north-western Canada and Alaska between 2000 and 2017 [23]. It is suggested that reindeer adjust departure dates and travel speed to local as well as large-scale cli- matic conditions [23], allowing them to pass through areas just prior to snowmelt [62], which facilitates easier passage over partly frozen soil and ice [152]. In addition, by pacing migration speed with local timing of snowmelt, reindeer may be able to optimally time their arrival on the calving grounds to match local dates of snow melt and vegetation green-up [23,62]. As a result, calving date has advanced in several populations of reindeer in response to a warming climate [153]. However, large variation exists in the extent to which reindeer appear to be able to keep up their timing of reproduction with the local climate. Domestic reindeer in Northern Finland have been able to advance timing of calving with earlier springs [154], which has benefitted reproductive success [155]. On the other hand, reindeer populations in Svalbard and Western Greenland do not seem to advance calving dates with earlier springs [15,156], and a mismatch with phenology of local forage plants has resulted in a reduction in reproductive success in Western Greenland [15,85,157,158]. While longer summer seasons could extend the summer period during which forage plants are available, reindeer have been found to advance autumn migration timing [59,151]. It is possible that such changes are a response to resource depletion, but it is yet unclear whether this is mainly driven by climate change or population dynamics 4.2.4 Pinnipeds The seasonal distribution of pinnipeds in the Arctic is heavily influenced by the spatial extent of the sea pack ice, which shows large seasonal and inter-annual varia- tion. Most pinnipeds associate with the ice well before the breeding season, which means that ice regions need to be accessible at the onset of the breeding season and remain stable throughout the breeding period in order to be suit- able [74]. Some species (e.g. ringed seals) breed on (more stable) fast-ice, as they have a relatively long nursing period (~ 6 weeks) [161,162]. In contrast, pinnipeds that rely on floating pack ice such as hooded seals generally have a shorter nursing period. Hooded seals breed several weeks later than harp seals, during the start of the sea- sonal ice break-up. To combat the effect of drift, their lactation period is extremely short (~ 4 days). Due to the strong association with sea pack ice, Arctic pinnipeds will be highly influenced by climatic changes in temperature. However, currently, little information is available about changes in phenology of pinnipeds in response to increas- ing temperatures and changes in ice cover [163]. We found only one study reporting on shifts in timing in pinnipeds, showing a long-term advancement in the arrival of walrus in their summering range, as observed by local Inuit hunters in the Canadian Arctic [29]. to higher sea-surface temperature [145]. In the Arctic, advancements in reproduction phenology over time have been found for surface-feeding species (gulls and tubenoses) in the Pacific ocean but not in the Atlantic ocean, while pursuit-diving species (alcids) showed no trends in either ocean [146]. While this may be the general pattern, some pursuit-diving and benthic-feeding species do show advancements in reproduction timing in response to local earlier ice break-up [147–149] and increases in air temperature [150]. We did not find studies reporting trends in timing of autumn migration in sea birds. 4.2.1 Terrestrial and marine birds For species for which data are available, advance- ments in migration timing are relatively small, with the exception of Arctic-breeding guillemots (Uria spp.) which have advanced arrival in breeding colonies all over the Arctic [144]. A large meta-analysis, that included many Arctic breeding seabirds, showed that seabirds in general have not adjusted their timing of reproduction in response In order to quantify how taxonomic groups differed in responses to changing climatic conditions, we compared the relative number of species per taxonomic group for 118 Lameris et al. 118 4.3.2 Ungulates Reindeer have often been considered to display strong site fidelity, especially during the calving season in summer [178,179]. This idea of site fidelity is under discussion, as reindeer can shift their wintering ranges following over- grazing of pastures [180], and recently, two reindeer herds have started to adjust their calving grounds to annual variation in forage quality, moving further westwards into Alaska in earlier springs [25,181]. Similarly, moose in Alaska have shifted their summering ranges northwards following shrub encroachment in tundra habitats [182]. 4.2.5 Polar bears Although pinnipeds, the main prey of polar bears, have not been reported to display major shifts in phenology, Migratory vertebrates shift migration timing and distributions in a warming Arctic 119 the Arctic. Nevertheless, so far there is little evidence that shifts in breeding distribution are already taking place. In part, this is because range shifts are typically picked up in long-term monitoring studies with high spatial coverage [176], which are rare in the Arctic. In Finland, an average northward shift of 0.8 km/year has been observed in a suite of Arctic bird species [24]. A long-term local study in Arctic Russia has revealed a strong decline in densi- ties of typical high-Arctic breeding shorebird species, while species typical for southern tundra habitats have increased [177]. climate warming is strongly reducing the seasonal avail- ability of pack ice [164,165], used by pinnipeds and polar bears as haul-out sites and foraging habitat, respectively. Polar bears time their migration towards terrestrial hab- itats with the break-up of pack ice in spring [166]. As a result, bears have advanced their arrival in terrestrial hab- itats with earlier ice break-up [78,165], as well as delayed the time when they travel back to the pack ice in winter, which also impacts their condition when entering mater- nity dens [167]. By shifting their migration timing, bears have increased the fasting period in terrestrial habitats during which they have no access to their pinniped prey [78]. In addition, due to reduced availability of pack ice, bears often have to travel longer distances on terrestrial habitats and swim larger distances in order to migrate back to the pack ice [75]. Longer fasting periods and higher travelling costs have been shown to cause reductions in population vital rates [75,79,168]. 4.3.1 Terrestrial and marine birds Northward shifts in wintering distribution of Arctic migra- tory birds, also named ‘short-stopping’, have in the past decades been shown for multiple species of Arctic-breed- ing waterfowl and shorebirds [126,169–171]. For birds wintering in Europe, this mostly translates to shifts in a north-easterly direction up to 13 km/year (as reported for Bewick’s swans, 126). Changing energetic requirements and prey availability under different scenarios of future climate are also expected to affect the winter distributions of the five most numerous species of seabirds in the North Atlantic, many of which breed in the Arctic [172], but shifts in winter distributions have not yet been shown for these species. 4.3.3 Cetaceans For cetacean species, it is predicted that the ranges of 88% of all cetaceans may be affected due to global warming [183]. In accordance, northward shifts in distribution have been revealed for several migratory baleen whales, including typical southern Arctic species [184,185]. Also short-distance migrants, bowhead whales and beluga whales, are shifting their distributions within the Arctic, likely in response to changes in sea ice cover [186,187]. Killer whales (Orcinus orca) typically avoid heavy ice con- centrations, and are increasingly occurring in the Arctic following reductions in sea ice cover which has opened up movement corridors [188,189]. The increase of this top predator might in turn influence the distribution of ceta- ceans and pinnipeds restricted to the Arctic. Sea ice reduc- tion might also provide opportunities for cetacean species to move between the North Pacific and North Atlantic, as is supported by recent reports of grey whales in the Medi- terranean Sea [190]. The investigation of shifts in breeding distributions of Arctic species is in its infancy, but theoretical exer- cises predicting shifts in winter and summer ranges are contributing a basis for forecasting potential changes. Shifts in breeding distribution have been predicted for Arctic-breeding shorebirds [173] as well as for Arctic sea- birds [174] given the northward shifts of their prey [175]. In this way, climate change may result in shifts in migration destinations and even flyways, for example the predicted establishment of wintering populations of little auks (Alle alle) in the Pacific, which would facilitate trans-Arctic migrations [174]. Likewise, Arctic seabirds may also cease migration completely and become year-round residents of 120 Lameris et al. 4.4.2 Shifts in timing Advancements in spring migration timing were reported for many terrestrial and marine birds, as well as for polar bear and some populations of reindeer, but less often for cetaceans and pinnipeds (Figure 4A). Although few studies were available, polar bears showed stronger advancements in spring migration timing compared to birds (Figure 4C). Unexpectedly, some cetacean species and populations of reindeer showed a delay in spring migration timing. Shifts in autumn migration timing also showed mixed results, with both delayed and advanced timing in birds and cetaceans, advancements in ungu- lates and a delay for one sub-population of polar bears (reported in one study, Figure 4C). 4.3.5 Polar bears Following reductions in pack ice and earlier ice break-up, polar bears have been observed to have shifted their winter ranges northward [199–201]. Also, polar bears have shifted their maternity dens more often to terrestrial coastal areas rather than on pack ice, in response to absence of stable old ice [202]. In summer, available habitat on pack ice has contracted for several populations of polar bears [199,201], and the number of polar bears spending the summer in terrestrial habitats is increasing [203]. In some regions polar bears are able to cope with sea ice loss by making use of coastal seasonal ice [200]. However, the increasing distance between wintering habitats on pack ice and ter- 4.4.1 Available data Most of the studies that we found were on shifts in the timing of migration, while much fewer studies were avail- able on changes in distribution (Figure 4A). Shifts in timing were more often studied for spring migrations and less often for autumn migrations. While terrestrial and marine birds, with data available for 46 out of 126 species, as well as ungulates and polar bear appeared to be well studied, much less studies were available for cetaceans and pinnipeds. Most studies originated from the American and Canadian Arctic, as well as from the Atlantic Arctic (Greenland and Svalbard) (Figure 4B). Much fewer studies were available for the European and West-Russian Arctic, and we found no studies reporting shifts in migration timing and distribution from the East-Russian Arctic. 4.3.4 Pinnipeds restrial summering habitats makes polar bears vulnerable to climate change [75,202]. Most species of pinnipeds restricted to the Arctic are heavily reliant on sea ice for reproduction, moult and resting. During the winter months the sea ice connects to all landmasses surrounding the Arctic Ocean (i.e. Russia, Alaska, Canada, Greenland). However, as a result of climate change, the Arctic sea ice extent, as well as its thickness and age, have decreased [191], with the largest changes during the summer months. Currently, the summer sea ice only connects to the shallow waters of Greenland and Northern Canada [192], and, somewhere between 2030 and 2050, it is expected that the Arctic will be completely ice-free during summer [21,193]. Since most arctic pinnipeds are reliant on sea ice and generally feed in shallower (and coastal) waters, the distribution of sea ice relative to the coastal waters will likely have a main impact on the distribution of Arctic pinnipeds. Probably in a result to changing sea ice conditions, range shifts in pupping grounds have been shown for harp seals [194,195], as well as in summering distributions of harp seal, bearded seal and ringed seal around Svalbard, which show a northward latitudinal trend [121]. In addition, some species (e.g. walrus) are forced to haul-out on land more often in the absence of sea ice, and this may impose additional safety and energy expenditure costs [196], also considering density-dependent effects as fewer haul-out sites are available [197]. The disappearance of sea ice may also provide opportunities for the more temperate seal species that rely on land to rest, moult and reproduce, and these species, like harbour seals, which show an increase in numbers in the Arctic [121,198]. 4.4.3 Shifts in distribution Despite the low number of studies, it appears that a north- ward shift in distribution was found for relatively more species of both marine (cetaceans, pinnipeds and polar bear) and terrestrial mammals (ungulates) compared to bird species (Figure 4A). Migratory vertebrates shift migration timing and distributions in a warming Arctic 121 5 Di i advancement of timing of arrival on their summering Figure 4: (A) The fraction of species per taxonomic group for which shifts in spring migration timing, autumn migration timing and distri- bution have been studied (light-coloured bars) and have been observed (dark-coloured bars). The total number of species for each group is noted in brackets in the legend. (B) The number of studies conducted within the four different geographic regions of the Arctic, shown per species group. (C) Histograms of reported trends in migration timing (as the slope in days of change over years), shown in bins of 0.25 as black bars. Grey bars show the number of studies reporting no change in migration timing over years without quantifying the slope. Histo- grams are shown for spring (left) and autumn migrations (right) and for terrestrial birds, sea birds, ungulates, cetaceans and polar bear. For pinnipeds no trends were reported. 4.4.3 Shifts in distribution −2 0 2 4 −2 0 2 4 0 10 20 0 10 20 0 10 20 0 10 20 0 10 20 0.00 0.25 0.50 0.75 1.00 Spring migration Autumn migration Distribution Fraction of species 0 5 10 15 American Arctic Atlantic Arctic European & West−Russian Arctic East−Russian Arctic Number of studies Terrestrial birds (86) Marine birds (40) Ungulates (2) Cetaceans (16) Pinnipeds (9) Polar bear (1) Slope (days / year) Spring migration Autumn migration Terrestrial birds Marine birds Ungulates Cetaceans Polar bear C A B Species groups (number of species) 0.00 0.25 0.50 0.75 1.00 Spring migration Autumn migration Distribution Fraction of species 0 5 10 15 American Arctic Atlantic Arctic European & t−Russian Arctic ast−Russian Arctic Number of studies Terrestrial birds (86) Marine birds (40) Ungulates (2) Cetaceans (16) Pinnipeds (9) Polar bear (1) A B Species groups (number of species) −2 0 2 4 −2 0 2 4 0 10 20 0 10 20 0 10 20 0 10 20 0 10 20 Wes E Slope (days / year) Spring migration Autumn migration Terrestrial birds Marine birds Ungulates Cetaceans Polar bear C C Terrestrial birds Marine birds Figure 4: (A) The fraction of species per taxonomic group for which shifts in spring migration timing, autumn migration timing and distri- bution have been studied (light-coloured bars) and have been observed (dark-coloured bars). The total number of species for each group is noted in brackets in the legend. (B) The number of studies conducted within the four different geographic regions of the Arctic, shown per species group. (C) Histograms of reported trends in migration timing (as the slope in days of change over years), shown in bins of 0.25 as black bars. Grey bars show the number of studies reporting no change in migration timing over years without quantifying the slope. Histo- grams are shown for spring (left) and autumn migrations (right) and for terrestrial birds, sea birds, ungulates, cetaceans and polar bear. For pinnipeds no trends were reported. advancement of timing of arrival on their summering grounds in a warming climate [15,16,204], our review shows that there are many examples of advancement of migration timing of especially terrestrial Arctic-breeding birds, as well as for polar bears and some populations of ungulates. Advancements are observed both over long time spans (several decades) and in association with First, Arctic regions differ in the rate of climate warming and local response in for example advancement of resource abundance [205], date of snow- melt [135] or sea ice dynamics [159], which can drive dif- ferentiation in responses in migration timing. Second, flexibility in migration timing may depend on reproduc- tion strategies, depending on whether animals reproduce in the Arctic or southern wintering grounds [159,160], or whether animals rely more on internal energy reserves or on local resources for successful reproduction [206–208]. Third, Arctic migrants may also be constrained to make advancements in timing by, for example, the time needed for fuel deposition [19,209], little potential to increase travel speed [17], a lack of relevant cues to time their migration [30], or physical barriers during migration, such as earlier ice break-up in rivers [152,210] or available light [119]. Our review suggests that few species of cetaceans 5.1 Shifts in timing While many studies highlight the potential for trophic mismatches for Arctic migrants due to their inadequate 122 Lameris et al. 122 Moreover, a publication bias might exists where studies finding no shifts are less often published. In theory, shifts in timing for marine mammals could be constrained by their relatively low travel speed [18], but given the short migration distances of Arctic pinnipeds, this should not form a major constraint for many species. While climate warming changes the trophic interactions between marine mammals and their prey resources, changes in abundance and distribution of resources may have a larger effect on populations than changes in timing. Therefore, as a primary response to a warming climate, marine mammals may be more likely to display shifts in distribution. The same may hold for marine Arctic-breeding birds, for which we find less evidence for shifts in migration timing as compared to terrestrial birds. changing environmental conditions, such as increasing temperatures and earlier snowmelt and ice break-up. This suggests that animals adjust their migration timing to locally changing conditions, either as they aim to match reproduction timing with local food abundance, or simply because warmer conditions allow [52,53], or even force, earlier migrations [78]. In comparison to most bird species, polar bears show especially rapid trends in migration timing, matching arrival and departure from pack ice with timing of ice freeze-up and break-up [78]. However, not all species show such flexible changes in timing, as shown by observations of multiple species at a single study site, showing advancements in reproduction timing in some species but not for others [139]. Differen- tial responses in migration timing may also occur within species, which is notable in the differential migration timing for different reindeer populations. Whereas we had expected to find stronger shifts in migration timing for short-distance migrants (ungulates, pinnipeds, polar bear and some cetaceans) compared to long-distance migrants (birds and most cetaceans), we find large variation in both short- and long-distance migrants. Whether or not species and populations advance migration timing therefore likely depends on other factors as well, for example (1) variation in the environmental change that species experi- ence, (2) strategies which animals use for reproduction, as well as (3) potential physiological constraints for making changes in the timing of migration. All these factors may differ between species. 5.4 Future outlook Remarkably, in the cases where shifts in distribu- tions are observed, these often show sub-Arctic species extending their range into the Arctic, such as common seals, southern cetacean species, and shorebird species of southern tundra regions [74,177,198]. At the same time, shifts in distribution for species within the Arctic are less often observed. This review suggests the potential for many Arctic migra- tory animals to make shifts in the timing of migration and in their distribution in the Arctic, potentially allowing them to adequately respond to changed resource distribu- tion in a warming Arctic. Yet, our review also highlights potential constraints for animals to make such shifts, which could eventually result in inadequate or no shifts, with possible negative effects on fitness. The potential for animal populations to make shifts in distribution and timing likely relies on the potential for making shifts in migration schedules and strategies, either by individual flexibility [127], or by changes in subsequent generations [225]. In the latter case, the ability of populations to shift in response to a warming climate is linked to its reproduc- tive success under current conditions. but see [223]. At the same time, not all species which show little change in timing of breeding and reproduction, expe- rience mismatches with reductions in reproductive success (e.g. [11]). While generally, timing of food availability may advance in a warming Arctic, and more rapidly when com- pared with temperate regions [4,224], the rate of warming and the responses of prey species can strongly differ between regions [205]. Such regional differences could be an important explanation for the absence of shifts in timing and distribution of migratory species, rather than it reflecting suboptimal behaviour. Fitness consequences for Arctic migratory species may also arise from increased competition with sub-Arctic species, extending their ranges into the Arctic. However, northward shifts in distri- bution by some species originating from outside the Arctic circle may be constrained by the unique light environment at high latitudes [118,119], complicating forecasts of future ranges of birds and mammals in the Arctic. As such, it is difficult to predict whether or not migratory populations will suffer from reproductive consequences in a warming climate, based on whether populations are showing shifts in their migratory behaviour and distributions. such diet shifts may be a possibility for generalist species, like certain cetacean and pinniped species [216], species with a more specialized diet (e.g. planktivorous marine birds) may not be able to switch prey, and face potentially severe fitness impacts following shifts in prey species dis- tribution, should they not be able to respond adequately by shifting their own distribution [217–219]. In addition, distribution shifts in marine birds are also potentially con- strained by available habitat for their breeding colonies at higher latitudes. For terrestrial bird species, our review suggests rela- tively few distribution shifts, which is possibly explained by adequate responses in migration timing, as well as no clear evidence for shifts in distribution ranges of prey resources. In addition, several terrestrial bird species have been shown to be rather flexible in choice of habitat within their current range [220] and their choice for prey species [221], which could also reduce the need for distri- bution shifts. For ungulates, several studies show shifts in calving grounds, presumably to locations with higher food abundance and more suitable habitats. Possibly, terrestrial ungulates possess such adaptive responses to changing conditions, as they continuously need to shift ranges in response to overgrazing events [210]. 5.2 Shifts in distribution Shifts in distribution over the past decades appear to occur more often in marine mammals, which is in confir- mation of our hypothesis. For cetaceans, pinnipeds and polar bears, relatively many species display northward distribution shifts, and it is likely that such shifts are a response to changes in sea ice cover [74] and associated shifts in suitable areas for feeding and reproduction. Most Arctic cetaceans and pinnipeds depend on high food abundance close to the edge of the pack ice, and as a result are expected to shift their ranges with retreating ice cover [186]. Moreover, pinnipeds also rely on sea ice as haul-out platforms for reproduction and moult, and their life-histories are strongly tied to sea ice [195]. The close association with pack ice is also evident for polar bears, for which a large extent of available data shows a combi- nation of northward shifts in winter, matching changes in pack ice, and shifts to terrestrial habitats during summer. With longer stays in terrestrial habitats, an increasing number of bears is preying on eggs of waterfowl and sea- birds [203,211], even though this prey is unlikely to com- pensate for the increasing periods of fasting under declin- ing sea ice [212]. Our review suggests distribution shifts to be less evident for marine and terrestrial bird species. Although this could be explained by the rarity of long-term moni- toring programmes with extensive cover, there are also ecological explanations. Marine birds, given observed dis- tribution shifts of their prey [117], would be expected to show shifts in distribution, similar to marine mammals. Instead, observed shifts in diet show that some species of marine birds may cope with shifts in prey species distri- bution by preying on different resources [213–215]. While Our review suggests that few species of cetaceans and pinnipeds display shifts in migration timing. Admit- tedly, very few data appear to be available to test for shifts in timing [74,163], and it is therefore a possibility that the low number of observed shifts is caused by the difficulty in observing migration timing in these animals. Migratory vertebrates shift migration timing and distributions in a warming Arctic 123 5.3 Implications of inadequate shifts in timing and distribution Many populations of Arctic migratory endothermic verte- brates appear able to shift their timing of migration and their distributions, yet not all species are making such shifts, nor do we know whether such shifts are in fact ade- quate responses to changes in distribution and abundance of food. As a result from inadequate responses in either the timing of migration and reproduction, or inadequate shifts in distribution, phenological mismatches between the period of offspring growth and timing of peak food abundance may arise. Such mismatches have been shown for several species of Arctic terrestrial birds [16,204], marine birds [149] and terrestrial ungulates [15], resulting in reductions in reproductive success. In temperate-breed- ing migratory songbirds, slow adjustments in migration timing have even been linked to population declines [222] Our review also suggests a severe lack of data, limit- ing our ability to identify shifts in timing and distribution. Data are especially lacking for migration timing in ceta- ceans and pinnipeds, but also in other taxonomic groups potential shifts in timing and distribution have not been studied. It is striking that shifts in spring migration timing have received much more attention than shifts in autumn migration timing [226], and also there are relatively few studies on shifts in distribution. Our review also suggests strong regional differences in available data, with most 124 Lameris et al. 124 [7] IPCC. IPCC Special Report on the Ocean and Cryosphere in a Changing Climate. Pörtner H-O, Roberts DC, Masson- Delmotte V, Zhai P, Tignor M, Poloczanska E, et al., editors. 2019. data available for Arctic vertebrates in Alaska, Northern Canada, Greenland, Svalbard and Scandinavia, and fewer data for the European and Russian Arctic. The need for long-term data collection over the entire Arctic region is well recognised [227], as advised in recent reports on pop- ulation monitoring for marine as well as terrestrial moni- toring [228,229]. Given the rate of climatic changes in the Arctic, it is likely that shifts in timing of migration and distribution of migratory animals will become more prom- inent. Better monitoring of migratory animals will allow an increased understanding of the responses of these animals to global warming, which may help to identify the possible limitations that restrict adaptations of animals to the globally changing conditions, and the potential impacts on their populations. 5.3 Implications of inadequate shifts in timing and distribution Such data will be essential for the conservation of migratory species in a warming climate, as well as for the persistence of Indigenous and local human communities in the Arctic, which are often culturally and nutritionally dependent on the presence of migratory vertebrates [230]. [8] ] Doiron M, Gauthier G, Lévesque E. Effects of experimental warming on nitrogen concentration and biomass of forage plants for an arctic herbivore. J Ecol. 2014;102:508–17. ] Lameris TK, Jochems F, van der Graaf AJ, Andersson M, Limpens J, Nolet BA. Forage plants of an Arctic-nesting herbivore show larger warming response in breeding than wintering grounds, potentially disrupting migration phenology. Ecol Evol. 2017;7:2652–60. 0] Høye TT, Loboda S, Koltz AM, Gillespie MAK, Bowden JJ, Schmidt NM. Nonlinear trends in abundance and diversity and complex responses to climate change in Arctic arthropods. Proc Natl Acad Sci U S A. 2021;118:1-8 1] Reneerkens J, Schmidt NM, Gilg O, Hansen J, Hansen LH, Moreau J, et al. Effects of food abundance and early clutch predation on reproductive timing in a high Arctic shorebird exposed to advancements in arthropod abundance. Ecol Evo 2016;6:7375–7386. 2] T l I S h kk H H il bilit f ti bi d Doiron M, Gauthier G, Lévesque E. Effects of experimental warming on nitrogen concentration and biomass of forage plants for an arctic herbivore. J Ecol. 2014;102:508–17. Lameris TK, Jochems F, van der Graaf AJ, Andersson M, Limpens J, Nolet BA. Forage plants of an Arctic-nesting herbivore show larger warming response in breeding than wintering grounds, potentially disrupting migration Doiron M, Gauthier G, Lévesque E. Effects of experimental warming on nitrogen concentration and biomass of forage plants for an arctic herbivore. J Ecol. 2014;102:508–17. [9] Lameris TK, Jochems F, van der Graaf AJ, Andersson M, Limpens J, Nolet BA. Forage plants of an Arctic-nesting herbivore show larger warming response in breeding than wintering grounds, potentially disrupting migration phenology. Ecol Evol. 2017;7:2652–60. [10] Høye TT, Loboda S, Koltz AM, Gillespie MAK, Bowden JJ, Schmidt NM. Nonlinear trends in abundance and diversity and complex responses to climate change in Arctic arthropods. Proc Natl Acad Sci U S A. 2021;118:1-8 [11] Reneerkens J, Schmidt NM, Gilg O, Hansen J, Hansen LH, Moreau J, et al. 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W4285068191.txt	https://zenodo.org/record/4008636/files/3_msa-2020-006.pdf	en		COMMUNITY AFFAIRS: REWRITING THE TEMPEST IN THE SERVICE OF ART AND CIVILISATION	Zenodo (CERN European Organization for Nuclear Research)	2,020	cc-by	7,371	Messages, Sages and Ages, Vol. 7, No. 1, (2020) DOI: 10.5281/zenodo.4008636  Marian Rebei Colegiul Tehnic Alexandru Ioan Cuza România, Suceava, 49 Calea Unirii E-mail: m_rebei@hotmail.com COMMUNITY AFFAIRS: REWRITING THE TEMPEST IN THE SERVICE OF ART AND CIVILISATION Abstract William Shakespeare’s The Tempest comes across as an inviting and resourceful ground that has prompted a range of (re-)reading perspectives and creative appropriations. The reworking of the original plot, characters, motifs, themes and ideas originates in geographical, cultural, social and ideological spaces in order to supply an alternative and even contesting perspective to the established meanings. The play has been appropriated to serve a range of causes that match particular agendas. Percy MacKaye appropriated partially The Tempest to fashion a plot through which to educate the US citizens in the spirit of art while fostering a national identity spirit. His masque, Caliban by the Yellow Sands, was meant to celebrate Shakespeare’s dramatic works and also to serve civilizing purposes and social ideals that would generate a sense of cultural and community belonging. In this way, MacKaye’s creative reworking of The Tempest is a means to an end. Caliban is appropriated as a symbol of aspiring humanity going through traumatic periods of vicious destruction and extreme suffering and serves to amplify conflicting or oppositional relationships in order to better point out ideas about civilisation, socio-cultural community, the creative potential and role of art as a civilizing agency, democracy, freedom and anti-colonialism. Thus, this rewriting of Caliban should invite reflections on a number of levels – personal, public, cultural, racial, educational and socio-political – and may trigger awareness against inhumane and harmful ideologies, policies and practices. Keywords: alternative reading, rewriting, creative appropriation, community, art, identity The twentieth-century rewriting of William Shakespeare’s The Tempest is more diversified in respect to theme, approach and literary genre than it was in the previous centuries and crosses over the geographical, cultural and socio-political frontiers of the Anglo-Saxon world.1 From 1667 to 1900,  Marian Rebei holds a B.A. and a M.A. from the Ştefan cel Mare University of Suceava and has been a teacher of English since 1999. 1 This is an informative list of research on the adaption, transformation and rewriting of The Tempest: Frederick W. Kilbourne. 1906. Alterations and Adaptations of Shakespeare. Boston: The Poet Lore Company; Peter Hulme and William H. Sherman (eds.). 2000. ‘The Tempest’ and Its Travels. London: Reaktion Books; Christine Dymkowski (ed.). 2000. The Tempest. Cambridge University Press; Michael Dobson. 1992. The Making of the National Poet: Shakespeare, Adaptation and Authorship, 1660-1769. Oxford: Clarendon Press; Gary Taylor. 1989. Reinventing Shakespeare: A Cultural History from the Restoration to the Present. London: The Hogarth Press; Jonathan Bate. 1989. Shakespearean Constitutions: Politics, Theatre, Criticism 1730-1830. Oxford: Clarendon Press; Jean I. Marsden. 1995. The Re-Imagined Text: Shakespeare, Adaptation, and Eighteenth Century Literary Theory. The University Press of Kentucky; Christopher Spencer (ed.). 1965. Five Restoration Adaptations of Shakespeare. Urbana: University of Illinois Press; George C. Branam, 1956. Eighteenth Century Adaptations of Shakespearean Tragedy. Berkeley and Los Angeles: University of California Press; Michael Dobson. 1991. ‘‘Remember/First to Possess His Books’: The Appropriation of The Tempest, 1700-1800’. Shakespeare Survey, 43; Mary M. Nilan. 1972. ‘The Tempest at the Turn of the Century: Cross-Currents in Production’, Shakespeare Survey, vol. 25; Sandra Clark (ed.). 1997. Shakespeare Made Fit: Restoration Adaptation of Shakespeare (London: J. M. Dent/Everyman; Richard W. Schoch. 2002. Not Shakespeare: Bardolatry and Burlesque in the Nineteenth Century. Cambridge University Press; Bill Ashcroft, Gareth Griffiths and Helen Tiffin. 1989. The Empire Writes Back: Theory and Practice in Post-Colonial Literature. London and New York: Routledge; Chantal Zabus. 2002. Tempests after Shakespeare. New Yord: Palgrave; Cary M. Mazer. 1981. Shakespeare Refashioned: Elizabethan Plays on Edwardian Stages. Ann Arbor, Michigan: UMI Research Press; Rob Nixon. 1987. ‘Caribbean and African Appropriations of The Tempest. Critical Inquiry, Vol. 13, No. 3, Spring; Marianne Novy (ed.). 1990. Women’s Re-Visions of Shakespeare. 28 Messages, Sages and Ages, Vol. 7, No. 1, (2020) DOI: 10.5281/zenodo.4008636 drama was the dominant genre through which authors adapted and transformed The Tempest, supplemented by a small number of prose narratives and poems.2 The expansion of the novel in the eighteenth century may account in part for the genre diversification of The Tempest revisions in the twentieth century. From a thematic point of view, the pre-twentieth century transformations originate from engaging and challenging perspectives that are pre-eminently relevant to events and matters in the British Isles and, only marginally, Europe and elsewhere. Starting with the second half of the nineteenth century, the works that rewrite The Tempest depart, through novel artistic and sociopolitical approaches, from the previous adaptations and transformations of the play. This is mainly due to the fact that rewriting occurs in various geographical, cultural and linguistic sites, emerging from a range of intellectual, artistic, social and political motives to serve a number of causes – the most frequent and prominent ones being anti-colonialism, decolonisation and feminism.3 This is to say that those who actively engage with the Shakespearean play seek to create flexible and distinct narratives through which to initiate freshly engaging dialogue that transcends the play’s borders of meaning and brings new insights to the foreground in terms of reading and interpretation. This engagement, both creative and critical, generates an alternative, supplementary or even oppositional space to the original play. In this space the rewriters set up new identities for the inherited characters and forge new plots in which the Shakespearean ideas, symbolism, motifs and themes are refashioned. In the twentieth century The Tempest triggered an impressive array of both fictional and nonfictional works because it has been subjected to new, alternative readings, i.e. re-readings. The fictional works are creative rewritings of the play’s aspects, motifs, ideas, characters and symbolism, while the non-fictional ones are alternative (re-)readings of the play. They rework, within a new frame, the entire play or they single out from the original a motif, such as the relationship between art and nature, master and servant, father and daughter, the absent women, and/or one or more characters – most often Prospero, Caliban, Miranda, Ariel, Ferdinand, and Sycorax. The non-fictional works interpret and engage critically with The Tempest’s plot, characters, motifs, ideas and symbolism from positions that are relevant to certain cultural and geographical locations and to certain socio-political and gender perspectives. The fictional and non-fictional works engage with the play, as a cultural text, through commentary, criticism and creative responses – the last group writes back or in reply to the play. These works foreground concepts, motifs and meanings pertaining to their originating agendas that situate them in contesting and oppositional relations and stances to the play. The fictional works that recreate The Tempest reposition the play while recuperating a complex of social, cultural, and political events, motifs, and ideas. This type of active engagement – fictional and non-fictional – shows that The Tempest is an inviting and resourceful ground that develops and absorbs other geographical, social, cultural or ideological spaces but it is also drawn into other such spaces. Thus, the characters undergo identity changes, complex interconnections surface and intercultural exchanges occur. Some transformations of The Tempest, including the one discussed hereinafter, focus on Caliban not so much by giving him more dramatic attention and space, but rather by emphasizing and amplifying his conflicting and oppositional relationship with Prospero in its different guises – artistic/cultural, social, political and racial – to the detriment of other relationships and matters. Caliban acquires prominent symbolic status and function to express ideas about civilization, the Urbana and Chicago: University of Illinois Press; Diana Brydon. 1984. ‘Re-Writing The Tempest’, World Literature Written in English, Vol. 23, No. 1. 2 Dramatic works that are adaptations and rewritings of The Tempest: The Tempest, or The Enchanted Island, (1667), by William Davenant and John Dryden; Thomas Shadwell’s operatic version of the former (1674) and The Mock Tempest; or The Enchanted Castle, (1674), by Thomas Duffett. In addition, there are the stage versions and transformations by David Garrick (1756-1757, 1757-1758), J. P. Kemble (1789), Charles Macready (1838), Charles Kean (1857), as well the domestic editions of Shakespeare’s works: Charles and Mary Lamb, Tales from Shakespeare (1807); Thomas Bowdler, The Family Shakespeare, in Eight Volumes (1818). 3 Cf. Aimé Césaire. 1969. UneTempête: D’apres “la Tempête” de Shakespeare. Paris: Seuil, translated as A Tempest. Based on Shakespeare’s The Tempest – Adaptation for a Black Theatre (Ubu Repertory Theatre Publications, 1985); David Wallace. 1977. Do You Love Me Master? Lusaka: National Educational Company of Zambia Ltd. For feminist rewriting of The Tempest see Marianne Novy (ed.). 1990. Women’s Re-Visions of Shakespeare. Urbana and Chicago: University of Illinois Press; Diana Brydon. 1984. ‘Re-Writing The Tempest’, World Literature Written in English, Vol. 23, No. 1; Chantal Zabus. 2002. Tempests after Shakespeare. 29 Messages, Sages and Ages, Vol. 7, No. 1, (2020) DOI: 10.5281/zenodo.4008636 power and role of art, democracy, freedom, racial subjugation, anti-colonialism and decolonization. This is to say that the practice of creative appropriation leads to new texts. Caliban has an extended role in Percy MacKaye’s Caliban by the Yellow Sands, an American transformation4 of The Tempest that “resembles Rénan’s Caliban in presenting Shakespeare’s savage and deformed slave as a symbol for the common people” (Cohn, 1976: 276). Caliban by the Yellow Sands is, in MacKaye’s words, a “Community Masque” which was purposely written and performed to commemorate in America the Tercentenary of Shakespeare’s Death in 1916 (MacKaye, 1916: XV).5The Celebration of the Shakespeare Tercentenary6 in the USA is considered to have links with the debates concerning the nature of American national identity and democracy in the face of increasing immigration at the beginning of the 20th century. MacKaye explained that the “function of the Celebration” was “to help unite all classes and all beliefs in a great coöperative [sic] movement for civic expression through dramatic art” (MacKaye, 1916: XX). For the aims of his masque, MacKaye appropriated only partially Shakespeare’s play by deriving from it four characters – Prospero, Miranda, Ariel, and Caliban – whom he re-imagined in a symbolic re-casting of a plot and conflict according to his own conception (MacKaye, 1916: XX). MacKaye devised his dramatic work to celebrate not only Shakespeare but also the art of the theatre at a time when, across the Atlantic, Europe was experiencing the destruction and suffering caused by World War I. In his masque, he opposes the creative power and civilizing force of art – for which reason he conceived of Prospero’s art “as the art of Shakespeare in its universal scope” (MacKaye, 1916: XV) – to the destructive force of the war, which he identified as the primitive manifestation of Setebos. According to Mel Gordon, MacKaye’s conception was such that through Caliban “America would find a substitute for war” (Gordon, 1976: 94). MacKaye developed his plot around Caliban, “that passionate child-curious part of us all [whether as individuals or as races], grovelling close to his aboriginal origins [sic]” (MacKaye, 1916: XV)7, who seeks to learn the art of Prospero. The plot dealing with the education of Caliban “from a bestial creature to a potentially civilized individual” is “deliberately not fixed at any point in time” (Greene, 1989: 62). MacKaye did not treat Caliban as a comic character but envisaged him as “the protagonist of aspiring humanity, not simply its butt of shame and ridicule” (MacKaye, 1916: XVI). In other words, MacKaye viewed Caliban as part of humanity. Despite his “aboriginal origins”, Caliban is drawn, albeit hesitantly “toward the serener plane of pity and love, reason and disciplined will, where Miranda and Prospero commune with Ariel and his Spirits” (MacKaye, 1916: XV). MacKaye developed the four characters transplanted from Shakespeare in accordance with his theme – “the slow education of mankind through the influence of cooperative art, that is, the art of the theatre in its full social scope” – and adapted the masque to “democratic expression and dedicated [it] to public service” (MacKaye, 1916: XVI). Although MacKaye takes dramatic licenses with Shakespeare’s text, his “Masque aims to accord its theme with the art and spirit of Shakespeare” (MacKaye, 1916: XVI). Caliban by the Yellow Sands is not MacKaye’s first or last dramatic work pertaining to “the American pageant drama in general and the American masque in particular” (Brock and Welsh, 1972: 68). From a theoretical point of view, the American pageantry of the early twentieth century “combined artistry and social work in attempts to build community unity and identity” (Mehler, 2010: 11). MacKaye was writing within the tradition of an American dramatic form that “flourished during the reform era, especially from 1900 to 1920, and rapidly became an American institution” (Kahn, 2000: 262). Brock and Welsh consider MacKaye’s theoretical and practical contribution to this dramatic genre in relation to Ben Jonson’s work, stating that MacKaye viewed his masques as MacKaye’s masque, written at the invitation of the New York Shakespeare Tercentenary Celebration Committee, is a re-imagination/re-creation of Shakespeare’s The Tempest originating from Act I, Scene 2, more specifically from Prospero’s declaration to Ariel, “It was mine art that set thee free”. 5 The masque premiered on May 24, 1916, in the Lewisohn stadium of the City College of New York and ran for ten nights. A year later it was produced in Harvard Stadium in Cambridge. 6 For an article that sheds light on the Tercentenary’s contribution to the crucial political debates of its time, see Monika Smialkowska, “A democratic art at a democratic price”: The American Celebrations of the Shakespeare Tercentenary, 1916, Transatlantica [Online], 1 \| 2010, Online since 27 September 2010, connection on 01 October 2016. URL: http://transatlantica.revues.org/4787. 7 MacKaye, ‘Preface’, 2000: xv. 4 30 Messages, Sages and Ages, Vol. 7, No. 1, (2020) DOI: 10.5281/zenodo.4008636 dramatic works of art that both reflect artistic and literary purposes and are adapted to democratic ideals and society (Brock and Welsh, 1972: 68). This is to say there emerged a “concept that art was integral to, and a basic element of, the democratic process” (Prevots, 1990: 1). In fact, most proponents of the community drama saw it play an important role in broader progressive social and political agendas of the time. MacKaye was interested in the social/civic potential and role of the dramatic pageants as mass performances that can reach the people. In 1910, MacKaye stated his intent “to link public leisure with national ideals” and further argued that “pageants and masques could serve to celebrate the contributions of the many cultures that had arrived in America and at the same time unite these diverse components under the single ideal of liberty” (Mehler, 2010: 11). MacKaye’s views signal the importance of promoting a national American identity instead of a local one. His masques, as means through which democracy can speak of and for the people, would produce a “civic-inspiring art” (Potter, 1996: 71) as they “would furnish individuals of all classes and trades the opportunity to work in and create a communal and democratic art, while ‘uplifting’ the moral and social values of the community” (Gordon, 1976: 95). One of the aims of his masques, Caliban included, was to transform the American community by breaking down social and cultural barriers so as “to create a solidarity transcending conflicting class and ethnic interests” (Kahn, 2000: 263). In other words, MacKaye claimed that the masque, as a community drama, promoted a “celebration of cultural pluralism” providing “an environment for [...] several cultures to interact and intermix in a positive manner” (Mehler, 2010: 15). Thus, with its blend of pageantry, poetry and dance, Caliban was meant to involve New York’s several races, religions and economic layers on a large and elaborate scale (Kahn, 2000: 256). However, the concept of community that MacKaye attempts to establish through Caliban is, to some extent, fraught with a number of ideological issues that have to do with the ethnic or national composition of the audience and the dramatic content or structure of the masque itself. Potter, Cartelli, and Kahn offer readings of Caliban that examine how MacKaye tries to deal with broader social issues such as immigration and Americanisation, i.e. the forging of a social and cultural American identity. In particular, Caliban explores the social conflicts manifest in the early twentieth century American society, namely the problem of integrating the immigrants into the American society (cf. Potter, 1996: 71-79; Cartelli, 1999: 63-64; Kahn, 2000: 256-266). Cartelli notes that MacKaye’s themes: as they emerge from the published text of the masque […], articulate his responsiveness to the anxieties of others of his class and caste regarding how best to “Americanize” the newly arrived masses of immigrants and introduce them to the standards and obligations of Anglo-Saxon culture (Cartelli, 1999: 63). The “Americanization” of the immigrants implied the creation of an American national consciousness at cultural and social levels, and MacKaye uses Shakespeare and his work to educate the immigrants in the spirit and tradition of Anglo-American culture which may have relied on a concept of an Anglo-American Protestant core. Cartelli comments that MacKaye uses the living heritage of Shakespeare to sustain and support the native American drama because he envisioned that Shakespeare’s work, with its deep roots into the English tradition and language, can be a “formative source or power” for an emerging American drama (Cartelli, 1999: 64-65). Kahn observes that MacKaye’s attempt to make Shakespeare “the poet who speaks to all Americans” presents a “cultural dissonance” because Shakespeare was pre-eminently England’s literary asset and icon that, at this “cultural moment”, becomes the switch point between two separate and distinct countries – England and America – sharing a certain cultural and linguistic legacy (Kahn, 2000: 258). As Kahn further argues, the “cultural dissonance” involved the question of “what transformed an immigrant into an American” – a dual question that raised philosophical and social issues dating back to the beginnings of the American republic (Kahn, 2000: 258). The ideological ambivalence of the question of Americanness lies in the opposition between the “set of universal principles” that the founding fathers envisioned as “the basis of nationhood and citizenship” and a later theory of an American identity based upon a sense of “a certain ethnic pride” in which a belief in the superiority of the Anglo-Saxon culture was a predominant element (Kahn, 2000: 258-262; Vaughan and Vaughan, 1991: 114). The 31 Messages, Sages and Ages, Vol. 7, No. 1, (2020) DOI: 10.5281/zenodo.4008636 opposition becomes obvious as long as the immigrants from a non-Anglo-Saxon background were expected to learn English and adopt the American culture as their own. In the light of these points of view, MacKaye’s declared aim of fostering a community feeling through Caliban may come across as problematic because he exposes the non-English speaking or non-Anglo-American cultural background segment of his (New York) audience to a staging of the English-speaking tradition represented by Shakespeare. MacKaye’s artistic choice most likely meant to bring Shakespeare to a mass audience beyond the artistic traditions and conventions of the commercial theatre. Actually, MacKaye was not fond of the commercial theatre tradition and looked favourably on contemporary texts that follow the “civic ideal of Greek tradition,” in which theatre is “the chief force of civilization and religion” (MacKaye, 1911: 122-128). The structure and the text of the play reveal that MacKaye makes: “Shakespeare encompass the historical diversity of theatrical art through the ages” (Kahn, 2000: 266). In other words, Shakespeare is both English and universal – a comprehensive dramatist for all seasons and cultural tastes. However, Caliban is likely to have posed problems of understanding even for spectators/readers with some (English) literary background. The action of the masque, whose eminently performative and visual aspects MacKaye stressed in his “Preface” to the text, takes place in three acts structured on three symbolic planes: in the cave of Setebos, in the mind of Prospero, and on the ground circle of “the Yellow Sands” (MacKaye 1916: XXIX). The acts, each with its own theme, are introduced by mime interludes reminiscent of three periods of theatre: Antiquity, Middle Ages and Elizabethan England.8 The acts are interwoven with nine inner scenes – eight of them from a number of Shakespeare’s plays – and a Morality play. The characters of the masque proper – the speaking persons – include Ariel, Sycorax, Caliban, Prospero, Miranda, Lust, Death, War, Caligula (impersonated by Lust), One in Gray (impersonated by Death), and Another One in Gray (impersonated by Caliban) (MacKaye, 1916: XXXI).9 It is worth noting that Michael Peter Mehler makes a useful comment regarding the spatiality of the masque. Drawing on theories of space (Soja, 1989: 120-122; Lefebvre, 1991: 14-38), Mehler notes that Caliban displays “separate spaces that reflected oppositional cultures” and contrasts “civilized protagonists with savage antagonists that [...] [are] conquered by force or benevolent reform” and aspire to the privileged space which the protagonist controls (Mehler, 2010: 25). The plot follows Caliban as the figure of Everyman tempted by Lust, Death and War but eventually saved by Prospero, Ariel and Miranda. It is an example of a journey from savagery to civilisation in which the enlightened characters manage to remain above the savage ones. Charles H. Shattuck rightly comments that MacKaye’s rather unsophisticated transposition of the Shakespearean characters actually stripped them “of their Shakespearean habiliments – their poetry and their magic – and reduc[ed] them to counters in a banal allegory of Good mastering Evil” (Shattuck, 1987: 307). The masque begins some time prior to the action of The Tempest when Caliban is still king of the island. In the “Prologue”, Ariel, fettered in the tiger-jaws of the idol Setebos, is the prisoner of Sycorax. Caliban fancies himself a god because he interprets his gesture of picking up an eelworm out of the mud as a creative action. Teased by Caliban for his imprisonment, Ariel describes to Caliban his vision of the coming of Prospero10, “one from the heart of the world”, and Miranda – a maid, “a child, all wonder” that comes before him: He bears A star-wrought staff and hooded cloak of blue, And on his right hand bursts the sun, and on 8 The first interlude, Antiquity, consists of an Egyptian symbolic ritual worshipping the god Osiris, the second chorus of Sophocles’s Antigone, and a Roman farcical comedy. The second interlude consists of episodes of the dramatic art of Europe in the Middle Ages as follows: pantomime of Doctor Faust, the French entitled The Field of the Cloth of Gold, and finally a fusion of Italian and Spanish groups performing Commedia dell’ Arte. The third interlude is folk festivals of Elizabethan England. 9 MacKaye, “Preface”, 2000: xxxi. MacKaye divided his Dramatis Personae into four groups according to the moments of the work: the masque proper, the scenes from Shakespeare’s plays, the interludes, and the epilogue. 10 The description of Prospero’s coming echoes the coming of Jesus Christ in the New Testament: Prospero is a Messiah-like figure who comes to set Ariel free from the dark power of Setebos, whilst Ariel resembles the figure of John the Baptist. 32 Messages, Sages and Ages, Vol. 7, No. 1, (2020) DOI: 10.5281/zenodo.4008636 His left, the moon; and these he makes his masks Of joy and sorrow. (MacKaye, 1916: 9) Caliban tries to persuade Ariel to join Lust, Death and War, the priests of Setebos, in praising the halftiger and half-toad idol of his father. Whereas Shakespeare’s Setebos is an unseen god that Sycorax worshipped, MacKaye’s Setebos is Caliban’s father with a physical onstage presence – “the evocation of all that is barbarian and inimical to western civilization” (Kahn, 2000: 266). The three priests perform rites of worship in the face of which Ariel feels powerless – “Death has closed/ My sight in darkness” (MacKaye, 1916: 13) – and prays for deliverance. At this point Miranda enters the cave and comforts Ariel by saying that Prospero, yet invisible, commands the isle with his great art. Caliban starts sniffing and peering at Miranda to whom he brags, in a language reminiscent of Robert Browning’s Caliban upon Setebos, that he is god of the isle and master of Ariel: Am seed of Setebos: Am Caliban: the world is all mine isle: Kill what I please, and play with what I please. (MacKaye, 1916: 16) Captivated by Miranda, Caliban desires her but his gross manner of courtship as well as his increasingly overt sexual attraction to her give her serious reasons to fear for her safety, more so when Sycorax incites Caliban to mate with Miranda at the altar of Setebos. Kahn points out that the dramatic action of the masque overtly emphasizes “Caliban’s primitive, unbridled sexuality, partly through the character and partly through an allegorical overplot involving Setebos” (Kahn, 2000: 267). Miranda’s honour is yet again conveniently saved as Prospero enters the scene on “a glowing, winged throne”, with a scroll in one hand and “a miraculous staff” (MacKaye, 1916: 23) in the other, and brings calmness and light to the cave.11 Although Sycorax lies dead by the altar, Prospero knows that in the struggle to rule the world his art is matched against the will of Setebos and, should he fail to prevail over Setebos, Miranda will not be safe. To achieve his end Prospero enlists Ariel’s help. As soon as Prospero frees Ariel from the tiger-jaws of the idol, he charges the spirit with the education of Caliban because Miranda’s freedom and honour depend on taming the beast: Prospero. Never Till this immortal Caliban shall rise To Lordly reason, can Miranda hold Her maiden gladness undismayed. (MacKaye, 1916: 26) William Green comments that “the allegorical nature of the masque” rests on its portrayal of “the struggles between the powers of Darkness represented by Setebos, Sycorax, and Caliban and those of Light, represented by Prospero, Miranda, and Ariel” (Green, 1989: 63). By the power of his art Prospero wants to turn the cave of Setebos into a temple dedicated to Miranda and a theatre for his own art. At the close of the “Prologue” Prospero invites Ariel onto the Yellow Sands – Prospero’s magic isles, the world itself (MacKaye, 1916: XVI) – to behold a pageant of his art in a succession of dramatic episodes beginning with the Antiquity. Prospero’s art is “a world/ Snatched from the womb of History, to survive/ Its mortal mother in imagination” (MacKaye, 1916: 31). Meanwhile, the priests of Setebos, Lust, Death, and War, who are reminiscent of Antonio and Sebastian, conspire with Caliban against Prospero and Ariel. The themes of the three acts are, in order, lust, death, and war. In each act Caliban watches scenes from a number of Shakespearean plays conjured by Prospero and Ariel. The scenes complement and exemplify the themes, influencing Caliban’s thoughts and actions. He is easily tempted and therefore shifts his allegiance as apprentice between Prospero and Setebos and their corresponding arts. This is to say that Caliban yields to his narrow-minded, egocentric desires “losing his way along the path to enlightenment set out for him by Miranda, Ariel, and Prospero” (Mehler, Cartelli, 68, notes that “Prospero’s grand entrance onstage, in a style that doubly echoes Christ’s harrowing of hell and Moses’s destruction of the idols at the base of Mt. Sinai, is preceded by that of Miranda, who advances as an overdetermined embodiment of grace, innocence, and beauty”. 11 33 Messages, Sages and Ages, Vol. 7, No. 1, (2020) DOI: 10.5281/zenodo.4008636 2010: 116). In each act Caliban fails to understand and learn the meaning and value of Prospero’s art – which can set Caliban free from Setebos – and every time he manages to get possession of Prospero’s magical staff for his own use disorder follows, consequently affecting Miranda’s safety as Prospero predicted in the “Prologue”. At various stages in the play, Caliban shows his inability to control power, or his misuse of it, with disastrous effects. His repeated falling back into his savagery and his slow progress toward a civilised condition portray a symbolic contest between enlightenment and savagery. In act one, at the end of the scene from Julius Caesar, Caliban grabs the magical staff and shouts aloud “Awake, Romans, awake!”, echoing Brutus in Shakespeare’s play. Next, he cries that his art “shaketh the throne of Prospero”, invokes Setebos, and calls upon the Roman emperor Caligula, the scene turning into mingled riot and orgy – “a sordid saturnalia, from the midst of which the masked form of Caligula rises dominant in splendor” (MacKaye, 1916: 68-69). A colossal burning cross appears from darkness before Caligula manages to place a crown upon the head of Miranda whose imminent rape by Caligula is prevented by the symbolic intervention of Christ. Kahn comments on a possible correspondent between “[t]his configuration of rape forestalled by the power of Christ” and D. W. Griffith’s landmark silent film The Birth of A Nation released in 1915: The pathbreaking film “builds to its sustained climax from two attempted rapes of white women by black men,” assaults that produce the famous “rides to the rescue” by the Ku Klux Klan. These rides anticipate the spectacular rescues of Miranda, first by Prospero and then symbolically by the intervention of a Christian God. (2000: 267) The following inner stage shows Saint Agnes holding a white lamb and then a shepherd – Prospero himself – orders Caligula, in fact Lust, to remove his mask and return to Setebos. The symbolism of this scene, with its allegorical Christian triumph, speaks for itself. It is played out in the contrast between lust and purity impersonated by historical or mythical characters that stand for, on the one hand, different types of lust, i.e., sexual lust and lust for power; and, on the other hand, purity and self-control. The antagonism of the act culminates with the Roman figures of Caligula and, respectively, Saint Agnes. Caligula’s appearance as Lust is not random as the Roman Emperor, known for his extravagance, eccentricity, depravity and cruelty, is remembered as a despot and was rumoured to have engaged in incestuous relationships with his sisters and to have set up a brothel at the palace.12 On the other hand, St Agnes, the patron saint of young girls, chastity and rape survivors, was a member of the Roman nobility, raised in a Christian family, who suffered martyrdom at the age of thirteen on January 21, 304, because she refused to marry the prefect Sempronius’ son.13 The contrast between lust and purity is also a contrast between two historical/chronological stances of the Roman Empire, the pagan and the Christian. Such scenes allow interpretations that take into consideration the audience’s response and their civic spirit and collective memory. For Cartelli, “MacKaye clearly expects his audience to construe Caliban’s overstimulated imagination as primitive and pornographic” (Cartelli, 1999: 69); while Potter attaches to the scene troubling concerns of unresolved social and political issues reading this scene of mingled riot and orgy as “a horrifying American image for this period of unpunished lynchers” (Potter, 1996: 76). Mehler considers that Caliban’s thinking and behaviour carry “long-established racial and ethnic prejudice” and such “stereotypical images of cultural others” may have attracted more the audience’s focus than MacKaye’s intended idealist principle and positivist message, namely social education and human development (Mehler, 2010: 116-117). In act two, Death tries to persuade Caliban to restore the temple of Setebos, tempting Caliban with a “gray” [sic] cloak that will assist him in snaring Miranda into bondage. However, when Death is ready to place the cloak upon Caliban, the latter recoils from the freezing touch of Death and cries aloud: “Prospero! I will serve thee” (MacKaye, 1916: 82). Prospero responds to Caliban’s cry and, to encourage him, produces the ghost scene from Hamlet, at the end of which Caliban forgetfully 12 See http://www.history.com/topics/ancient-history/caligula, accessed 9 http://ancientrome.wikia.com/wiki/Caligula, accessed 29 May 2006. 13 See http://www.catholic.org/saints/saint.php?saint_id=106, accessed 29 http://www.passionistnuns.org/Saints/StAgnes/index.htm, accessed 29 May 2006. 34 May 2005, and May 2006, and Messages, Sages and Ages, Vol. 7, No. 1, (2020) DOI: 10.5281/zenodo.4008636 follows Death after asking Prospero for the wonder scroll to bear it against Death and free his father’s spirit. Eventually, Death prevails and, after taking Prospero’s scroll from Caliban, asks his followers to take Miranda to Setebos. In act three a remorseful Caliban appeals to Miranda to release him from Death’s power saying he is ready to forgo Death’s gray cloak. Moved by Caliban’s repentant attitude, Miranda appeals to a reluctant Prospero on Caliban’s behalf. Ariel conjures a “dream of fairy laughter” with scenes from Henry IV and Merry Wives of Windsor. Caliban feels that Prospero mocks him since the former saw similarities between him and Falstaff, but in fact Caliban was supposed to recognize the impropriety of his lustful desire for Miranda. Consequently, he becomes enraged with the last pageant and is incited by War to possess Miranda who comes in to show Caliban another vision – Henry V’s speech to his soldiers by the walls of Harfleur – so that he may learn the meaning of honour and thus “[may] recognize an image of yourself/ And so recoil to reason and to love” (MacKaye, 1916: 136). As soon as the scene ends, Caliban snatches from Ariel the hood of Prospero and shouts: “Ho, God for Caliban and Setebos!/War, War for Prospero’s throne! Miranda’s shrine” (MacKaye, 1916: 139). The masque nears its climax when Prospero, whose unhooded features reveal likeness to Shakespeare’s, makes his way to the throne where Caliban confronts him with a martial attitude proclaiming the fall of Prospero’s art. In the clash between the priests and powers of Setebos and the spirits of Ariel, War takes Miranda, Prospero and Ariel captive. Caliban, wearing Prospero’s hood and raising his staff, exults because he now considers himself the master-artist who wields the world. However, Prospero cautions Caliban that although he possesses the magic hood and staff he does not possess the will for creation – “Thy will and War/ May break, but cannot build the world” – because only Prospero/Shakespeare’s art immortal “builds the beauty of the world” (MacKaye, 1916:141). In the “Epilogue”, the Spirit of Time announces the supremacy and endurance of art in the face of destructive forces: “So, out of War up looms unconquered Art” (MacKaye, 1916: 143). In the pageant of the great theatres of the world that follows, “the modest figure of Shakespeare, at first unemphasized,” emerges from the group of the Elizabethan dramatists, approaches Prospero and the two figures exchange places (MacKaye, 1916: 145). Coming out of the darkness, Caliban approaches Shakespeare, accompanied by Ariel and Miranda, and pleads for more visions as he admits to his inability to create and yearns to be an artist of Shakespeare’s art. Caliban is now not only reconciled with Prospero/Shakespeare, but also submits to the power of his craft. Miranda seconds Caliban’s plea to which the figure of Shakespeare replies with Prospero’s “Our revels are now ended” speech. MacKaye’s “Preface” is self-explanatory about his intentions in producing this masque which is both a pro-art and an anti-war manifesto. Caliban by the Yellow Sands emerges from a rather reductive reading of The Tempest that MacKaye envisioned as “a master narrative concerned with the central role art and the artist play in the eternal struggle of mind and matter, spirit and body, civility and savagism” (Cartelli, 1999: 68). Art is the medium through which humankind can be educated and the means to bring races or individuals together in community and fellowship. Humankind can share in the common inheritance and tradition of art that comes down to the present from the past. The raging Great War in 1916 Europe may be one of the reasons why the masque considers the cultural tradition and legacy of Antiquity and Western Europe. However, as far as MacKaye’s aim to foster a feeling of community goes, Kahn and Cartelli maintain that this was not properly achieved in at least two ways. On the one hand, African-Americans did not support or participate in the masque of Caliban (cf. Potter, 1996: 74; Cartelli, 1999: 73-74; Kahn, 2000: 269) and, on the other hand, MacKaye produces Shakespearean drama as the culmination of “the historical diversity of theatrical art through the ages” for a culturally and ethnically diverse audience living in an Anglo-Saxon America that, at that historical moment, required them “to adopt English tradition as the foundation of American culture” (Kahn, 2000: 271). Cartelli similarly points out the “contradictions between the democratic claims advanced on behalf of the masque and the largely anti-democratic bias of its themes and organization”, and adds that MacKaye’s masque “eschews specific social and political references, apart from those made to the Great War in the preface which are reprised in the triumph of Setebos at the end of Act III” (Cartelli, 1999: 72-74). Depicted as Everyman, MacKaye’s Caliban does not dismiss, let alone curse, Prospero’s art in the way that Shakespeare’s Caliban rejects Prospero’s language. It is true, though, that in both plays Prospero educates Caliban more for his and Miranda’s benefit rather than for the benefit of Caliban himself. Kahn maintains that in MacKaye’s masque the real point of the education of Caliban “isn’t so much to assure Miranda’s purity as to interpellate 35 Messages, Sages and Ages, Vol. 7, No. 1, (2020) DOI: 10.5281/zenodo.4008636 Caliban, the cultural – and racial – Other, into Anglo-American culture. That is accomplished by making him a spectator of Shakespeare rather than the playwright’s rival” (Kahn, 2000: 268). Whereas Shakespeare’s Caliban perceives Prospero’s language as a burden and a means to his own enslavement, MacKaye’s Caliban is fascinated by Prospero’s art that he wants to replicate, but he lacks the desire or will for real creation. Caliban’s rather childish fascination with Prospero’s art and his destructive impulses once he seizes the instruments to produce art, distinguish him from Herbert Beerbohm Tree’s sensitive and possibly noble Caliban. If MacKaye’s Caliban succeeds, albeit temporarily, in seizing Prospero’s magic staff in order to establish himself as a creator of art, Shakespeare’s Caliban unsuccessfully plans and leads an insurgency against Prospero’s life through which he seeks to rid himself of his master. In both cases, however, Caliban fails to practically overthrow or overcome Prospero, let alone master his art. Mehler invites one to view Caliban by the Yellow Sands as “a national pageant with the universal theme of human development”, which is unique among the American pageant movement owing to its allegorical framework that brings together community formation and national unity. He also draws one’s attention to reading the masque as a cultural document only in relation “to early twentieth-century American understanding of race and culture” (Mehler, 2010: 118). In the twentieth century rewrites of The Tempest, the figure of Caliban is appropriated for socio-political and cultural causes. Thus, his identity acquires polyvalent traits through the interconnections achieved by rewriting. He is less of a beast-like, undefined being with primitive appearance, thinking and conduct and more of an indigenous native with distinct human(e) traits who climbs the evolutionary scale to self-determination and emancipation. Caliban symbolizes those who feel or are usurped, dislocated and enslaved by, for instance, imperialism and colonization in particular or other inappropriate ideological practices at large. Thus, the Prospero-Caliban relationship has been turned into an archetype of anti-colonialism in a number of geographical and cultural contexts. With Caliban’s rise to eminence, he becomes more reflective and mindfully assertive on a range of levels – personal, linguistic, cultural, ethnical, educational, social and political – and serves to raise awareness against inhumane and harmful ideologies, policies and practices. In Caliban by the Yellow Sands, Prospero endeavours to teach Caliban his art, but Caliban fails to understand or to “conjure” art and thus falls short of becoming a counterpart rival creator to Prospero. Caliban’s desire to learn means that he yields to Prospero/Shakespeare – learning is a process of submission to the master. In the masque, the pupil manages to overcome the master but only by seizing the magical staff, not by the power of his artistic craft. The act of creation can be liberating and yet frustrating in the sense it requires the repressions of one’s primitive impulses. In the “Prologue” and “Epilogue” – the frames of the masque – the cultural and political authority of Prospero/Shakespeare contain Caliban’s cultural or political virtual threat. Outside MacKaye’s masque, Caliban is tamed or rather forced into submission because he lacks the ability and will to create; in Shakespeare he lacks the knowledge. MacKaye’s Caliban is victorious within the fictive world of the work, but is eventually restrained outside the fictive plot. The practice of rewriting establishes a dialogic relationship between a master text – quite often a culturally known text – and its ensuing transformations. The interconnections that arise out of alternative, active (re-)readings of the original work lead to the development of new meanings and provide new insights. The rewrites emerge and exist as distinct creations that stand on their own in relation to the text they draw on as they bring to the foreground alternative readings and interpretations. This type of engagement, which takes place at different levels and is triggered by geographical, cultural, social and ideological perspectives that aim to challenge the already established viewpoints and meanings, involves refashioning the original plot, characters, symbolism, motifs, ideas and themes, facilitating intercultural relationships between texts. Caliban by the Yellow Sands falls within the category of rewrites “intended for a particular historical moment and specific socio-political purposes” and “not only does it comment on the contemporary situation, but also intervenes in it, proposing solutions to current problems” (Śmiałkowska, 2007: 17). It is possible to sense an ideological ambivalence in the way MacKaye, on the one hand, appropriated The Tempest, envisaged and staged Caliban by the Yellow Sands and, on the other hand, the context in which his masque was used, for what purpose and what meanings one could draw out of it. The ambivalence may arise from “the play’s representations of history and its uses of Shakespeare and the Shakespearean canon” (Śmiałkowska, 2007: 18), the temporal and spatial 36 Messages, Sages and Ages, Vol. 7, No. 1, (2020) DOI: 10.5281/zenodo.4008636 dimensions of the play, the promotion of an allegedly better culture and civilisation – typified by Shakespeare – in order to educate and integrate the ignorant or immigrant masses and the alternative representations of Shakespeare – a significant cultural icon – as prominent creator and surprisingly weak figure. Works cited Brock, H. D. and Welsh, J. M. 1972. ‘Percy MacKaye: Community Drama and the Masque Tradition’. Comparative Drama, Vol. VI, No. 1. Cartelli, Th. 1999. Repositioning Shakespeare: National Formations, Postcolonial Appropriations. London and New York: Routledge. Cohn, R. 1976. Modern Shakespeare Offshoots. Princeton University Press. Gordon, Mel. 1976. ‘Percy Mackaye’s Masque of “Caliban” (1916)’. The Drama Review, Vol. 20, No. 2. Green, W. 1989. ‘Caliban by the Yellow Sands: Percy MacKaye’s Adaptation of The Tempest’. Maske und Kothurn, 35: 1. Kahn, C. 2000. ‘Caliban at the Stadium: Shakespeare and the Making of Americans’. The Massachusetts Review, 41: 2. Lefebvre, H. 1974. The Production of Space. Translation by Smith, Donald Nicholson. 1991. Oxford: Blackwell. MacKaye, P. 1911. ‘The Development of the Humanities in the Modern Poetic Drama’. In Herts, Alice Minnie. The Children’s Educational Theatre. New York: Harper & Brothers Publishers. MacKaye, P. 1916. Caliban by the Yellow Sands. New York: Doubleday, Page & Company. Mehler, M. P. 2010. Percy MacKaye: Spatial Formations of a National Character. (PhD dissertation submitted to the Graduate Faculty of The School of Arts and Sciences). University of Pittsburgh. http://d-scholarship.pitt.edu/6567/1/MehlerM_etd2010.pdf Potter, V. R. 1996. ‘Percy MacKaye’s Caliban for a Democracy’. Journal of American Culture, 19:4. 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https://openalex.org/W4396625748	https://link.springer.com/content/pdf/10.1007/s00247-024-05939-z.pdf	English	null	Photon-counting computed tomography for paediatric congenital heart defects yields images of high diagnostic quality with low radiation doses at both 70 kV and 90 kV	Pediatric radiology	2,024	cc-by	7,653	Pediatric Radiology (2024) 54:1187–1196 https://doi.org/10.1007/s00247-024-05939-z Pediatric Radiology (2024) 54:1187–1196 https://doi.org/10.1007/s00247-024-05939-z ORIGINAL ARTICLE ORIGINAL ARTICLE Fredrik Stålhammar1,2 · Marie‑Louise Aurumskjöld3,4 · Sofie Meyer1,2 · Marie Wiklund1,2 · Pär Wingren1,2 · Petru Liuba5,6 · Erik Hedström1,2,7,8 Received: 30 December 2023 / Revised: 23 April 2024 / Accepted: 24 April 2024 / Published online: 3 May 2024 © The Author(s) 2024 * Erik Hedström erik.hedstrom@med.lu.se Abstract Background Photon-counting computed tomography (PCCT) is a new clinical method that may show better diagnostic quality at lower radiation doses than conventional CT. Objective To investigate the diagnostic quality and radiation dose of paediatric cardiovascular PCCT for diagnosis of con- genital heart defects at 70 kV and 90 kV. Materials and methods This retrospective assessment included clinical non-gated paediatric PCCT examinations for assess- ment of congenital heart defects. Radiation doses were recorded, and overall and specific diagnostic quality (1–4) were scored by four paediatric radiologists. Agreement, differences, and trends were assessed by percent rater agreement, intraclass cor- relation, Mann–Whitney tests, and Jonckheere-Terpstra tests. Results Seventy children with congenital heart defects were examined at 70 kV (n = 35; age 2 days–16 years; 63% boys) or 90 kV (n = 35; age 2 days–17 years; 51% boys). All observers gave a median score of 4 (high diagnostic quality) for both 70 kV and 90 kV, with no difference in median values between tube voltages (all P > 0.06). Agreement for overall scores was 66–94% for 70 kV and 60–77% for 90 kV. Agreement for specific scores was 80–97% for 70 kV and 83–89% for 90 kV. Size-dependent dose estimate was 0.68 mGy (0.25–2.02 mGy) for 70 kV and 1.10 mGy (0.58–2.71 mGy; P < 0.001) for 90 kV. Effective dose was 0.30 mSv (0.15–0.82 mSv) for 70 kV and 0.39 mSv (0.22–1.51 mSv; P = 0.01) for 90 kV. Conclusion Paediatric cardiovascular PCCT yields images for congenital heart defects of high diagnostic quality with low radiation dose at both 70 kV and 90 kV. Results Seventy children with congenital heart defects were examined at 70 kV (n = 35; age 2 days–16 years; 63% boys) or 90 kV (n = 35; age 2 days–17 years; 51% boys). All observers gave a median score of 4 (high diagnostic quality) for both 70 kV and 90 kV, with no difference in median values between tube voltages (all P > 0.06). Agreement for overall scores was 66–94% for 70 kV and 60–77% for 90 kV. Agreement for specific scores was 80–97% for 70 kV and 83–89% for 90 kV. Size-dependent dose estimate was 0.68 mGy (0.25–2.02 mGy) for 70 kV and 1.10 mGy (0.58–2.71 mGy; P < 0.001) for 90 kV. Effective dose was 0.30 mSv (0.15–0.82 mSv) for 70 kV and 0.39 mSv (0.22–1.51 mSv; P = 0.01) for 90 kV. Photon‑counting computed tomography for paediatric congenital heart defects yields images of high diagnostic quality with low radiation doses at both 70 kV and 90 kV Fredrik Stålhammar1,2 · Marie‑Louise Aurumskjöld3,4 · Sofie Meyer1,2 · Marie Wiklund1,2 · Pär Wingren1,2 · Petru Liuba5,6 · Erik Hedström1,2,7,8 Material and methods The regional ethics committee approved this retrospective study using pseudonymised data from patients referred for a clinical examination, waiving the need for individual con- sent. Paediatric non-gated cardiovascular PCCT (Naeotom Alpha, Siemens Healthineers, Erlangen, Germany) exami- nations with intravenous contrast agent completed between 30th September 2021 and 1st March 2023 in children with suspected or confirmed congenital heart defects were eligible. Initially, only 90 kV protocols were available as this was an early installation. After 70 kV was made available by the ven- dor, all clinical examinations were performed at 70 kV, which also limited the number of 90 kV examinations in the current study. The aim was to include the same number of examina- tions using 70 kV as had been completed using 90 kV. Photon-counting CT (PCCT) was recently made avail- able for clinical use. It yields images with negligible electronic noise and improved contrast between soft tissue and iodinated contrast agents [6]. However, this means that the images look different from conventional CT images, further dependent on the chosen reconstruc- tion algorithm. This new CT method may potentially impact diagnostic accuracy because the observer’s expe- rience is different. Abstract Conclusion Paediatric cardiovascular PCCT yields images for congenital heart defects of high diagnostic quality with low radiation dose at both 70 kV and 90 kV f Conclusion Paediatric cardiovascular PCCT yields images for congenital heart defects of high diagnostic quality with low radiation dose at both 70 kV and 90 kV. Keywords Diagnosis · Heart defects, congenital · Paediatrics · Photon-counting computed tomography · Radiation dosage * Erik Hedström erik.hedstrom@med.lu.se 5 Paediatric Cardiology, Department of Clinical Sciences Lund, Lund University, Lund, Sweden 6 Department of Paediatric Cardiology, Skåne University Hospital, Lund, Sweden 7 Clinical Physiology, Department of Clinical Sciences Lund, Lund University, Lund, Sweden 8 Department of Clinical Physiology, Skåne University Hospital, Lund, Sweden 5 Paediatric Cardiology, Department of Clinical Sciences Lund, Lund University, Lund, Sweden 6 Department of Paediatric Cardiology, Skåne University Hospital, Lund, Sweden 7 Clinical Physiology, Department of Clinical Sciences Lund, Lund University, Lund, Sweden 8 Department of Clinical Physiology, Skåne University Hospital, Lund, Sweden 5 Paediatric Cardiology, Department of Clinical Sciences Lund, Lund University, Lund, Sweden 1 Diagnostic Radiology, Department of Clinical Sciences Lund, Lund University, Lund, Sweden 6 Department of Paediatric Cardiology, Skåne University Hospital, Lund, Sweden 2 Department of Radiology, Skåne University Hospital, S‑22185 Lund, Sweden 7 Clinical Physiology, Department of Clinical Sciences Lund, Lund University, Lund, Sweden 3 Medical Radiation Physics, Department of Clinical Sciences Malmö, Lund University, Lund, Sweden 8 Department of Clinical Physiology, Skåne University Hospital, Lund, Sweden 4 Radiation Physics, Department of Haematology, Oncology and Radiation Physics, Skåne University Hospital, Lund, Sweden :(0123 3456789) 1188 Pediatric Radiology (2024) 54:1187–1196 be investigated to what extent radiation dose is reduced when moving from 90 kV to 70 kV using PCCT. Introduction The aim of this study was therefore to compare the diag- nostic quality and radiation dose in paediatric non-gated car- diovascular PCCT for diagnosis of congenital heart defects at 70 kV and 90 kV. Computed tomography (CT) is used in paediatric patients with congenital heart defects for diagnosis, preoperative planning, and postoperative evaluation [1–3]. CT is widely available and fast. It can often be used without sedation or with feed-and-sleep meth- ods, with the benefit of avoiding general anaesthe- sia. It also has high spatial resolution and enables simultaneous assessment of extracardiac structures such as the pulmonary parenchyma, pleura, and skel- eton. The relative disadvantage is that the patient is exposed to radiation and receives an iodine contrast agent with potential risk for renal function impairment (although this risk is low when using iso-osmolar con- trast agents) [4, 5]. Table 1 Photon-counting computed tomography parameters Photon‑counting computed tomography examination and settings PCCT provides higher spatial resolution than conven- tional CT, as well as intrinsic spectral information in every scan [7]. Thus, image quality may be improved with a lower radiation dose. A study on small children that used PCCT at 90 kV for diagnosis of congenital heart defects achieved higher image quality than conventional CT but yet reported a similar effective dose (Eeff) [8]. Neonates and infants were positioned in a vacuum pillow and imaged during free breathing. Small children between 1 year and 3 years were sedated with Propofol (Sandoz AS, Novartis, Stockholm, Sweden) according to clinical routine, and imaged during free breathing. Children above 3 years were generally not sedated, and images were acquired during breathhold when possible. ff It is unknown if the essentially noise-free PCCT images impact observers impression of diagnostic quality, and it is unknown which PCCT radiation dose is adequate for children with congenital heart defects. It also remains to Tube voltage was 70 kV or 90 kV. Table 1 shows PCCT parameters. Different monoenergetic levels were tested Bv body-vascular, CARE combined applications to reduce exposure, D dimensional Tube voltage 70 kV 90 kV Automated tube current modulation CARE dose 4-D CARE dose 4-D Monoenergetic level (keV) 55 55 Rotation time (s) 0.25 0.25 Slice thickness (mm) 0.6 0.6 Increment (mm) 0.4 0.4 Kernel Bv44 Bv40, Bv44 Quantum iterative reconstruction level 4 2 (57%), 3 (17%), 4 (26%) Acquisition 144 × 0.4 144 × 0.4 Pitch 3.2 3.2 Field of view (mm) 300 300 Image quality level 70 70 Table 1 Photon-counting computed tomography parameters 1189 Pediatric Radiology (2024) 54:1187–1196 before the current study as part of clinical optimisation, and 55 keV was considered the optimal monoenergetic level for visualisation of the cardiac chambers and the thoracic ves- sels. Therefore, the current study used 55 keV throughout.f before the current study as part of clinical optimisation, and 55 keV was considered the optimal monoenergetic level for visualisation of the cardiac chambers and the thoracic ves- sels. Therefore, the current study used 55 keV throughout.f cases were randomised, although perceived image differ- ences related to the essentially noise-free images at 70 kV could not be overcome. Four blinded paediatric cardiobas- cular radiologists (F.S., P.W., M.W., and S.M. Results Figure 1 shows the inclusion and exclusion flow chart. Table 2 shows patient characteristics including distribu- tion of cardiovascular malformations. Although there were differences in the distribution of cardiovascular defects between the 70 kV and 90 kV groups, there was no difference between groups for age (P > 0.99), height (P = 0.86), or weight (P = 0.83). However, scan length differed (P = 0.002) between 70 kV and 90 kV groups, and therefore comparisons were made between radiation doses for 70 kV and 90 kV both using all available data Radiation exposure and contrast agent administration Statistical analyses were performed in Prism 9.5.1 (Graph- Pad Software, San Diego, CA) and in R [10, 11]. Values are reported as median (range) or median [interquartile range; IQR]. Due to the low score variability, standard interrater reli- ability measures for ordinal data are misleading. As observers were aligned in scoring before the study, chance is not the main driver for agreement. Therefore, percent rater agreement is presented. However, for comparison with other studies, intraclass correlation is also reported, assessed using a two- way, consistency, average-measures model. Mann–Whitney’s and Jonckheere-Terpstra’s tests were applied to test for score differences and for trends between Eeff and scores, respectively [12]. Differences between groups for 70 kV and 90 kV were assessed, and radiation dose differences were also assessed after correction for confounders. P < 0.05 was considered to show statistically significant differences. The radiation dose parameters CT dose index (CTDIvol), dose-length product (DLP), and size-specific dose estimate (SSDE) were extracted from the PCCT system, and Eeff was calculated based on DLP using age-dependent conversion factors [9]. Iodixanol 270 mg I/ml (GE Healthcare, Stockholm, Swe- den) was administered using a MEDRAD Centargo injector (Bayer Pharmaceuticals, Leverkusen, Germany) with 15 s bolus length. For bodyweight ≤ 10 kg, a lower extremity peripheral vein was used for contrast agent administration, and for bodyweight > 10 kg an upper extremity peripheral vein was used. Preloading of contrast agent was applied in patients with bodyweight ≤ 10 kg for the programmed injection of contrast agent to be delivered directly without intermediate saline. For 70 kV, an optimal dilute concentration of 189 mg I/ml was determined as part of clinical optimisation, with constant injected volume and rate. For 90 kV, the clinical routine dose of 270 mg I/ml was used. Photon‑counting computed tomography examination and settings with 30, 28, 24, and 20 years’ experience, respectively) independently assessed the PCCT examinations with regard to overall and specific diagnostic quality.i During the initial phase of clinical acquisitions, different reconstruction algorithms were tested to find the optimal quantum iterative reconstruction (QIR) level as this was the first release of the system. Furthermore, reconstruction algo- rithms also changed with software upgrades. This explains why different QIR levels are present for 90 kV in the current study (Table 1), whereas 70 kV protocols applied the same QIR level throughout. The clinical optimisation process for choosing QIR 4 as the superior level was by consensus dis- cussions among all paediatric cardiovascular radiologists in the department (including those not acting as observers in the current study), in a side-by-side comparison of QIR 2, QIR 3, and QIR 4 reconstructed images. i Overall diagnostic quality was defined as how well the examination in total could answer the clinical questions. Specific diagnostic quality was defined as how well the anatomical structures of clinical value to the individual con- genital heart defect case were visualised for diagnosis. The examinations were scored 1–4 where 4 corresponded to “high diagnostic quality”, 3 “acceptable diagnostic quality”, 2 “low diagnostic quality”, and 1 “insufficient diagnostic quality”. fi All examinations were evaluated as per clinical routine with four image stacks prepared for the observers’ convenience: 0.6 mm transverse images, and 2 mm images in the transverse, coronal, and sagittal planes. Multiplanar reconstruction and volume rendering based on the 0.6 mm images were used as in clinical routine. Finally, the selected image quality level 70 means that the system’s dose modulation compensated for the lower voltage, leading to less impact of noise that would otherwise increase with lower voltage. Image analysis Before the evaluation of study cases, the observers partici- pated in a session with other PCCT congenital heart defect cases to obtain a common assessment basis. All study cases were fully anonymised, including the removal of all personal identifying information, date and time of acquisition, kV level, QIR level, contrast density, and all other scan information. Also, 70 kV and 90 kV 1190 Pediatric Radiology (2024) 54:1187–1196 Fig. 1 Inclusion and exclusion flowchart. As this was an early instal- lation of the clinical photon-counting computed tomography system, only 90 kV protocols were initially available. Therefore, consecutive patients were included at 90 kV until 70 kV protocols were made available by the vendor. Thereafter, as the clinical standard was to use the then available 70 kV protocols, the aim was to include as many examinations using 70 kV as already included using 90 kV. For 90 kV, one patient was excluded due to failed contrast agent timing and no possibility to reacquire the scan on the same occasion, and for 70 kV two patients were excluded due to corrupt dose data reported by the system many examinations using 70 kV as already included using 90 kV. For 90 kV, one patient was excluded due to failed contrast agent timing and no possibility to reacquire the scan on the same occasion, and for 70 kV two patients were excluded due to corrupt dose data reported by the system Fig. 1 Inclusion and exclusion flowchart. As this was an early instal- lation of the clinical photon-counting computed tomography system, only 90 kV protocols were initially available. Therefore, consecutive patients were included at 90 kV until 70 kV protocols were made available by the vendor. Thereafter, as the clinical standard was to use the then available 70 kV protocols, the aim was to include as Table 3 shows the quality scores for all observers, with a median of 4 (high diagnostic quality) for all observers for both 70 kV and 90 kV. However, all observers scored some 90 kV cases below 3, whereas for 70 kV there were no scores below 3. Sum- of-ranks was different between 70 kV and 90 kV for observer 2 and corrected for different scan lengths by matching. As expected, there was a numerical change in the P-value for DLP when adapting for scan length, but not for CTDIvol, SSDE, or Eeff. Table 2 Patient characteristics Image analysis for overall diagnostic scoring (median 4 vs 4, sum-of-ranks 1,032 vs 1,454; P = 0.001; all other P-values > 0.34), and for observer 3 for specific diagnostic scoring (median 4 vs 4, sum-of-ranks 1,129 vs 1,357; P = 0.03; all other P-values > 0.06). All cases were of high diagnostic quality and could answer the clinical questions. Observers gave scores of 3 and 4 to the 70 kV images, but scores from 1 to 4 for the 90 kV images. This indicates that the essentially noise- free images at 70 kV do not impact observers negatively. Also, agreement between observers was generally high. In regard to image quality, this is similar to a 90 kV PCCT study showing a 97% success rate; however, that study only showed moderate or poor interreader agreement [8]. The average percent rater agreement for 70 kV was 79% (range 66–94%) for overall scores, and 89% (range 80–97%) for specific scores. For 90 kV, the average percent rater agree- ment for overall scores was 72% (range 60–77%) and for spe- cific scores 86% (range 83–89%). i The intraclass correlation for 70 kV was 0.58 (95% con- fidence interval [CI] 0.26–0.75) for overall scores, and 0.71 (95% CI 0.51–0.84) for specific scores. For 90 kV, intraclass correlation for overall scores was 0.84 (95% CI 0.72–0.91) and for specific scores 0.85 (95% CI 0.75–0.92). The current study showed a median Eeff of 0.39 mSv at 90 kV for all patients between birth and 17 years. For those below 1 year of age, median Eeff was 0.30 mSv. Both values are lower than those in the only previous comparable PCCT study, where mean Eeff was 0.50 mSv (± 0.23 mSv) at 90 kV, even though the children in that study (66 [10–161] days) were much younger than in the current study [8]. Further, Dirrichs et al. [8] showed a mean DLP at 90 kV of 14.3 (± 6.6; range 3–42) mGy × cm for their very young children, compared with the current study’s mean DLP of 4 (range 3–8) mGy × cm. It is unclear why the previous study showed such high radiation exposure, but possible explanations include larger scan length or the use of a lead apron which may increase radiation dose unless automated tube current modulation is turned off [13]. i Figures 2 and 3 show clinical examples of 70 kV and 90 kV examinations. Image analysis Table 2 Patient characteristics a Number, bmedian (range) Tube voltage 70 kV 90 kV P-value Number of patientsa 35 (22 male) 35 (18 male) Ageb 24 months (2 days– 16 years) 8 months (2 days– 17 years) > 0.99 Weight (kg)b 12 (2–53) 8 (3–75) 0.83 Height (cm)b 89 (46–170) 78 (51–190) 0.86 Scan length (cm)b 22 (14–32) 17 (11–39) 0.002 Heart defectsa Transposition of the great arteries 5 0 Atrioventricular septal defect 1 5 Coarctation of the aorta 5 5 Pulmonary atresia 8 3 Arteriopathy 0 3 Single ventricle 8 3 Double outlet right ventricle 1 2 Ventricular septal defect 0 2 Tetralogy of Fallot 2 2 Vascular ring 0 2 Total or partial anomalous pulmonary venous drainage 1 2 Valvular stenosis 0 2 Other 4 4 Pediatric Radiology (2024) 54:1187–1196 1191 dose at 70 kV, with scoring equal to or higher than the full contrast dose used for 90 kV. Table 3 Overall and specific diagnostic quality scores a Median [interquartile range] (range) Overall diagnostic scorea Specific diagnostic scorea Tube voltage 70 kV Observer 1 4 [4–4] (3–4) 4 [4–4] (3–4) Observer 2 4 [4–4] (3–4) 4 [4–4] (3–4) Observer 3 4 [4–4] (3–4) 4 [4–4] (3–4) Observer 4 4 [3–4] (3–4) 4 [3.5–4] (3–4) Tube voltage 90 kV Observer 1 4 [4–4] (2–4) 4 [4–4] (2–4) Observer 2 4 [3–4] (2–4) 4 [4–4] (2–4) Observer 3 4 [3.75–4] (2–4) 4 [3.75–4] (2–4) Observer 4 4 [3–4] (1–4) 4 [4–4] (1–4) Table 3 Overall and specific diagnostic quality scores Paediatric cardiovascular PCCT therefore has the poten- tial to become the primary complementary modality to echocardiography in congenital heart defects due to its high diagnostic quality, low radiation dose, and low con- trast agent dose. This is particularly important in complex congenital heart defects where repeat examinations are needed in small radiosensitive children.i Although magnetic resonance imaging has the benefit of providing both functional and anatomical information, it has limited spatial resolution. CT will thus remain the gold standard for anatomical assessment of small ves- sels in conditions such as congenital heart defects with pulmonary atresia, pulmonary vein stenosis, collaterals, or coronary artery anomalies, where precise delineation of anatomy is central to planning and guiding surgical or interventional treatment. Image analysis Maximum intensity projections or 0.6 mm images are presented to more clearly show relevant anatomi- cal structures. Radiation dose parameters are shown in Table 4, and radia- tion dose parameters versus age and body surface area in Fig. 4 for CTDIvol, in Fig. 5 for SSDE, and in Fig. 6 for Eeff. Radiation dose parameters are shown in Table 4, and radia- tion dose parameters versus age and body surface area in Fig. 4 for CTDIvol, in Fig. 5 for SSDE, and in Fig. 6 for Eeff. Jonckheere-Terpstra’s tests showed that there was no trend between Eeff and overall scores (70 kV: P = 0.55; 90 kV: i Radiation dose parameters are shown in Table 4, and radia- tion dose parameters versus age and body surface area in Fig. 4 for CTDIvol, in Fig. 5 for SSDE, and in Fig. 6 for Eeff. f Jonckheere-Terpstra’s tests showed that there was no trend between Eeff and overall scores (70 kV: P = 0.55; 90 kV: P = 0.98) or between Eeff and specific scores (70 kV: P = 0.55; 90 kV: P = 0.29). f For 70 kV, the current study showed a median Eeff of 0.30 mSv with all ages up to 17 years included, and for those below 1 year of age, the median Eeff was 0.22 mSv. In comparison, a recent 70 kV dual-source conventional CT study that only included young children (3.5 [0.2–6.6] months) showed a median Eeff of 0.20 mSv [14].f Discussion This study shows that PCCT yields images of high diag- nostic quality for paediatric congenital heart defect exami- nations with a concomitant low radiation dose. Further, vessels were well-defined with a diluted contrast agent f Different CT methods, dose calculations, and use of weighting factors make direct comparison between studies challenging. There is nevertheless a trend towards reduced 1192 Pediatric Radiology (2024) 54:1187–1196 Fig. 2 Examples of contrast-enhanced photon-counting computed tomography (PCCT) images using the 70 kV protocol. a Coronal maximum intensity projection with a slice thickness of 6 mm in a 14-day-old boy with pulmonary atresia, who had received a modi- fied Blalock-Thomas-Taussig shunt and later desaturated. The image shows an open shunt (arrowhead) between the right subclavian and pulmonary arteries with a slight narrowing in the proximal and dis- tal anastomosis and a stenosis (arrow) in the right pulmonary artery. This case had a median overall diagnostic score of 4 and a median specific diagnostic score of 4 with a size-specific dose estimate of 0.32 mGy and an effective dose of 0.15 mSv. b Sagittal oblique image with a slice thickness of 0.6 mm in a 5-year-old boy with tetralogy of Fallot, corrected at four months of age with a pulmonary conduit. Echocardiography showed a suspected stenosis of the pulmonary con- duit, which was clearly shown using PCCT (arrow). This case had a median overall diagnostic score of 4 and a median specific diagnos- tic score of 4 with a size-specific dose estimate of 1.16 mGy and an effective dose of 0.52 mSv. c Transaxial image with a slice thickness of 0.6 mm in a 3-month-old boy in whom echocardiography showed a suspected aortopulmonary window and anomalous origin of pulmo- nary artery branches. The PCCT image shows a large aortopulmonary window between the ascending aorta and the distal part of the pulmo- nary trunk (asterisk). This case had a median overall diagnostic score of 4 and a median specific diagnostic score of 4 with a size-specific dose estimate of 0.41 mGy and an effective dose of 0.30 mSv. d Cor- onal oblique image with a slice thickness of 0.6 mm in a 16-year-old girl born with pulmonary atresia, ventricular septal defect, and major aortopulmonary collateral arteries, unifocalised to a pulmonary con- duit. Multiple stents were inserted due to stenosis of the pulmonary artery branches. Discussion The PCCT image shows a heavily calcified pulmo- nary conduit (arrows) and a pulmonary artery stent with intimal pro- liferation (arrowheads). Note the good visualisation of vessel lumens despite the presence of dense calcifications and a metal stent. This case had a median overall diagnostic score of 4 and a median specific diagnostic score of 4 with a size-specific dose estimate of 1.81 mGy and an effective dose of 0.80 mSv Fig. 2 Examples of contrast-enhanced photon-counting computed tomography (PCCT) images using the 70 kV protocol. a Coronal maximum intensity projection with a slice thickness of 6 mm in a 14-day-old boy with pulmonary atresia, who had received a modi- fied Blalock-Thomas-Taussig shunt and later desaturated. The image shows an open shunt (arrowhead) between the right subclavian and pulmonary arteries with a slight narrowing in the proximal and dis- tal anastomosis and a stenosis (arrow) in the right pulmonary artery. This case had a median overall diagnostic score of 4 and a median specific diagnostic score of 4 with a size-specific dose estimate of 0.32 mGy and an effective dose of 0.15 mSv. b Sagittal oblique image with a slice thickness of 0.6 mm in a 5-year-old boy with tetralogy of Fallot, corrected at four months of age with a pulmonary conduit. Echocardiography showed a suspected stenosis of the pulmonary con- duit, which was clearly shown using PCCT (arrow). This case had a median overall diagnostic score of 4 and a median specific diagnos- tic score of 4 with a size-specific dose estimate of 1.16 mGy and an effective dose of 0.52 mSv. c Transaxial image with a slice thickness of 0.6 mm in a 3-month-old boy in whom echocardiography showed a suspected aortopulmonary window and anomalous origin of pulmo- nary artery branches. The PCCT image shows a large aortopulmonary window between the ascending aorta and the distal part of the pulmo- nary trunk (asterisk). This case had a median overall diagnostic score of 4 and a median specific diagnostic score of 4 with a size-specific dose estimate of 0.41 mGy and an effective dose of 0.30 mSv. d Cor- onal oblique image with a slice thickness of 0.6 mm in a 16-year-old girl born with pulmonary atresia, ventricular septal defect, and major aortopulmonary collateral arteries, unifocalised to a pulmonary con- duit. Multiple stents were inserted due to stenosis of the pulmonary artery branches. Discussion The PCCT image shows a heavily calcified pulmo- nary conduit (arrows) and a pulmonary artery stent with intimal pro- liferation (arrowheads). Note the good visualisation of vessel lumens despite the presence of dense calcifications and a metal stent. This case had a median overall diagnostic score of 4 and a median specific diagnostic score of 4 with a size-specific dose estimate of 1.81 mGy and an effective dose of 0.80 mSv radiation doses for non-gated cardiovascular CT, where the current PCCT study shows a further dose reduction with Eeff of 0.15–0.82 mSv for 70 kV and 0.22–1.51 mSv for 90 kV in children between 0 and 17 years. This is lower than previous conventional non-gated CT studies in small children between 0 years and 4.5 years, where Eeff was between 0.20 and 1.95 mSv [15–17]. doses, which is supported by the current results. For comparisons between studies, SSDE may be preferred as a reliable tool to estimate average radiation dose depending both on CT parameters and size of the spe- cific patient, even though it does not include organ or tissue weighting factors [18]. So far, relatively few stud- ies have presented SSDE, and none of them have used PCCT for congenital heart defects. The current study, by presenting SSDE, provides an opportunity for future comparisons. Both CTDIvol and DLP are commonly used to estimate radiation exposure. They are however relatively blunt measures—especially DLP—when comparing radiation 1193 Pediatric Radiology (2024) 54:1187–1196 Fig. 3 Examples of contrast-enhanced photon-counting computed tomography (PCCT) images using 90 kV protocols. a Sagittal oblique image with a slice thickness of 0.6 mm in an 8-day-old girl with sus- pected coarctation of the aorta by echocardiography. PCCT shows a hypoplastic arch (asterisk) with hypoplasia of the aortic isthmus and a large arterial duct (plus sign). This case had a median overall diag- nostic score of 4 and a median specific diagnostic score of 4 with a size-specific dose estimate of 0.63 mGy and an effective dose of 0.22 mSv. b Transaxial image with a slice thickness of 0.6 mm in an 8-year-old girl with arterial vasculopathy, status post dilatation of the pulmonary arteries and the ascending aorta due to stenosis. Note the streak artefact from the relatively dense contrast medium (asterisk). Despite this, the pulmonary arteries and the ascending aorta were suf- ficiently visualised for diagnostic purposes. Discussion This case had a median overall diagnostic score of 3 and a median specific diagnostic score of 4 with a size-specific dose estimate of 1.09 mGy and an effective dose of 0.24 mSv. c, d Coronal (c) and transaxial (d) oblique images with a slice thickness of 0.6 mm depict the right (c; asterisk), and left (d; asterisk) pulmonary arteries in a 5-month-old prematurely born girl with idiopathic pulmonary arterial hypertension. The pulmonary arteries were well visualised without stenosis. This case had a median overall diagnostic score of 3 and a median specific diagnostic score of 3 with an effective dose of 0.30 mSv Despite this, the pulmonary arteries and the ascending aorta were suf- ficiently visualised for diagnostic purposes. This case had a median overall diagnostic score of 3 and a median specific diagnostic score of 4 with a size-specific dose estimate of 1.09 mGy and an effective dose of 0.24 mSv. c, d Coronal (c) and transaxial (d) oblique images with a slice thickness of 0.6 mm depict the right (c; asterisk), and left (d; asterisk) pulmonary arteries in a 5-month-old prematurely born girl with idiopathic pulmonary arterial hypertension. The pulmonary arteries were well visualised without stenosis. This case had a median overall diagnostic score of 3 and a median specific diagnostic score of 3 with an effective dose of 0.30 mSv Fig. 3 Examples of contrast-enhanced photon-counting computed tomography (PCCT) images using 90 kV protocols. a Sagittal oblique image with a slice thickness of 0.6 mm in an 8-day-old girl with sus- pected coarctation of the aorta by echocardiography. PCCT shows a hypoplastic arch (asterisk) with hypoplasia of the aortic isthmus and a large arterial duct (plus sign). This case had a median overall diag- nostic score of 4 and a median specific diagnostic score of 4 with a size-specific dose estimate of 0.63 mGy and an effective dose of 0.22 mSv. b Transaxial image with a slice thickness of 0.6 mm in an 8-year-old girl with arterial vasculopathy, status post dilatation of the pulmonary arteries and the ascending aorta due to stenosis. Note the streak artefact from the relatively dense contrast medium (asterisk). Despite this, the pulmonary arteries and the ascending aorta were suf- ficiently visualised for diagnostic purposes. Discussion This case had a median overall diagnostic score of 3 and a median specific diagnostic score of 4 with a size-specific dose estimate of 1.09 mGy and an effective dose of 0.24 mSv. c, d Coronal (c) and transaxial (d) oblique images with a slice thickness of 0.6 mm depict the right (c; asterisk), and left (d; asterisk) pulmonary arteries in a 5-month-old prematurely born girl with idiopathic pulmonary arterial hypertension. The pulmonary arteries were well visualised without stenosis. This case had a median overall diagnostic score of 3 and a median specific diagnostic score of 3 with an effective dose of 0.30 mSv Therefore, neither accuracy nor potentially missed diag- noses were assessed. It is however unlikely that accuracy is decreased using PCCT compared to conventional CT either for congenital heart defects as studied here, or for other possible associated cardiovascular abnormalities. Finally, there is no international consensus regarding which QIR level to use for congenital heart defects, but QIR 4 in its present form as used in the current study is now preferred in several centres. This study has limitations. The PCCT software was updated during the study. However, image scores did not change with time or stepwise after software updates, and so the impact of the updates is considered small. Even though diagnostic quality was assessed it would be appro- priate for clinical impact to study anatomy by PCCT, and compare it to anatomy during surgery. Surgery was not performed in all patients and there is no other examina- tion more accurate than PCCT available for comparison. Pediatric Radiology (2024) 54:1187–1196 1194 Table 4 Radiation dose parameters at 70 kV and 90 kV CTDIvol computed tomography dose index, DLP dose-length product, Eeff effective dose, SSDE size-specific dose estimate. Values are reported for all patients, and for the smallest and most radiosensitive patients, i.e. Discussion those aged 0–12 months 70 kV 90 kV P-value CTDIvol (mGy) All patients 0–17 years 0.32 (0.09–1.32) 0.50 (0.23–1.96) 0.01 0–12 months 0.17 (0.09–0.28) 0.32 (0.23–0.59) < 0.0001 SSDE (mGy) All patients 0–17 years 0.68 (0.25–2.02) 1.1 (0.58–2.71) < 0.001 0–12 months 0.40 (0.25–0.63) 0.74 (0.58–1.35) < 0.0001 Eeff (mSv) All patients 0–17 years 0.30 (0.15–0.82) 0.39 (0.22–1.51) 0.01 0–12 months 0.22 (0.15–0.37) 0.30 (0.22–0.59) 0.001 DLP (mGy × cm) not matched for scan length All patients 0–17 years 7 (2–40) 7 (3–74) 0.22 0–12 months 3 (2–5) 5.5 (4–8) < 0.0001 DLP (mGy × cm) matched for scan length All patients 0–17 years 5 (2–40) 8 (4–53) 0.02 0–12 months 3 (2–5) 4 (3–8) 0.001 Table 4 Radiation dose parameters at 70 kV and 90 kV Table 4 Radiation dose parameters at 70 kV and 90 kV CTDIvol computed tomography dose index, DLP dose-length product, Eeff effective dose, SSDE size-specific dose estimate. Values are reported for all patients, and for the smallest and most radiosensitive patients, i.e. those aged 0–12 months Fig. 4 Computed tomography dose index (CTDIvol) versus age and body surface area (BSA). For all patients below 1 year of age, CTDIvol was less than 0.3 mGy for 70 kV, and less than 0.6 mGy for 90 kV. For patients between 1 year and 17 years of age, CTDIvol increased with age for both 70 kV and 90 kV. It also increased with BSA for both 70 kV and 90 kV, but to a larger degree for 90 kV 0 3 6 9 12 0.0 0.2 0.4 0.6 0.8 Age [months] CTDIvol [mGy] CTDIvol vs age up to 1 year 90 kV 70 kV 0.0 0.1 0.2 0.3 0.4 0.5 0.0 0.2 0.4 0.6 0.8 BSA [m2] CTDIvol [mGy] CTDIvol vs BSA up to 1 year 90 kV 70 kV 5 10 15 1 18 0.0 0.5 1.0 1.5 2.0 Age [years] CTDIvol [mGy] CTDIvol vs age from 1 year 90 kV 70 kV 0.0 0.5 1.0 1.5 2.0 2.5 0.0 0.5 1.0 1.5 2.0 BSA [m2] CTDIvol [mGy] CTDIvol vs BSA from 1 year 90 kV 70 kV A B C D Fig. 4 Computed tomography dose index (CTDIvol) versus age and body surface area (BSA). For all patients below 1 year of age, CTDIvol was less than 0.3 mGy for 70 kV, and less than 0.6 mGy for 90 kV. CTDIvol computed tomography dose index, DLP dose-length product, Eeff effective dose, SSDE size-specific dose estimate. Values are reported for all patients, and for the smallest and most radiosensitive patients, i.e. those aged 0–12 months Discussion Eeff increased with BSA for both 70 kV and 90 kV, but to a larger degree for 90 kV A B C D 0 3 6 9 12 0.0 0.2 0.4 0.6 0.8 Age [months] Eeff [mSv] Eeff vs age up to 1 year 90 kV 70 kV 5 10 15 1 18 0.0 0.5 1.0 1.5 2.0 Age [years] Eeff [mSv] Eeff vs age from 1 year 90 kV 70 kV 0.0 0.1 0.2 0.3 0.4 0.5 0.0 0.2 0.4 0.6 0.8 BSA [m2] Eeff [mSv] Eeff vs BSA up to 1 year 90 kV 70 kV 0.0 0.5 1.0 1.5 2.0 2.5 0.0 0.5 1.0 1.5 2.0 BSA [m2] Eeff [mSv] Eeff vs BSA from 1 year 90 kV 70 kV Pediatric Radiology (2024) 54:1187–1196 1195 B A 0 3 6 9 12 0.0 0.5 1.0 1.5 Age [months] SSDE [mGy] SSDE vs age up to 1 year 90 kV 70 kV A B 5 10 15 1 18 0 1 2 3 Age [years] SSDE [mGy] SSDE vs age from 1 year 90 kV 70 kV SSDE vs age from 1 year C 0.0 0.1 0.2 0.3 0.4 0.5 0.0 0.5 1.0 1.5 BSA [m2] SSDE [mGy] SSDE vs BSA up to 1 year 90 kV 70 kV C D 0.0 0.5 1.0 1.5 2.0 2.5 0 1 2 3 BSA [m2] SSDE [mGy] SSDE vs BSA from 1 year 90 kV 70 kV C D SSDE vs BSA from 1 year A 0 3 6 9 12 0.0 0.2 0.4 0.6 0.8 Age [months] Eeff [mSv] Eeff vs age up to 1 year 90 kV 70 kV B 5 10 15 1 18 0.0 0.5 1.0 1.5 2.0 Age [years] Eeff [mSv] Eeff vs age from 1 year 90 kV 70 kV Fig. 6 Effective dose (Eeff) ver- sus age and body surface area (BSA). For most patients below 1 year of age, Eeff was less than 0.5 mSv for 70kV, and less than 1 mSv for all patients between 1 year and 17 years of age. Discussion For patients between 1 year and 17 years of age, CTDIvol increased with age for both 70 kV and 90 kV. It also increased with BSA for both 70 kV and 90 kV, but to a larger degree for 90 kV 0 3 6 9 12 0.0 0.2 0.4 0.6 0.8 Age [months] CTDIvol [mGy] CTDIvol vs age up to 1 year 90 kV 70 kV A 5 10 15 1 18 0.0 0.5 1.0 1.5 2.0 Age [years] CTDIvol [mGy] CTDIvol vs age from 1 year 90 kV 70 kV B B A A 0.0 0.1 0.2 0.3 0.4 0.5 0.0 0.2 0.4 0.6 0.8 BSA [m2] CTDIvol [mGy] CTDIvol vs BSA up to 1 year 90 kV 70 kV C 0.0 0.5 1.0 1.5 2.0 2.5 0.0 0.5 1.0 1.5 2.0 BSA [m2] CTDIvol [mGy] CTDIvol vs BSA from 1 year 90 kV 70 kV D C D C 1195 Pediatric Radiology (2024) 54:1187–1196 Fig. 5 Size-specific dose estimate (SSDE) versus age and body surface area (BSA). For all patients below 1 year of age, SSDE was less than 0.65 mGy for 70 kV, and less than 1.4 mGy for 90 kV. For patients between 1 year and 17 years of age, SSDE was less than 2.05 mGy for 70 kV and less than 2.75 mGy for 90 kV. SSDE increased with BSA for both 70 kV and 90 kV, but to a larger degree for 90 kV A B C D 0 3 6 9 12 0.0 0.5 1.0 1.5 Age [months] SSDE [mGy] SSDE vs age up to 1 year 90 kV 70 kV 5 10 15 1 18 0 1 2 3 Age [years] SSDE [mGy] SSDE vs age from 1 year 90 kV 70 kV 0.0 0.1 0.2 0.3 0.4 0.5 0.0 0.5 1.0 1.5 BSA [m2] SSDE [mGy] SSDE vs BSA up to 1 year 90 kV 70 kV 0.0 0.5 1.0 1.5 2.0 2.5 0 1 2 3 BSA [m2] SSDE [mGy] SSDE vs BSA from 1 year 90 kV 70 kV Fig. 6 Effective dose (Eeff) ver- sus age and body surface area (BSA). For most patients below 1 year of age, Eeff was less than 0.5 mSv for 70kV, and less than 1 mSv for all patients between 1 year and 17 years of age. Conflicts of interest None 13. Yu L, Bruesewitz MR, Vrieze TJ, McCollough CH (2019) Lead shielding in pediatric chest CT: effect of apron placement outside the scan volume on radiation dose reduction. AJR Am J Roent- genol 212:151–156 Open Access This article is licensed under a Creative Commons Attri- bution 4.0 International License, which permits use, sharing, adapta- tion, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. 14. Kravchenko D, Hart C, Garbe S, Luetkens JA, Isaak A, Mesropyan N, Vergnat M, Leyens J, Attenberger U, Kuetting D (2022) Image quality and radiation dose of dual source high pitch computed tomography in pediatric congenital heart disease. Sci Rep 12:9934 15. Ben Saad M, Rohnean A, Sigal-Cinqualbre A, Adler G, Paul JF (2009) Evaluation of image quality and radiation dose of thoracic and coronary dual-source CT in 110 infants with congenital heart disease. Pediatr Radiol 39:668–676 16. Han BK, Lindberg J, Grant K, Schwartz RS, Lesser JR (2011) Accuracy and safety of high pitch computed tomography imaging in young children with complex congenital heart disease. Am J Cardiol 107:1541–1546 Conclusion Paediatric cardiovascular PCCT yields images for congenital heart defects of high diagnostic quality with low radiation dose at both 70 kV and 90 kV. 4. Aspelin P, Aubry P, Fransson SG, Strasser R, Willenbrock R, Berg KJ (2003) Nephrotoxicity in high-risk patients study of isoosmo- lar, low-osmolar non-ionic contrast media study I Nephrotoxic effects in high-risk patients undergoing angiography. N Engl J Med 348:491–499 5. Bruce RJ, Djamali A, Shinki K, Michel SJ, Fine JP, Pozniak MA (2009) Background fluctuation of kidney function versus contrast- induced nephrotoxicity. AJR Am J Roentgenol 192:711–718 Author contribution Guarantor of integrity of entire study, E.H.; study concept and design, F.S., M-L.A., S.M., M.W., P.W., E.H.; data acquisi- tion, F.S.; data analysis and interpretation, all authors; approval of final manuscript, all authors. 6. Leng S, Bruesewitz M, Tao S, Rajendran K, Halaweish AF, Cam- peau NG, Fletcher JG, McCollough CH (2019) Photon-counting detector CT: system design and clinical applications of an emerg- ing technology. Radiographics 39:729–743 Funding Open access funding provided by Lund University. The Swed- ish Heart–Lung Foundation, the Swedish governmental funding of clinical research (ALF), Region Skåne, and Skåne University Hospital. 7. Willemink MJ, Persson M, Pourmorteza A, Pelc NJ, Fleischmann D (2018) Photon-counting CT: technical principles and clinical prospects. Radiology 289:293–312f Data availability Data supporting the findings of this study are not openly available due to reasons of sensitivity. Data are located in con- trolled access data storage at Skåne University Hospital. Data may be made available upon reasonable request to the corresponding author. 8. Dirrichs T, Tietz E, Ruffer A, Hanten J, Nguyen TD, Dethlefsen E, Kuhl CK (2023) Photon-counting versus dual-source CT of congenital heart defects in neonates and infants: initial experience. Radiology 307:e223088 9. Deak PD, Smal Y, Kalender WA (2010) Multisection CT proto- cols: sex- and age-specific conversion factors used to determine effective dose from dose-length product. Radiology 257:158–166fi Discussion Eeff increased with BSA for both 70 kV and 90 kV, but to a larger degree for 90 kV A B C g 0.0 0.1 0.2 0.3 0.4 0.5 0.0 0.2 0.4 0.6 0.8 BSA [m2] Eeff [mSv] Eeff vs BSA up to 1 year 90 kV 70 kV D 0.0 0.5 1.0 1.5 2.0 2.5 0.0 0.5 1.0 1.5 2.0 BSA [m2] Eeff [mSv] Eeff vs BSA from 1 year 90 kV 70 kV D D C 1196 Pediatric Radiology (2024) 54:1187–1196 3. Saengsin K, Pickard SS, Prakash A (2022) Utility of cardiac CT in infants with congenital heart disease: diagnostic performance and impact on management. J Cardiovasc Comput Tomogr 16:345–349 Declarations Consent to participate The regional ethics committee approved this retrospective study using pseudonymised data from patients referred for clinical purposes, waiving individual consent. f 10. Gamer M, Lemon J (2019) irr: various coefficients of interrater reliability and agreement. R package version 0.84.1. IFPS 11. Team RC (2023) R: a language and environment for statistical com- puting. R Foundation for Statistical Computing, Vienna, Austria 11. Team RC (2023) R: a language and environment for statistical com- puting. R Foundation for Statistical Computing, Vienna, Austria 12. Jonckheere AR (1954) A distribution-free k-sample test against ordered alternatives. Biometrika 41:133–145 12. Jonckheere AR (1954) A distribution-free k-sample test against ordered alternatives. Biometrika 41:133–145 Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. References 17. Young C, Taylor AM, Owens CM (2011) Paediatric cardiac com- puted tomography: a review of imaging techniques and radiation dose consideration. Eur Radiol 21:518–529 1. Prakash A, Powell AJ, Geva T (2010) Multimodality noninvasive imaging for assessment of congenital heart disease. Circ Cardio- vasc Imaging 3:112–125 18. Boone J, Strauss K, Cody D, McCollough C, McNitt-Gray M, Toth T (2011) AAPM Report No. 204: Size-specific dose esti- mates (SSDE) in pediatric and adult body CT examinations. Am Assoc Physicists Med 2. Han BK, Rigsby CK, Hlavacek A, Leipsic J, Nicol ED, Siegel MJ, Bardo D, Abbara S, Ghoshhajra B, Lesser JR, Raman S, Crean AM, Society of Cardiovascular Computed Tomography, Soci- ety of Pediatric Radiology, North American Society of Cardiac Imaging (2015) Computed tomography imaging in patients with congenital heart disease part I: Rationale and utility. An expert consensus document of the Society of Cardiovascular Computed Tomography (SCCT): Endorsed by the Society of Pediatric Radi- ology (SPR) and the North American Society of Cardiac Imaging (NASCI). J Cardiovasc Comput Tomogr 9:475–492 Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
https://openalex.org/W2043099854	https://zenodo.org/records/2171761/files/article.pdf	German	null	Die Frage des Geschichtsstudiums	Deutsche medizinische Wochenschrift/Deutsche Medizinische Wochenschrift	1,920	public-domain	445	Die Frage des tleschichtsstudiums. n cand. med. ilubert Beisele in Tutzing (Ob..Bayern). Herr Oberstabsarzt H a berlin g fordert in seinem Artikel die Einführung von Vorlesungen über die Geschichte der Medizin und vertritt damit nicht nur die Meinung zahlreicher F-lochscliullehrer, sondern auch die vieler Medizinstudierender. Vielleicht dient es als Ergänzung zu dem angeführten Aufsatz, wenn hier diese Frage vom Standpunkt des Studierenden aus kurz beleuchtet wird. I d b d M di i f k d ß i Es Ist dem angebenden Mediziner oft anzumerken, daß sein medizinischer Ehrgeiz fast ausschließlich nach baldigster Approba - tion trachtetl), sein Streben also darauf ausgeht, nur den Exaniens- bedarf in möglichst kurzer Frist zu decken. Auf diese Weise ist es ihm nicht möglich, den aufgenommenen Lehrstoff zu einer ärzt- lichen Weltanschauung zu verarbeiten. Er hat keine Zeit, Interesse au seiner Wissenschaft im weiteren Sinne zu ge\vinnen. Daß dem wirklich so ist, kann man leider sehr häufig sowohl in den Nörsälen als auch in den Versammlungen refornilustiger Mediziner beobachten. Hi klä i h d ß i M di i l di k M g g Hieraus erklärt es sich, daß wir Mediziner als die krassen Mate- rialisten unter den Akademikern gelten, und dies nicht ganz mit Un- recht. Denn die oben angeführten Tatsachen könnten den Eindruck erwecken, als ob wir nicht Aerzte dem innersten Wesen nach werden, sondern nur eine gewisse Fertigkeit in der Therapie erreichen vollten. So kommt es, daß als Reaktion hierauf eine Reihe von Medizin- studierenden mehr denn je an unklare und spekulative Richtungen in der Medizin Anschluß sucht, zumal solche, die nicht einsehen, daß die Schule der Medizin nur mit exakt wissenschaftlicher Arbeit sich befassen darf und nicht mit subjektiver Naturphilosophie. G d di Ei füh d G hi h l P ül f h ( d j p p Gerade die Einführung der Geschichte als Prülungsfach (oder doch größere Berücksichtigung des Historischen in den einzelnen Prüfungsfachern) verspricht nun in diesem Punkt einen guten Er- folg, denn auch die zwangsweise Beschäftigung mit der Geschichte unserer Wissenschaft erschliellt das Interesse für die Zusammen- hänge und wird sicher auf fruchtbaren Boden fallen. Wenigstens herrscht diese Ansicht bei eineni nicht unbeträchtlichen Teil der Medïziuerschaft. DEUTSCHE MEDIZINISCHE WOC1-IENSCHRIFT DEUTSCHE MEDIZINISCHE WOC1-IENSCHRIFT Nr.4 102 Die Frage des tleschichtsstudiums. Zum Schlusse möchte ich noch darauf hinweisen, daß das tiefere Verstandnis für die Entwicklung unserer Wissenschaft wohl erst in den klinischen Semestern richtig erwacht; denn da beginnt die Be- schäftigung mit der Therapie, die in der alten Geschichte so ziem- lieb die Hauptrolle spielt. Ob es so leicht sein wird, die Geschichte der vorklinisclien Disziplinen von der der klinischen zu trennen, wie Herr Oberstahsarzt H aberling vorschlägt, wage ich nicht zu beurteilen.
https://openalex.org/W3000683042	https://europepmc.org/articles/pmc7020159?pdf=render	English	null	The Effect of Caffeine and Trifluralin on Chromosome Doubling in Wheat Anther Culture	Plants	2,020	cc-by	9,115	Received: 3 December 2019; Accepted: 10 January 2020; Published: 15 January 2020 Abstract: Challenges for wheat doubled haploid (DH) production using anther culture include genotype variability in green plant regeneration and spontaneous chromosome doubling. The frequency of chromosome doubling in our program can vary from 14% to 80%. Caﬀeine or triﬂuralin was applied at the start of the induction phase to improve early genome doubling. Caﬀeine treatment at 0.5 mM for 24 h signiﬁcantly improved green plant production in two of the six spring wheat crosses but had no eﬀect on the other crosses. The improvements were observed in Trojan/Havoc and Lancer/LPB14-0392, where green plant numbers increased by 14% and 27% to 161 and 42 green plants per 30 anthers, respectively. Caﬀeine had no signiﬁcant eﬀect on chromosome doubling, despite a higher frequency of doubling in several caﬀeine treatments in the ﬁrst experiment (67–68%) compared to the control (56%). In contrast, triﬂuralin signiﬁcantly improved doubling following a 48 h treatment, from 38% in the control to 51% and 53% in the 1 µM and 3 µM triﬂuralin treatments, respectively. However, triﬂuralin had a signiﬁcant negative eﬀect on green plant regeneration, declining from 31.8 green plants per 20 anthers (control) to 9–25 green plants per 20 anthers in the triﬂuralin treatments. Further work is required to identify a treatment regime with caﬀeine and/or anti-mitotic herbicides that consistently increases chromosome doubling in wheat without reducing green plant regeneration. Keywords: wheat doubled haploids; anther culture; chromosome doubling; caﬀeine; triﬂuralin Article Department of Primary Industries and Regional Development, 3 Baron-Hay Court, South Perth 6151, Western Australia, Australia; Marieclaire.Castello@dpird.wa.gov.au (M.C.); Li.Liu@dpird.wa.gov.au (L.L.); Julie.Killen@dpird.wa.gov.au (J.K.); Anna.Hepworth@dpird.wa.gov.au (A.H.); Rebecca.O'Leary@dpird.wa.gov.au (R.O.) Department of Primary Industries and Regional Development, 3 Baron-Hay Court, South Perth 6151, Western Australia, Australia; Marieclaire.Castello@dpird.wa.gov.au (M.C.); Li.Liu@dpird.wa.gov.au (L.L.); Julie.Killen@dpird.wa.gov.au (J.K.); Anna.Hepworth@dpird.wa.gov.au (A.H.); Rebecca.O'Leary@dpird.wa.gov.au (R.O.) * Correspondence: Sue.Broughton@dpird.wa.gov.au * Correspondence: Sue.Broughton@dpird.wa.gov.au plants plants plants plants 1. Introduction In wheat, however, the frequency of spontaneous doubling can vary more widely, with reports of 25% to 70% [23]. In our program, which predominantly handles Australian spring wheat crosses, we have observed frequencies from 14% to 80%. Anti-mitotic chemicals, such as colchicine, can also be applied in vitro to promote chromosome doubling at an early stage. This can be labor- and cost-effective and avoid some of the problems of colchicine rootdipping.Theadditionofcolchicinetoantherandmicrosporeinductionmediahassuccessfullyimproved doubling in wheat [18,24,25] as well as other species, including Brassica napus [26,27], triticale [28], rice [29] and red pepper [30]. Colchicine also has a positive eﬀect on embryogenesis and can replace the heat pretreatment normally required for embryogenesis in B. napus [31]. Increases in embryo and/or green plant numbers following in vitro colchicine application have also been reported in wheat, although the eﬀects varied with genotype [18,24,25]. While colchicine has been widely used to induce polyploidy in many plant species, it also has negative aspects. It has been shown to have a low aﬃnity for plant microtubules and therefore must be used at relatively high concentrations [32,33]. It is also toxic to humans and has a high aﬃnity for vertebrate microtubules [34]. g y Several herbicides also target mitosis as a primary mechanism of action. They belong to a range of chemically diverse classes, including dinitroanilines (triﬂuralin and oryzalin), phosphorothioamidates (aminoprophos-methyl or APM), benzamides (pronamide), carbamates (chlorpropham and isopropyl N-3-chlorophenyl carbamate) and others [34]. Studies on the mechanism of oryzalin and APM have shown they bind to tubulin proteins, inhibiting microtubule polymerization and promoting depolymerization of the anaphase spindle [35,36]. Mitosis and cell division are inhibited, and aﬀected cells may contain polyploid nuclei. Like colchicine, these chemicals have been applied to a range of plant species to induce polyploidy [34]. Triﬂuralin, oryzalin and APM have also been used to induce chromosome doubling during androgenesis in wheat [37], B. napus [38–40], maize [41] and cork oak [42] and during parthenogenesis in beet [43] and cucumber [44]. Because these chemicals have a much higher aﬃnity for plant microtubules than colchicine, they can be applied at micromolar concentrations [35,45]. Additionally, these chemicals do not bind to animal microtubules [35,36,45], reducing the toxicity risk to humans. There is evidence that these chemicals can also stimulate embryogenesis, which is not surprising, given that their eﬀects on plant microtubules are similar to colchicine. 1. Introduction The production of doubled haploid (DH) lines remains an important tool for the rapid generation of ﬁxed lines for breeding and research purposes. In self-pollinating crops such as wheat, the time to variety release can be reduced by three to four years when DHs are used. Additionally, phenotypic evaluation and selection is more reliable and accurate in DH populations. The beneﬁts and applications of DHs have been reviewed extensively, and the large numbers of wheat DH varieties attest to the success of the technology [1–3]. In addition to variety development, DH populations are a signiﬁcant research tool for mapping single locus genes and QTL controlling traits of interest [4–6]. In their recent review of wheat DHs, Devaux and Cistué [3] also indicated the usefulness of DHs for their application in genome wide association studies (GWAS), genomic selection (GS) and sequencing activities. Doubled haploid technology can also be used in tandem with transformation and gene-editing technologies, where isolated microspores or microspore-derived embryos can be used as targets. For these technologies, the totipotent microspore is a valuable tool, and homozygous DH transgenic or gene-edited plants can be generated in a single step [7–10]. www.mdpi.com/journal/plants Plants 2020, 9, 105; doi:10.3390/plants9010105 www.mdpi.com/journal/plants 2 of 14 Plants 2020, 9, 105 In Western Australia, large numbers of spring bread wheat (Triticum aestivum L.) DH lines are produced each year using anther culture. In 2018, approximately 15,000 wheat DH lines were produced. Improvements in wheat DH production have resulted from co-culture with ovaries [11] and n-butanol treatment [12,13]. Approximately 90% of the DH lines developed in our program are destined for plant breeding companies, with the remaining populations used for research projects throughout Australia [14,15]. Genotype variability in key production parameters, such as embryo production, green plant regeneration and chromosome doubling, is an ongoing challenge, especially for the high-throughput production of breeding populations. We rely on spontaneous chromosome doubling to restore fertility. This avoids the necessity to screen regenerant plants for ploidy and treat haploid plants with colchicine using root dipping “immersion” treatments [16,17], and the associated issues of plant mortality, ploidy chimeras and variable seed set that can occur as a result of this treatment [18]. In microspore-derived wheat and barley DHs, nuclear fusion is widely accepted as the mechanism responsible for spontaneous chromosome doubling [19–21]. In barley, the frequency of spontaneous doubling is relatively high, 60% to 90% [22,23]. 1. Introduction The cell plate is synthesized by the phragmoplast, which expands centrifugally. Dynamic microtubules depolymerize in the central region, where cell plate synthesis is completed, and re-polymerize at the expanding phragmoplast front, where cell plate synthesis will next take place. Golgi-derived vesicles fuse into a continuous membrane network in the center of the phragmoplast, and callose forms a coat-like structure on the membrane surface, later replaced by cell wall polysaccharides [49,50]. In the presence of caﬀeine, however, both the deposition of callose and the redistribution of phragmoplast microtubules is completely inhibited, and the deposition of callose in the cell plate appears tightly related to the depolymerization of microtubules at the central region of the phragmoplast [49,51]. Although the exact mechanism of caﬀeine remains unclear, it has been proposed that Ca2+ gradients and the reduction of Ca2+ levels near the cell plate play a role [48,49,52]. There is a limited number of studies in which caﬀeine has been used to induce polyploidy for practical purposes. Espino and Vazquez [53] applied caﬀeine and colchicine to detached cultured leaves of African violet, Saintpaulia ionantha, to induce polyploidy; however, the frequency of polyploids following caﬀeine treatment was very low. Lim et al. [54] injected caﬀeine solution into the buds of interspeciﬁc Lilium crosses to restore gametic fertility to obtain 2n gametes. In wheat, caﬀeine has been tested as an alternative to colchicine to induce chromosome doubling in haploids generated from interspeciﬁc (wheat × maize) crosses [55]. In that study, caﬀeine was tested in immersion/root dipping treatments over a range of concentrations (0.3–10 g/L) and times (3–24 h). Various treatments resulted in pollen shedding and substantial seed set compared with the untreated haploid controls. To our knowledge, caﬀeine has not been tested as an in vitro doubling agent following androgenesis. There is a limited number of studies in which caﬀeine has been used to induce polyploidy for practical purposes. Espino and Vazquez [53] applied caﬀeine and colchicine to detached cultured leaves of African violet, Saintpaulia ionantha, to induce polyploidy; however, the frequency of polyploids following caﬀeine treatment was very low. Lim et al. [54] injected caﬀeine solution into the buds of interspeciﬁc Lilium crosses to restore gametic fertility to obtain 2n gametes. In wheat, caﬀeine has been tested as an alternative to colchicine to induce chromosome doubling in haploids generated from interspeciﬁc (wheat × maize) crosses [55]. 1. Introduction In that study, caﬀeine was tested in immersion/root dipping treatments over a range of concentrations (0.3–10 g/L) and times (3–24 h). Various treatments resulted in pollen shedding and substantial seed set compared with the untreated haploid controls. To our knowledge, caﬀeine has not been tested as an in vitro doubling agent following androgenesis. This study aimed to determine whether chemicals such as caﬀeine or triﬂuralin, applied in vitro during anther culture, could improve green plant regeneration and/or the frequency of chromosome doubling in wheat. The eﬀects of caﬀeine and triﬂuralin on albino plant production were also considered. 1. Introduction Triﬂuralin and APM stimulated embryogenesis in wheat microspores and oryzalin, triﬂuralin and APM stimulated embryogenesis in B. napus [37,38]. In these studies, the herbicides were applied at concentrations of 0.1–10 µM (wheat) or 0.3–30 µM (B. napus) for either 24 or 48 h. Stimulatory eﬀects were observed at low concentrations (0.3–1.0 µM) of the herbicides, while higher concentrations inhibited embryo formation in both species. In contrast, plant fertility improved with increasing herbicide concentration. In wheat, the highest percentage of fertile plants was obtained with 10 µM triﬂuralin or APM applied for 48 h. Colchicine was also included in the B. napus study, although at much higher concentrations (3–3000 µM) and for shorter Plants 2020, 9, 105 3 of 14 exposure times (6–24 h). The response curves for colchicine were similar to the herbicides, with small improvements in embryo numbers at low colchicine concentrations, while concentrations above 300 µM were considered toxic. Again, plant fertility increased with increasing colchicine concentration and exposure time. Data from these studies indicate that anti-mitotic herbicides such as oryzalin, triﬂuralin and APM can have similar in vitro eﬀects to colchicine. Exposing plant cells to caﬀeine can also result in binucleate and multi-nucleate cells by impeding cytokinesis. Although cell plate formation commences normally in the presence of caﬀeine, it is never completed [46–48]. The cell plate is synthesized by the phragmoplast, which expands centrifugally. Dynamic microtubules depolymerize in the central region, where cell plate synthesis is completed, and re-polymerize at the expanding phragmoplast front, where cell plate synthesis will next take place. Golgi-derived vesicles fuse into a continuous membrane network in the center of the phragmoplast, and callose forms a coat-like structure on the membrane surface, later replaced by cell wall polysaccharides [49,50]. In the presence of caﬀeine, however, both the deposition of callose and the redistribution of phragmoplast microtubules is completely inhibited, and the deposition of callose in the cell plate appears tightly related to the depolymerization of microtubules at the central region of the phragmoplast [49,51]. Although the exact mechanism of caﬀeine remains unclear, it has been proposed that Ca2+ gradients and the reduction of Ca2+ levels near the cell plate play a role [48,49,52]. Exposing plant cells to caﬀeine can also result in binucleate and multi-nucleate cells by impeding cytokinesis. Although cell plate formation commences normally in the presence of caﬀeine, it is never completed [46–48]. 2. Results 2.1. Experiment 1: The Eﬀect of Caﬀeine on Green and Albino Plant Regeneration and Chromosome Doubling (One Cross) 2.1. Experiment 1: The Eﬀect of Caﬀeine on Green and Albino Plant Regeneration and Chromosome Doubling (One Cross) Caﬀeine application for 48 h had a signiﬁcant negative eﬀect on green plant regeneration in the (Yr57/3Gladius#60)/2Trojan cross (p < 0.001). In both 48 h treatments, the number of green plants decreased signiﬁcantly, from 38.6 green plants per 20 anthers in the control to 24.3 and 27.5 green plants in the 0.5 mM and 1.5 mM treatments, respectively (Table 1). The 24 h caﬀeine treatments had no eﬀect on green plant production, despite higher green plant numbers following the 0.5 mM/24 h treatment. Caﬀeine treatments also aﬀected albino plant regeneration (p < 0.001), both positively and negatively. The number of albino plants decreased signiﬁcantly in the 1.5 mM/24 h treatment, from 16.6 (control) to 11.0 (caﬀeine), and increased signiﬁcantly in the 0.5 mM/48 h treatment, to 22.0 albino plants per 20 anthers (Table 1). The frequency of chromosome doubling was higher in three of the four caﬀeine treatments compared to the control, but the increases were not signiﬁcant (p = 0.327) (Table 1). Despite the lack of signiﬁcance, the results were encouraging, with three treatments resulting in 67–68% doubling, compared to 56% in the control. When green plant regeneration and chromosome doubling were combined in a success index, the 0.5 mM/24 h caﬀeine treatment was the most successful, with 29.5 DHs per 20 anthers compared with 20.1 in the control (Table 5). The increase warrants further investigation in a larger experiment (Experiment 2). 4 of 14 Plants 2020, 9, 105 Table 1. Means (± standard error [SE]) of green and albino plant numbers, expressed per 20 anthers, and chromosome doubling (% doubled haploid (DH)) following caﬀeine treatment during anther culture on the (Yr57/3Gladius#60)/2Trojan cross in 2017. LSD: least signiﬁcant diﬀerence. Treatment Green Plants per 20 Anthers Albino Plants per 20 Anthers No. of Plants Transplanted % DH Control 38.6 (±3.42) 16.6 (±1.97) 100 56 (±5.0) 0.5 mM caﬀeine for 24 h 41.5 (±3.93) 17.0 (±2.33) 50 68 (±6.6) 0.5 mM caﬀeine for 48 h 24.3 (±3.68) 22.0 (±2.44) 50 54 (±7.1) 1.5 mM caﬀeine for 24 h 36.6 (±3.81) 11.0 (±2.10) 50 67 (±6.8) 1.5 mM caﬀeine for 48 h 27.5 (±3.67) 13.1 (±2.15) 45 67 (±7.0) 5% LSD 5.56 3.72 18.4 2.2. 2. Results Plants were grown to maturity at DPIRD South Perth (DPI) or Virginia, South Australia (VIR). The number of plants transplanted for each treatment and cross is indicated in parentheses. Cross Grow-Out Location % DH Control Caﬀeine (0.5 mM/24 h) 5% LSD Lancer/LPB14-0392 DPI 31 (±2.6) 31 (±2.5) 9.6 (309) (351) Coolah/LPB16-3182 DPI 29 (±2.7) 35 (±3.0) (285) (249) Scepter/05PN240 DPI 62 (±4.4) 61 (±4.7) (125) (108) Trojan/Havoc VIR 64 (±3.5) 58 (±3.6) 10.0 (200) (190) Trojan/Chief CL VIR 47 (±3.6) 42 (±3.5) (200) (200) 2.3. Experiment 3: The Eﬀect of Triﬂuralin on Green and Albino Plant Regeneration and Chromosome Doubling periment 3: The Eﬀect of Triﬂuralin on Green and Albino Plant Regeneration and Chromosome Doubling All triﬂuralin treatments had a signiﬁcant negative eﬀect on green plant regeneration in Tammarin Rock (p < 0.001). The control treatment yielded 31.8 green plants per 20 anthers (approximately 95 green plants per spike) compared with 9–25 green plants per 20 anthers in the triﬂuralin treatments (Table 4). When anthers were exposed to 1 µM triﬂuralin for 24 h, the number of green plants per 20 anthers decreased signiﬁcantly, from 31.8 in the control treatment to 25.1 in the triﬂuralin treatment (21% reduction). Extending the exposure time to 48 h caused further signiﬁcant reductions in green plant numbers, to 20.0 green plants per 20 anthers (37% reduction). Larger reductions in green plant numbers occurred when anthers were exposed to 3 µM triﬂuralin, declining from 31.8 to 10.8 green plants per 20 anthers (66% reduction) after 24 h exposure and from 9.2 green plants per 20 anthers (71% reduction) after 48 h. Triﬂuralin also had a signiﬁcant eﬀect on albino plant numbers (p < 0.001), with signiﬁcantly more albino plants produced in the 1 µM/48 h treatment and signiﬁcantly fewer albino plants produced in both the 3 µM treatments, compared to the control (Table 4). Table 4. Means (±SE) of green and albino plant numbers, expressed per 20 anthers, and chromosome doubling (% DH) following triﬂuralin treatment during anther culture on Tammarin Rock in 2018. Treatment Green Plants per 20 Anthers Albino Plants per 20 Anthers No. 2. Results Experiment 2: The Eﬀect of Caﬀeine on Green Plant Regeneration and Chromosome Doubling (Five Crosses) 2.2. Experiment 2: The Eﬀect of Caﬀeine on Green Plant Regeneration and Chromosome Doubling (Five Crosses) Caffeine treatment resulted in significantly higher green plant regeneration in Trojan/Havoc (p < 0.001, full analysis) and Lancer/LPB14-0392 (p < 0.001, post-hoc analysis). In Trojan/Havoc, green plants per 30 anthers increased by 14%, from 141.1 in the control to 161.0 following caffeine treatment (Table 2). In Lancer/LPB14-0392, green plants per 30 anthers increased by 27%, from 32.8 (control) to 41.8 (caffeine). Caffeine had no significant effect on green plant numbers in the remaining three crosses (p = 0.672). Genotype eﬀects were signiﬁcant in all analyses (p < 0.001), reﬂecting the variation in green plant regeneration between crosses. Green plant regeneration varied widely between the ﬁve crosses, ranging from 6 to 151 green plants per 30 anthers (approximately 12–302 green plants per spike) (Table 2). Caﬀeine treatment did not signiﬁcantly aﬀect the frequency of chromosome doubling in any of the ﬁve crosses in this experiment (Table 3). The diﬀerences between treatments were small (5% or less) and not signiﬁcant at either of the two grow-out locations, the Department of Primary Industries and Regional Development, South Perth (DPI) (p = 0.584) or Virginia, South Australia (VIR) (p = 0.245). The GT interaction was also non-signiﬁcant at both locations (p = 0.52 (DPI) and p = 0.868 (VIR)), indicating that the crosses responded in a similar manner. The frequency of chromosome doubling between crosses, however, varied signiﬁcantly at both locations (p < 0.001), with genotype means ranging from 31% in Lancer/LPB14-0392 to 61% in Trojan/Havoc and Scepter/05PN240. Table 2. Means (±SE) of green plant numbers, expressed per 30 anthers, following caﬀeine treatment during anther culture on ﬁve crosses in 2018. Table 2. Means (±SE) of green plant numbers, expressed per 30 anthers, following caﬀeine treatment during anther culture on ﬁve crosses in 2018. Cross Green Plants per 30 Anthers Control Caﬀeine (0.5 mM/24 h) 5% LSD Trojan/Havoc 141.1 (±4.71) 161.0 (±4.88) 10.94 Lancer/LPB14-0392 32.8 (±2.14) 41.8 (±2.25) 5.21 Coolah/LPB16-3182 14.9 (±1.91) 14.4 (±1.90) Trojan/Chief CL 28.7 (±2.10) 25.3 (±2.05) Scepter/05PN240 5.9 (±1.81) 6.4 (±1.81) 5 of 14 Plants 2020, 9, 105 Table 3. Means (±SE) of chromosome doubling (% DH) following caﬀeine treatment during anther culture on ﬁve crosses in 2018. 3. Discussion Exposing anthers to caﬀeine or triﬂuralin at the start of the induction phase yielded mixed results in this study. Caﬀeine treatment at 5 mM for 24 h signiﬁcantly improved green plant production in two of the six spring wheat crosses but had no eﬀect on the other four crosses. The improvements were observed in two responsive crosses, Trojan/Havoc and Lancer/LPB14-0392, where green plant numbers increased by 14% and 27% to 161 and 42 green plants per 30 anthers, respectively. Increasing the time anthers were exposed to caﬀeine from 24 to 48 h signiﬁcantly reduced the number of green plants in Experiment 1, where only one cross was tested. Caffeine had no significant effect on the frequency of chromosome doubling despite higher doubling in Experiment 1, from 56% in the control to 67–68% in three of the four caffeine treatments. When the results from Experiment 1 were expressed as a success index (DHs per 20 anthers), the 0.5 mM/24 h caffeine treatment yielded sufficient improvement to warrant further investigation, with 29.5 DHs per 20 anthers, compared with 20.1 in the control. However, when this treatment was tested on five crosses in Experiment 2, there was little difference between the control and caffeine treatment means (5% or less). p To our knowledge, caﬀeine has not been tested as an in vitro doubling agent in anther or microspore culture. Although caﬀeine did not signiﬁcantly improve doubling in this study, it did result in chromosome doubling and restored fertility in wheat haploids when applied as immersion/root dipping treatments [55]. Caﬀeine may also promote embryogenesis, given that it can aﬀect phragmoplast microtubules during cell division and cytokinesis [51] and its application resulted in modest improvements in green plant production in some genotypes in this study. It is widely accepted that the cytoskeleton is involved in reprogramming microspores toward androgenesis [1,20]. The disruption of spindle microtubules by colchicine and anti-mitotic herbicides has stimulated microspore embryogenesis in several species (see Section 1) and the disruption of cortical microtubules by n-butanol has stimulated embryogenesis in wheat [12,13]. In terms of this study, the concentration of caﬀeine may have been too low. The selected concentrations were based on previous studies with colchicine and caﬀeine. 2. Results of Plants Transplanted % DH Control 31.8 (±1.73) 20.1 (±2.27) 225 38 (±3.3) 1 µM triﬂuralin for 24 h 25.1 (±2.15) 24.7 (±3.47) 100 49 (±5.1) 1 µM triﬂuralin for 48 h 20.0 (±2.02) 27.8 (±3.68) 100 51 (±5.1) 3 µM triﬂuralin for 24 h 10.8 (±1.77) 4.0 (±1.47) 97 46 (±5.3) 3 µM triﬂuralin for 48 h 9.2 (±1.72) 6.1 (±1.82) 78 53 (±5.8) 5% LSD 4.16 7.44 10% LSD 1 11.5 1 A 10% LSD was used for % DH in this experiment as the omnibus p-value is between 0.05 and 0.1. Table 4. Means (±SE) of green and albino plant numbers, expressed per 20 anthers, and chromosome doubling (% DH) following triﬂuralin treatment during anther culture on Tammarin Rock in 2018. In contrast to green plant regeneration, trifluralin had a significant positive effect on the frequency of chromosome doubling in Tammarin Rock (p < 0.10). All trifluralin treatments had higher rates of chromosome doubling than the control, with significant improvements over the control in both 48 h treatments (Table 4). In these treatments, doubling increased from 38% (control) to 51% and 53% in the 1 µM and 3 µM trifluralin treatments, respectively. Despite improved doubling frequencies following treatment with trifluralin, the reductions in green plant regeneration meant that the success index values (DHs per 20 anthers) for trifluralin treatments were either similar to or less than the control (Table 5). 6 of 14 Plants 2020, 9, 105 Table 5. Success index, expressed as DHs per 20 anthers, following caﬀeine treatment on the (Yr57/3Gladius#60)/2Trojan cross and triﬂuralin treatment on Tammarin Rock. Caﬀeine Treatment DHs per 20 Anthers Triﬂuralin Treatment DHs per 20 Anthers Control 20.1 Control 10.7 0.5 mM caﬀeine for 24 h 29.5 1 µM triﬂuralin for 24 h 11.2 0.5 mM caﬀeine for 48 h 13.4 1 µM triﬂuralin for 48 h 9.7 1.5 mM caﬀeine for 24 h 25.5 3 µM triﬂuralin for 24 h 5.1 1.5 mM caﬀeine for 48 h 18.4 3 µM triﬂuralin for 48 h 4.6 3. Discussion For example, colchicine is generally applied at 0.1% (w/v) (2.5 mM) for immersion/root dipping treatments in cereals [16,17] but at lower concentrations (0.3 to 1.0 mM) when applied in vitro to anther and microspore cultures [18,25]. When caﬀeine was tested in a series of immersion/root dipping treatments in wheat, 3 g/L (15.4 mM) for 24 h was the most successful treatment in terms of seed recovery and the size and incidence of fertile sectors [55]. To test the in vitro application of caﬀeine in this study, concentrations of 0.5 and 1.5 mM were selected. The fact that there were no signiﬁcant improvements in doubling, however, indicates that higher concentrations of caﬀeine may be required. Anther walls may also act as a ﬁlter, preventing the absorption of caﬀeine. Soriano et al. [18] applied colchicine during anther and microspore culture to the wheat variety Pavon and obtained smaller improvements in doubling with anther culture compared to microspore culture. Based on the results of Pulido et al. [56], they proposed that the anther wall may act as a ﬁlter, preventing colchicine absorption. We may be seeing the same eﬀect, especially as dimethyl sulfoxide (DMSO) was not included in our experiments with caﬀeine. Given the preliminary results from this study, it would be beneﬁcial to test higher concentrations of caﬀeine for 24 h or less and include DMSO in the treatment. It would also be useful to test more than one genotype so that a more robust treatment can be identiﬁed. Plants 2020, 9, 105 7 of 14 In contrast to caﬀeine, triﬂuralin signiﬁcantly decreased green plant regeneration and signiﬁcantly increased chromosome doubling in the variety Tammarin Rock. The control treatment yielded 31.8 green plants per 20 anthers (approximately 95 green plants per spike), compared with 9–25 green plants per 20 anthers in the triﬂuralin treatments. Green plant numbers were reduced by 21% to 71% in the triﬂuralin treatments, with increasing concentration and exposure times resulting in stepwise signiﬁcant reductions in the number of green plants. However, every triﬂuralin treatment had higher rates of chromosome doubling than the control, with signiﬁcant improvements in both treatments where anthers were exposed to triﬂuralin for 48 h. In these treatments, doubling improved from 38% (control) to 51% and 53% in the 1 µM and 3 µM triﬂuralin treatments, respectively. 3. Discussion Despite the improved doubling frequencies following triﬂuralin treatment, the reductions in green plants meant that the success indices for the triﬂuralin treatments were either similar to the control (~10–11 DH per 20 anthers) or less than the control (~5 DHs per 20 anthers). The concentrations and exposure times of triﬂuralin selected for use in this study (1 and 3 µM) were based on the results of Hansen and Andersen [37], who tested both triﬂuralin and APM at concentrations ranging from 0.1 to 10 µM in a wheat microspore study. They observed that low concentrations could stimulate embryo production (relative to the treated controls) and plant regeneration, but higher concentrations reduced embryo and plant numbers. In contrast, the percentage of fertile diploid plants increased steadily with increasing concentrations of triﬂuralin or APM. When the results were combined in a success index (DHs per spike), the best results were obtained at concentrations between 1 and 3 µM. In the present anther culture study, we did not observe any positive eﬀects of triﬂuralin on green plant production, even with low concentrations (1 µM) of triﬂuralin. This might reﬂect the fact that we used anther culture and not microspore culture. In our experiment, the triﬂuralin (dissolved in DMSO) solution would have penetrated the anthers and remained in contact with microspores after the treatment ﬁnished. In contrast, Hansen and Andersen [37] rinsed their microspores following treatment with the herbicides. Given our results, it may be useful to try a rinse step, as well as more exposure time/concentration combinations. Additionally, we can test other culture phases such as embryos and alternative solvents such as acetone [34]. This study was a preliminary investigation into the application of in vitro doubling agents to improve the frequency of chromosome doubling following anther culture. Further work is required to identify a treatment regime with caﬀeine and/or anti-mitotic herbicides that consistently increases chromosome doubling but does not signiﬁcantly reduce green plant production. 4.2.1. Anther Culture Twenty spikes were harvested from three F1 donor plants of the cross, (Yr57/3Gladius#60)/2Trojan (6–7 spikes per plant). The spikes were harvested over 4 days and stored at 4 ◦C for up to 6 days. Spikes were harvested when microspores were at the late uninucleate stage, although in some spikes, mitosis was visible. Microspore stage was determined by squashing anthers in 2% acetocarmine stain (w/v) and examining the microspores under 400× magniﬁcation. All spikes were sterilized and processed on the same day using the protocol described in Broughton et al. [57]. The media used were (1) solid 1.0 M mannitol pretreatment medium, (2) liquid induction medium (LIM) and (3) solid regeneration medium, as described in Broughton et al. [57] with iron-source ethylenediaminetetraacetic acid ferric sodium salt (FeNaEDTA). The anther culture protocol involved a number of steps, including (1) anther pretreatment to induce embryogenesis (mannitol (5 days) and n-butanol (5 h)); (2) an induction phase, in which anthers and ovaries were co-incubated in LIM for 4–5 weeks, and (3) a regeneration phase, in which plants were grown on solid regeneration medium prior to transfer to soil (4–5 weeks). Following spike sterilization, 60 anthers were removed from each spike and placed on a 90 × 14 mm Petri dish containing mannitol pretreatment medium. Each 90 mm dish was divided into three sections by marking the base of the dish. The anthers were taken from ﬁve spikelets (10 ﬂorets) on each side of the spike, making 10 spikelets (20 ﬂorets) in total. Each ﬂoret contained three anthers, which were separated after removal from the ﬂoret and placed on a diﬀerent section of the dish so they could later be allocated to the three treatments (20 anthers per spike/treatment combination). This design was used to ensure that anthers from each spike were evenly divided between the three treatments. Immediately following anther removal, 20 ovaries were removed from each spike and placed in two 55 × 14 mm Petri dishes containing 4 mL LIM (10 ovaries per dish). Additional spikes at the same stage of development were also processed for ovary removal, so there was a dish of ovaries available for each of the 60 sets of anthers. All dishes were sealed with Paraﬁlm™and incubated in the dark at 25 ◦C for 5 days. 4.1. Germplasm and Donor Plant Growth A range of spring wheat (Triticum aestivum L.) crosses and the variety, Tammarin Rock, was used in this study (Table 6). The F1 seed for all crosses was provided by LongReach Plant Breeders Management Pty Ltd. (Adelaide, Australia). Donor plant growth was as described in Broughton et al. [57] with plants grown in controlled environment rooms at 18/13 ◦C (day/night) with a 12 h photoperiod. 8 of 14 Plants 2020, 9, 105 Table 6. Spring wheat crosses/varieties used in this study. Table 6. Spring wheat crosses/varieties used in this study. Table 6. Spring wheat crosses/varieties used in this study. Experiment Cross or Variety Sowing Date No. of Donor Plants Exp 1: The eﬀect of caﬀeine on green and albino plant regeneration and chromosome doubling (one cross) [Yr57/3Gladius#60]/2Trojan 3/3/17 3 Exp 2: The eﬀect of caﬀeine on green plant regeneration and chromosome doubling (ﬁve crosses) Trojan/Havoc 2/3/18 5 Trojan/Chief CL 2/3/18 5 Lancer/LPB14-0392 23/3/18 6 Coolah/LPB16-3182 23/3/18 6 Scepter/05PN240 6/4/18 6 Exp 3: The eﬀect of triﬂuralin on green and albino plant pant regeneration and chromosome doubling Tammarin Rock 13/7/18 & 27/7/18 8 4.2. Experiment 1. The Eﬀect of Caﬀeine on Green and Albino Plant Regeneration and Chromosome Doubling (One Cross) 4.2. Experiment 1. The Eﬀect of Caﬀeine on Green and Albino Plant Regeneration and Chromosome Doubling (One Cross) 4.2.1. Anther Culture Following the 5 day mannitol pretreatment, each of the three sets of 20 anthers (per spike) were transferred to three 55 × 14 mm Petri dishes containing 4 mL LIM and denoted “A”, “B” and “C”. When anthers from all spikes had been transferred, 8 µL n-butanol (0.2% v/v) was added to each dish (60 dishes in total). Dishes were covered with alfoil (but left unsealed) and incubated in the dark at 25 ◦C for 5 h. After 5 h, the LIM plus n-butanol solution was removed from each dish using a sterile pipette. The 20 dishes denoted “A” were treated as controls, and the standard protocol was applied with a dish of ovaries and ovary-conditioned LIM tipped into each dish of pre-treated anthers. If any ovaries remained in the dish and did not get tipped, they were gently transferred with forceps. Dishes Plants 2020, 9, 105 9 of 14 were sealed with Paraﬁlm™, and the anthers and ovaries were co-incubated in the dark at 25 ◦C for 4–5 weeks. Fresh LIM (4 mL) was added to the anthers in the “B” and “C” dishes, and caﬀeine stock (0.5 mL) was added to each dish. Caﬀeine stocks were prepared by dissolving caﬀeine (C0750, Sigma-Aldrich, Sydney, Australia) in deionized water, followed by ﬁlter sterilization. Diﬀerent concentrations of caﬀeine stocks were prepared, so the same amount of caﬀeine stock was added to the LIM for each treatment. For spikes 1–10, the ﬁnal caﬀeine concentration was 0.5 mM, while for spikes 11–20, the caﬀeine concentration was 1.5 mM. The anthers were exposed to caﬀeine for either 24 (“B” dishes) or 48 h (“C” dishes) (Table 6). There were 10 dishes (replicates) for each spike/treatment combination. After 24 or 48 h, the caﬀeine plus LIM solution was removed, and a dish of ovaries and ovary-conditioned LIM was added to each dish of anthers. Dishes were sealed with Paraﬁlm™, and the anthers and ovaries were co-incubated in the dark at 25 ◦C for 4–5 weeks. 4.2.2. Plant Regeneration and Grow-Out After 4–5 weeks, each dish of embryos (60) was tipped onto one dish of regeneration medium in a 90 × 20 mm Petri dish. Excess LIM was tipped oﬀor removed by pipette. Dishes were sealed with Paraﬁlm™and incubated in the dark at 25 ◦C for 7 days, then transferred to a constant temperature room with a 12 h photoperiod (light/dark) at 25 ◦C for a further 7–14 days. Individual green plants were then sub-cultured to fresh regeneration medium (approximately 5–10 plants per dish) in 90 × 20 mm Petri dishes. At this stage, any albino plants were counted and discarded as well as any excess green plants (>10). The total number of green and albino plants for each treatment/spike combination was recorded. Dishes of sub-cultured green plants were returned to the constant temperature room for a further 2–3 weeks until root growth was suﬃcient, and the plants were ready to be transplanted to soil. Five green plants from each treatment/spike combination were transplanted to soil (50 plants per treatment). There were more plants transplanted from the control treatment (100), as a control was included in each set of 10 spikes. Plants were transplanted to soil at DPIRD greenhouses in South Perth and grown to maturity to obtain DH plants. Ploidy was determined visually, and plants that set seed were classed as DHs while sterile plants were classed as haploids. 4.3. Experiment 2. The Eﬀect of Caﬀeine on Green Plant Regeneration and Chromosome Doubling (Five Crosses) 4.3. Experiment 2. The Eﬀect of Caﬀeine on Green Plant Regeneration and Chromosome Doubling (Five Crosses) 4.4.1. Anther Culture The application of triﬂuralin was tested on the variety Tammarin Rock. This variety was screened in an earlier study, in which it exhibited good green plant regeneration but a relatively low frequency of chromosome doubling (30%) [11]. Eight donor plants were sown over two sowing dates (Table 5). Spikes 1–10 were harvested from the ﬁrst sowing date, and spikes 11–20 were harvested from the second sowing date. Spikes were harvested over 6 days and stored at 4 ◦C for up to 2 days. The experimental design and protocol was the same as Experiment 1 with anthers from spikes 1–10 exposed to three treatments (control, 1 µM triﬂuralin for 24 h and 1 µM triﬂuralin for 48 h) and anthers from spikes 11–20 exposed to diﬀerent treatments (control, 3 µM triﬂuralin for 24 h and 3 µM triﬂuralin for 48 h) (Table 7). As per Experiment 1, anthers were exposed to triﬂuralin immediately following mannitol and n-butanol pretreatment at the start of the induction phase. Triﬂuralin (N13689, Sigma-Aldrich) was dissolved in sterile dimethyl sulfoxide (DMSO) (D2650, Sigma-Aldrich), and 10 or 30 µL of triﬂuralin stock was added to 4 mL LIM to provide ﬁnal concentrations of 1 or 3 µM, respectively. 4.4.2. Plant Regeneration and Grow-Out Embryos and regenerant plants were processed in the same manner as Experiment 1 except that 10 plants per treatment/spike combination (100 plants per treatment) were transplanted to soil, where possible. Plant numbers were reduced slightly in the 3 µM triﬂuralin treatments (due to the negative eﬀect of the treatments) and, as per Experiment 1, there were more plants transplanted from the control treatment, as a control was included in each set of 10 spikes. 4.3.2. Plant Regeneration and Grow-Out Embryos and regenerant plants were processed and harvested in the same manner as Experiment 1 with minor modiﬁcations. Firstly, albino plants were not counted. Secondly, a diﬀerent grow-out location was used in South Australia for two of the crosses (Trojan/Havoc and Trojan/Chief CL) due to limitations with greenhouse space at DPIRD South Perth (DPI). Due to the space constraints, other alternatives for regenerant plant grow-out were explored for our program. For this study, a small, specialized company that provides grow-out and seed-bulking services for cereals in Virginia (VIR), South Australia was used, and plants were transferred to this location. Ten to ﬁfteen plants for each treatment/spike combination (400–600 plants per cross) were transplanted to soil. This was not possible for Scepter/05PN240, as green plant numbers were low for this cross, so only 233 regenerant plants were transplanted to soil (Table 3). Where possible, we attempted to balance plant numbers across treatments and spikes. 4.4. Experiment 3. The Eﬀect of Triﬂuralin on Green and Albino Plant Regeneration and Chromosome Doubling 4.3.1. Anther Culture The 0.5 mM/24 h caﬀeine treatment was selected for further evaluation of ﬁve crosses that were being processed for DH production (Table 7). Spikes from F1 donor plants were harvested over 5 days and stored at 4 ◦C for up to 3 days. The anther culture protocol was the same as Experiment 1, but the design was simpliﬁed to two treatments: control and caﬀeine (0.5 mM/24 h). For each cross, 60 anthers were removed from each spike, and anthers from each side of the spike (30) were placed on separate sections of a 90 × 14 mm Petri dish containing mannitol pretreatment medium. Following anther removal, 20 ovaries were removed from each spike and placed in two 55 × 14 mm Petri dishes containing 4 mL LIM (10 ovaries per dish). All dishes were sealed with Paraﬁlm™and incubated in the dark at 25 ◦C for 5 days. As per Experiment 1, anthers were exposed to caﬀeine immediately following mannitol and n-butanol pretreatment at the start of the induction phase. Following the removal of LIM plus n-butanol, anthers were either processed using the standard protocol (control) or treated with 0.5 mM caﬀeine in LIM, for 24 h, as per Experiment 1. 10 of 14 Plants 2020, 9, 105 Table 7. Caﬀeine and triﬂuralin treatments for Experiments 1 and 3. Anthers were exposed to the chemicals at the start of the induction phase. Experiment Spike Numbers Treatment A Treatment B Treatment C Experiment 1 Spikes 1–10 Control 0.5 mM caﬀeine for 24 h 0.5 mM caﬀeine for 48 h Spikes 11–20 Control 1.5 mM caﬀeine for 24 h 1.5 mM caﬀeine for 48 h Experiment 3 Spikes 1–10 Control 1 µM triﬂuralin for 24 h 1 µM triﬂuralin for 48 h Spikes 11–20 Control 3 µM triﬂuralin for 24 h 3 µM triﬂuralin for 48 h 4 3 2 Plant Regeneration and Grow Out Table 7. Caﬀeine and triﬂuralin treatments for Experiments 1 and 3. Anthers were exposed to the chemicals at the start of the induction phase. 4.3.2. Plant Regeneration and Grow-Out 4.5. Data Analysis All analyses were performed using Genstat Edition 19 (http://genstat.com). The HGLM procedure was used to ﬁt hierarchical generalized linear models for green and albino plants, and generalized linear models for % DH. A ﬁxed eﬀect of treatment was considered for all experiments. For Experiment 2, genotype and genotype by environment interactions (GT) were also considered. Random eﬀects of 11 of 14 Plants 2020, 9, 105 source spike were considered for all responses; these were not signiﬁcant for % DH and not included in the reported models. Over-dispersion occurs when the data are more variable than the standard assumptions of a generalized linear model allow. This can be accounted for by allowing the dispersion parameter to be estimated as part of the modeling process. All models were run with and without (ﬁxed value at 1) an estimation of the dispersion parameter. When the dispersion parameter was signiﬁcant, outputs were reported from the model with an estimated dispersion parameter. p-values reported were from Wald tests for the dropping of ﬁxed terms. pp g The numbers of green and albino plants were analyzed using a Poisson model. An identity link ction was used to generate symmetrical LSDs. For green plants in Experiment 2, genotype and GT diﬀerences were explored using an iterative post-hoc analysis. At each stage, the genotype with the largest detected eﬀect was omitted, and the GT analysis was rerun. This was repeated until no signiﬁcant genotype eﬀects were identiﬁed. One genotype (Trojan/Havoc) was particularly responsive in terms of green plant production; removing it from the analysis enabled us to detect diﬀerences between treatments in another genotype. For albino plants in Experiment 3, one spike for Tammarin Rock was omitted from the analysis, as very low numbers of albino plants caused model ﬁtting to fail. The percentage (proportion) of DH plants was calculated as the total number of DHs/number of transplanted plants that survived to maturity × 100 and analyzed using a Binomial model with the identity link function. For Experiment 2, analyses were done separately for the two grow-out locations, as the locations diﬀered for several variables, including pot size and fertilizer regime, and environments, including temperature and radiation. Treatment means were compared using 5% least signiﬁcant diﬀerences (LSDs). In one instance (Experiment 3, % DH), diﬀerences were only observed at the 10% level and a 10% LSD was reported. 4.5. Data Analysis LSDs were calculated by multiplying the average standard error of diﬀerence (SED) by 2 (5% LSD) or 1.645 (10% LSD). The success index for Experiments 1 and 3 was calculated for each treatment/spike combination by multiplying the number of green plants by % DH to obtain the number of DHs per 20 anthers. Values were then averaged for each treatment. Author Contributions: S.B. designed the experiments, prepared an original draft of the manuscript, managed project administration and funding acquisition; M.C., L.L. and J.K. carried out the experiments and M.C. reviewed and edited the manuscript; A.H. and R.O. carried out statistical analyses and assisted with editing the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: This research was co-funded by an Australian Federal Government Innovations Grant, ICG Ref: RC55487, LongReach Plant Breeders Management Pty Ltd. and the Western Australian Department of Primary Industries and Regional Development. Acknowledgments: Plant grow-out services provided by Andrew Mathews (Red Earth Vegies) in Virginia, South Australia, are gratefully acknowledged. 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. 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https://openalex.org/W4372334165	https://www.nature.com/articles/s41408-023-00838-2.pdf	English	null	Characteristic immunophenotype and gene co-mutational status orchestrate to optimize the prognosis of CEBPA mutant acute myeloid leukemia	Blood cancer journal	2,023	cc-by	2,899	Dear Editor, Mutation on CEBPA (CEBPAmut) is one of the most common molecular abnormalities in acute myeloid leukemia (AML), especially in east Asian population [1]. As recently reported, in- frame mutations in bZIP domain of CEBPA (CEBPAbZIP-inf) exerted higher potency in favorable-risk prediction than biallelic mutated CEBPA (CEBPAbi), although cases were highly overlapped between the two categories [2–4]. However, about 30–50% CEBPAbi AML cases consolidated with chemotherapy alone suffered from disease relapse [5, 6], retaining the same CEBPAmut patterns as diagnosis [7, 8]. Hence, there might be clinically and biologically heterogeneous under current context of CEBPAmut grouping, and a comprehensive assessment of CEBPAmut AML prognosis remains to be established. t p y pp y The distribution of co-mutations was illustrated in Fig. 1A. The co-mutations of the 117/124 CR patients were categorized into Transcriptional Factors (TFs, 31/117, 26.5%), Chromatin/Cohesion/ Spliceosome (CCS, 54/117, 46.2%), Receptor Tyrosine Kinases (RTKs, 49/117, 41.9%), Tumor Suppressor (TS, only WT1 mutation in this group, 23/117, 19.7%) and Nucleolar (only NPM1 mutation in this group, 7/117, 6.0%). Notably, CEBPAbZIP-inf/CD7 + AML were more frequently accompanied with mutations in TFs than other CEBPAmut AML (32.1% vs. 12.1%, p = 0.027). Whereas mutations in CCS were highly enriched in other CEBPAmut AML compared to CEBPAbZIP-inf/CD7 + AML (60.6% vs. 40.5%, p = 0.049). No Nucleolar (NPM1) mutations were found in CEBPAbZIP-inf/CD7 + AML, while 21.2% of the rest CEBPAmut patients harboring NPM1 mutations (p < 0.001; Supplementary Table 3). In this study, a total of 293 de novo CEBPAmut AML patients were enrolled, with biological data available in 124 patients (Supple- mentary Fig. 1A). Usually, CEBPAbZIP-inf AML patients were diagnosed at younger age, with higher white blood cell counts, hemoglobin levels and lower platelet counts compared with other CEBPAmut AML patients (CEBPAother); while risk classiﬁcation of karyotypes according to the ELN 2022 showed no differences between CEBPAbZIP-inf and CEBPAother AML (Supplementary Table 1). pp y Consistence with previous reports [2, 3], CEBPAbZIP-inf AML correlated with higher CR rate (Supplementary Table 1), which could translate into improved overall survival (2-year OS: 86% vs. 53.1%, p = 0.0019) and event-free survival (2-year EFS: 64.7% vs. 37.5%, p = 0.01) (Supplementary Fig. 1B). However, the OS, EFS and relapse rate (Supplementary Table 1) of the patients who achieved CR showed no difference between CEBPAbZIP-inf and CEBPAother AML (2-year OS: 87% vs. 63.1%, p = 0.07; 2-year EFS: 65.5% vs. Blood Cancer Journal Blood Cancer Journal www.nature.com/bcj Blood Cancer Journal (2023) 13:70 ; https://doi.org/ 10.1038/s41408-023-00838-2 CD7-negative CEBPAmut AML patients also had a shorter disease- free survival compared with CD7-positive patients (2-year DFS: CD7-positive CEBPAmut AML 63.1% vs. CD7-negative CEBPAmut AML 39.4%, p < 0.0001; Supplementary Fig. 3C). p pp y g Given the prognostic signiﬁcance of CD7 in CEBPAmut AML, survival analysis of 117 CR patients demonstrated that the combine of CD7 with the CEBPAmut locus could discriminate disease prognosis, with distinguished 2-year OS and EFS: CEBPAbZIP-inf/ CD7 + AML, 90.4% and 68.8% vs. other CEBPAmut AML, 56.6% and 41.3%, respectively (p < 0.0001 and = 0.0076; Supplementary Fig. 3D, E). Besides, the 2-year DFS of CEBPAmut AML was as follows: CEBPAbZIP-inf/CD7 + AML 72.5% vs. other CEBPAmut AML 40.6%, (p = 0.0028; Supplementary Fig. 3F). Multivariable analysis further conﬁrmed CEBPAbZIP-inf/CD7+ as an independent risk factor that favors the prognosis of CEBPAmut AML, with hazard ratio of 0.16 (p = 0.001), 0.45 (p = 0.034), and 0.39 (p = 0.018) in OS, EFS and DFS, respectively (Supplementary Table 2). Received: 12 February 2023 Revised: 4 April 2023 Accepted: 12 April 2023 CORRESPONDENCE OPEN Characteristic immunophenotype and gene co-mutational status orchestrate to optimize the prognosis of CEBPA mutant acute myeloid leukemia © The Author(s) 2023 Blood Cancer Journal (2023) 13:70 ; https://doi.org/ 10.1038/s41408-023-00838-2 Dear Editor, 47.7%, p = 0.19) (Supplementary Fig. 1C). It seemed that the current risk stratiﬁcation based on CEBPAmut locus could not sufﬁciently distinguish certain patients who may develop disease progression. b f The corresponding clinical impacts of co-mutations were involved into prognosis evaluation. We categorized the co- mutations into groups (CCS, RTKs, TS, Nucleolar) to avoid the interference of low-frequency mutations as independent variables for hazard analysis. Multivariate Cox regression analysis showed TS (WT1 mutations, WT1mut) signiﬁcantly affected the OS of CEBPAmut AML, with risk ratio (RR) of 3.275, p = 0.0223 (Fig. 1B). Further analysis indicated WT1mut could signiﬁcantly shorten the survival of CEBPAbZIP-inf/CD7 + AML, with 2-year OS and EFS of 96.6% and 78.6% vs. 60.7% and 27.8%, respectively (p = 0.0016 and <0.0001, respectively) (Fig. 1C). p g Most patients with CEBPAbZIP-inf (85/89, 95.5%) displayed cross- lineage expression of CD7, while only 20/35 (57.1%) patients with CEBPAother harboring CD7-positive immunophenotype (p < 0.001) (Supplementary Table 1). CD7-positive cases showed distinct gene expression patterns compared with CD7 negative cases (Supple- mentary Fig. 2). Survival analysis further indicated CD7 could signiﬁcantly distinguish the clinical outcome in the whole cohort of 293 CEBPAmut AML cases, with improved 2-year OS and EFS of 81.8% and 66.4% respectively in CD7-positive CEBPAmut AML vs. 48.8% and 33.0% respectively in CD7-negative CEBPAmut AML (p < 0.0001 and <0.0001; Supplementary Fig. 3A, B). Moreover, Net reclassiﬁcation improvement (NRI) was then performed and indicated that the outcome was signiﬁcantly improved after the integration of CD7 expression and WT1 status into the clinical nomogram, with the value of 39.2% improvement [95% CI: 0.000–1.059]. Thus, we deﬁned CEBPAbZIP-inf AML patients char- acterized by immunophenotypic CD7-positive and wild-type WT1 as low-risk group (LR group: CEBPAbZIP-inf/CD7 + /WT1wt); Received: 12 February 2023 Revised: 4 April 2023 Accepted: 12 April 2023 Correspondence ig. 1 Integrating CD7 expression and WT1 mutation status for revised risk stratiﬁcation of CEBPAbZIP-inf AML patients. A The distribu of co-mutations within the cohort of 124 CEBPAmut AML patients. Genes were categorized into groups as labeled on the left. B C proportional hazard regression analysis for the categorized co-mutations independently affecting OS of 117 CR-achieved CEBPAmut A patients. TF transcriptional factor, CCS chromatin/cohesion/spliceosome, RTK receptor tyrosine kinase, TS tumor suppressor. C Kaplan–M urves for the survival of 84 CR-achieved CEBPAbZIP-inf/CD7+AML patients according to WT1 mutation status. Dear Editor, D Sankey plot for reclassiﬁca of 117 CR-achieved CEBPAmut AML patients from CEBPAbZIP-inf/CEBPAother grouping to the revised risk stratiﬁcation. E Kaplan–Meier curves he survival of 117 CR-achieved CEBPAmut AML patients according to the revised risk stratiﬁcation. F Kaplan–Meier survival curves for OS o CEBPAmut AML patients within the BeatAML cohort according to the revised risk stratiﬁcation. Correspondence 2 2 Fig. 1 Integrating CD7 expression and WT1 mutation status for revised risk stratiﬁcation of CEBPAbZIP-inf AML patients. A The distribution of co-mutations within the cohort of 124 CEBPAmut AML patients. Genes were categorized into groups as labeled on the left. B Cox- proportional hazard regression analysis for the categorized co-mutations independently affecting OS of 117 CR-achieved CEBPAmut AML patients. TF transcriptional factor, CCS chromatin/cohesion/spliceosome, RTK receptor tyrosine kinase, TS tumor suppressor. C Kaplan–Meier curves for the survival of 84 CR-achieved CEBPAbZIP-inf/CD7+AML patients according to WT1 mutation status. D Sankey plot for reclassiﬁcation of 117 CR-achieved CEBPAmut AML patients from CEBPAbZIP-inf/CEBPAother grouping to the revised risk stratiﬁcation. E Kaplan–Meier curves for the survival of 117 CR-achieved CEBPAmut AML patients according to the revised risk stratiﬁcation. F Kaplan–Meier survival curves for OS of 17 CEBPAmut AML patients within the BeatAML cohort according to the revised risk stratiﬁcation. Blood Cancer Journal (2023) 13:70 Correspondence 3 correspondingly, 19/89 patients of CEBPAbZIP-inf AML were re- stratiﬁed into the high-risk group (HR group, Fig. 1D). With the compared with HR patients (n = 13), although the differe was not signiﬁcant due to the limited sample size (n = 17, Fig. Fig. 2 Differentially expressed genes and survival analysis of two distinct sub-cohorts of patients with CEBPAmut AML. A Top ten D identiﬁed as up- (red) or down- (blue) regulated were ranked by the the magnitude of expression value change. B Volcano plot showing D according to the two distinct sub-cohorts clustered by unsupervised hierarchy. C Kaplan–Meier curves for the survival of LR (upper panel) HR (lower panel) CEBPAmut AML patients according to treatment of allo-HSCT in CR1 or chemotherapy-only. D Kaplan–Meier curves for the of HR CEBPAmut AML patients within the BeatAML cohort according to treatment of allo-HSCT or chemotherapy-only. Fig. 2 Differentially expressed genes and survival analysis of two distinct sub-cohorts of patients with CEBPAmut AML. A Top ten DEGs identiﬁed as up- (red) or down- (blue) regulated were ranked by the the magnitude of expression value change. ADDITIONAL INFORMATION 1Shanghai Institute of Hematology, State Key Laboratory of Medical Genomics, National Research Center for Translational Medicine at Shanghai, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China. 2Department of Hematology, The Second People’s Hospital of Kashi, Kashi, Xinjiang, China. 3Department of Hematology, Tongji Hospital, Tongji University School of Medicine, Shanghai, China. 4These authors contributed equally: Xinjie Chen, Diyaer Abuduaini, Yuliang Zhang. ✉email: zhm11931@rjh.com.cn; ljf12500@rjh.com.cn; yang_shen@sjtu.edu.cn Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41408-023-00838-2. Correspondence and requests for materials should be addressed to Hongming Zhu, Jianfeng Li or Yang Shen. Reprints and permission information is available at http://www.nature.com/ reprints Reprints and permission information is available at http://www.nature.com/ reprints Reprints and permission information is available at http://www.nature.com/ reprints Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. Dear Editor, B Volcano plot showing DEGs according to the two distinct sub-cohorts clustered by unsupervised hierarchy. C Kaplan–Meier curves for the survival of LR (upper panel) and HR (lower panel) CEBPAmut AML patients according to treatment of allo-HSCT in CR1 or chemotherapy-only. D Kaplan–Meier curves for the OS of HR CEBPAmut AML patients within the BeatAML cohort according to treatment of allo-HSCT or chemotherapy-only. correspondingly, 19/89 patients of CEBPAbZIP-inf AML were re- stratiﬁed into the high-risk group (HR group, Fig. 1D). With the revised stratiﬁcation, patients in LR group had a superior outcome than the patients in HR group (2-year OS: 96.6% vs. 64.4%, p < 0.0001; 2-year EFS: 78.6% vs. 36.6%, p < 0.0001; Fig. 1E). We also validate the revised stratiﬁcation in patients from BeatAML cohort. Improved OS was observed in LR patients (n = 4) correspondingly, 19/89 patients of CEBPAbZIP-inf AML were re- stratiﬁed into the high-risk group (HR group, Fig. 1D). With the revised stratiﬁcation, patients in LR group had a superior outcome than the patients in HR group (2-year OS: 96.6% vs. 64.4%, p < 0.0001; 2-year EFS: 78.6% vs. 36.6%, p < 0.0001; Fig. 1E). We also validate the revised stratiﬁcation in patients from BeatAML cohort. Improved OS was observed in LR patients (n = 4) compared with HR patients (n = 13), although the difference was not signiﬁcant due to the limited sample size (n = 17, Fig. 1F). The transcriptomic data was available in 122 (data of 2 patients were missing) CEBPAmut patients from our cohort. With unsuper- vised cluster analysis, HOXA/B family genes were identiﬁed to be highly associated with poor prognosis in CEBPAmut AML (Fig. 2A). Besides, the differentially expressed genes (DEGs) analysis also compared with HR patients (n = 13), although the difference was not signiﬁcant due to the limited sample size (n = 17, Fig. 1F). The transcriptomic data was available in 122 (data of 2 patients were missing) CEBPAmut patients from our cohort. With unsuper- vised cluster analysis, HOXA/B family genes were identiﬁed to be highly associated with poor prognosis in CEBPAmut AML (Fig. 2A). Besides, the differentially expressed genes (DEGs) analysis also Blood Cancer Journal (2023) 13:70 Correspondence 4 showed that HOXA/B family genes were highly enriched in the up- regulated gene patterns of HR patients compared with LR patients (Fig. DATA AVAILABILITY The data that support the ﬁndings of this study are available from the corresponding author upon reasonable requests. More details are provided in Supplementary Information. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http:// creativecommons.org/licenses/by/4.0/. Dear Editor, 2B); HR patients were usually accompanied with remarkably higher expression of HOXA/B family genes compared with LR patients (Supplementary Fig. 4). 5. Schlenk RF, Taskesen E, van Norden Y, Krauter J, Ganser A, Bullinger L, et al. The value of allogeneic and autologous hematopoietic stem cell transplantation in prognostically favorable acute myeloid leukemia with double mutant CEBPA. Blood. 2013;122:1576–82. 6. Su L, Tan Y, Lin H, Liu X, Yu L, Yang Y, et al. Mutational spectrum of acute myeloid leukemia patients with double CEBPA mutations based on next-generation sequencing and its prognostic signiﬁcance. Oncotarget. 2018;9:24970–9. y In addition, survival analysis revealed LR patients may not beneﬁt from allo-HSCT in CR1, with 2-year OS and EFS of 100% and 94.4% vs. 95.2% and 71.7% in chemotherapy-only, respec- tively (p = 0.23 and 0.10, respectively); whereas allo-HSCT in CR1 could signiﬁcantly improve the outcome of HR patients, with 2-year OS and EFS of 100% and 84.6% vs. 53.6% and 23.1% in chemotherapy-only, respectively (p = 0.019 and 0.0065, respec- tively) (Fig. 2C). The therapeutic efﬁcacy of allo-HSCT was also validated in patients from BeatAML cohort. There were 13 patients eligible for the criteria of HR CEBPAmut AML as we deﬁned. The survival curves were different although the small sample size limited the statistical signiﬁcance (p = 0.048, Fig. 2D). Therefore, not only CEBPAother AML patients, CEBPAbZIP-inf AML patients with negative CD7 expression or WT1mut may also be recommended for allo-HSCT as soon as CR achieved. 7. Pulikkan JA, Tenen DG, Behre G. C/EBPalpha deregulation as a paradigm for leu- kemogenesis. Leukemia. 2017;31:2279–85. 7. Pulikkan JA, Tenen DG, Behre G. C/EBPalpha deregulation as a paradigm for leu- kemogenesis. Leukemia. 2017;31:2279–85. 8. Shih LY, Liang DC, Huang CF, Wu JH, Lin TL, Wang PN, et al. AML patients with CEBPalpha mutations mostly retain identical mutant patterns but frequently change in allelic distribution at relapse: a comparative analysis on paired diagnosis and relapse samples. Leukemia. 2006;20:604–9. ACKNOWLEDGEMENTS The authors thank all the members of the Shanghai Institute of Hematology and National Research Center for Translational Medicine at Shanghai, and HOME for Researchers (https://www.home-for-researchers.com) for online analysis tools. This study was funded by the National Natural Science Foundation of China (82270116, 82000143), the Shanghai Municipal Education Commission-Gaofeng Clinical Medicine Grant Support (20161406) and Innovative research team of high-level local universities in Shanghai. All authors consent to publication. Conclusively, CD7 immunophenotype and WT1mut status is convenient for clinicians to acquire for the identiﬁcation of CEBPAbZIP-inf AML patients who are in risk of disease relapse (Supplementary Fig. 5). Evidences in our cohort are provided to support the necessity of allo-HSCT in CR1 for high-risk cases, with further validation in an independent cohort from BeatAML. For the limitation of the retrospective nature in this study, relative clinical trial may be conducted in the future to validate our results and explore the therapeutic efﬁcacy of allo-HSCT in CEBPAmut AML. COMPETING INTERESTS Xinjie Chen1,4, Diyaer Abuduaini1,2,4, Yuliang Zhang1,4, Jun Long3, Xiaojing Lin1, Hongming Zhu1✉, Jianfeng Li 1✉and Yang Shen1✉ The authors declare no competing interests. AUTHOR CONTRIBUTIONS SY, CXJ conceptualized the study; CXJ, ADY collected the original data. CXJ, ADY, ZYL, LJF, LXJ designed and performed the statistical analyses; SY, CXJ, ZYL, LJ, ZHM wrote the ﬁrst draft of the manuscript. All authors participated in revised and approved the ﬁnal manuscript. REFERENCES 1. Su L, Shi YY, Liu ZY, Gao SJ. Acute myeloid leukemia with CEBPA mutations: current progress and future directions. Front Oncol. 2022;12:806137. 1. Su L, Shi YY, Liu ZY, Gao SJ. Acute myeloid leukemia with CEBPA mutations: current progress and future directions. Front Oncol. 2022;12:806137. 2. Taube F, Georgi JA, Kramer M, Stasik S, Middeke JM, Rollig C, et al. CEBPA muta- tions in 4708 patients with acute myeloid leukemia: differential impact of bZIP and TAD mutations on outcome. Blood. 2022;139:87–103. 2. Taube F, Georgi JA, Kramer M, Stasik S, Middeke JM, Rollig C, et al. CEBPA muta- tions in 4708 patients with acute myeloid leukemia: differential impact of bZIP and TAD mutations on outcome. Blood. 2022;139:87–103. 3. Wakita S, Sakaguchi M, Oh I, Kako S, Toya T, Najima Y, et al. Prognostic impact of CEBPA bZIP domain mutation in acute myeloid leukemia. Blood Adv. 2022;6:238–47. 3. Wakita S, Sakaguchi M, Oh I, Kako S, Toya T, Najima Y, et al. Prognostic impact of CEBPA bZIP domain mutation in acute myeloid leukemia. Blood Adv. 2022;6:238–47. 4. Döhner H, Wei AH, Appelbaum FR, Craddock C, DiNardo CD, Dombret H, et al. Diagnosis and management of AML in adults: 2022 recommendations from an international expert panel on behalf of the ELN. Blood. 2022;140:1345–77. 4. Döhner H, Wei AH, Appelbaum FR, Craddock C, DiNardo CD, Dombret H, et al. Diagnosis and management of AML in adults: 2022 recommendations from an international expert panel on behalf of the ELN. Blood. 2022;140:1345–77. © The Author(s) 2023 © The Author(s) 2023 Blood Cancer Journal (2023) 13:70
https://openalex.org/W2919583668	https://sajhivmed.org.za/index.php/hivmed/article/download/982/1549	English	null	A descriptive analysis of the role of a WhatsApp clinical discussion group as a forum for continuing medical education in the management of complicated HIV and TB clinical cases in a group of doctors in the Eastern Cape, South Africa	Southern African journal of HIV medicine	2,019	cc-by	7,378	Southern African Journal of HIV Medicine ISSN: (Online) 2078-6751, (Print) 1608-9693 Original Research Page 1 of 9 Dates: Results: The analysis found the majority of participants had gained new clinical confidence from group participation. This was associated with the increased group engagement in group follow-up (odds ratio [OR] 48.13 [95% confidence interval [CI] 4.99–464.49]); in posting questions (OR 3.81 [95% CI 1.02–18.48]); in reports of ‘new’ clinical insights (OR 23.75 [95% CI 3.95–142.88]); in referencing old case material (OR 21.42 [95% CI 4.39–104.84]) and in the use of peer guidance to manage cases (OR 48.13 [95% CI 4.99–464.49]). However, there was a discrepancy in participants’ knowledge and actual use of informed consent when posting patient details on social media. How to cite this article: Woods J, Moorhouse M, Knight L. A descriptive analysis of the role of a WhatsApp clinical discussion group as a forum for continuing medical education in the management of complicated HIV and TB clinical cases in a group of doctors in the Eastern Cape, South Africa. S Afr J HIV Med. 2019;20(1), a982. https://doi. org/10.4102/sajhivmed. v20i1.982 Conclusions: Our study findings support the use of WhatsApp in a medical setting as an effective means of communication, long distance learning and support between peers and specialists. Keywords: Continuing medical education; HIV/TB; Eastern Cape; WhatsApp; Clinician. Copyright: © 2019. The Authors. Licensee: AOSIS. This work is licensed under the Creative Commons Attribution License. Read online: Scan this QR code with your smart phone or mobile device to read online. Copyright: © 2019. The Authors. Licensee: AOSIS. This work is licensed under the Creative Commons Attribution License. Read online: Scan this QR code with your smart phone or mobile device to read online. http://www.sajhivmed.org.za 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. A descriptive analysis of the role of a WhatsApp clinical discussion group as a forum for continuing medical education in the management of complicated HIV and TB clinical cases in a group of doctors in the Eastern Cape, South Africa Read online: Scan this QR code with your smart phone or mobile device to read online. Authors: Joana Woods1 Michelle Moorhouse1 Lucia Knight2 Affiliations: 1Wits Reproductive Health and HIV Institute (WRHI), Johannesburg, South Africa 2School of Public Health, University of the Western Cape, Cape Town, South Africa Corresponding author: Joana Woods, jwoods@wrhi.ac.za Dates: Received: 06 May 2019 Accepted: 13 June 2019 Published: 01 Aug. 2019 How to cite this article: Woods J, Moorhouse M, Knight L. A descriptive analysis of the role of a WhatsApp clinical discussion group as a forum for continuing medical education in the management of complicated HIV and TB clinical cases in a group of doctors in the Eastern Cape, South Africa. S Afr J HIV Med. 2019;20(1), a982. https://doi. org/10.4102/sajhivmed. v20i1.982 Copyright: © 2019. The Authors. Licensee: AOSIS. This work is licensed under the Creative Commons Attribution License. Read online: Scan this QR code with your smart phone or mobile device to read online. Background: As South Africa’s (SA) HIV programme increases in size, HIV/TB cases occur that are often beyond the clinical scope of primary healthcare clinicians. In SA’s Eastern Cape (EC) province, health facilities are geographically widespread, with a discrepancy in specialist availability outside of academic institutions. The aim of this study is to describe WhatsApp and its use as an alternative learning tool to improve clinicians’ access to specialised management of complicated HIV/TB cases. Objectives: To analyse clinicians’ use of the WhatsApp chat group as a learning tool; to assess clinicians’ confidence in managing complicated HIV and TB patients after participating in the WhatsApp case discussion group; to describe the perceived usefulness of the chat group as a learning tool; to understand clinicians’ knowledge and use of informed consent when sharing patient case details on a public platform such as WhatsApp. Method: An observational, cross-sectional study was conducted among a group of clinicians from the EC that formed part of a WhatsApp HIV/TB clinical discussion group. Data were collected using a structured anonymous Internet questionnaire and analysed with Epi Info, using descriptive and analytic statistics. Copyright: © 2019. The Authors. Licensee: AOSIS. This work is licensed under the Creative Commons Attribution License. Open Access Background These are often difficult to attend which limits training to attendees only.1 This is particularly applicable to clinicians working in the Eastern Cape (EC), a predominantly rural province, where the clinicians enrolled in this study are working. In South Africa, 46% of the population live in rural areas, but only 19% of the nursing workforce and 12% of physicians practise in those areas.2 The EC has a population of 7 million and an HIV prevalence of 12.1%.4 Approximately 4.1 million of the population live in rural communities.5 In this setting, district hospitals and public health clinics are often geographically widespread, with only three academic or tertiary centres servicing these facilities.6 In addition, the province has only four infectious disease (ID) specialists to provide expert care to its seriously ill HIV and TB patients. Per population size, South Africa (SA) has the largest HIV epidemic in the world. The overall HIV prevalence rate is approximately 12.6%. Similarly, the country’s TB burden is large. In 2016, SA recorded 438 000 new TB infections.7 TB was the leading cause of death in the country. HIV treatment and care is often complicated by the emergence of drug resistance, drug–drug interactions and the advanced immune suppression of newly diagnosed patients. A WhatsApp messenger chat group was created in 2016 for doctors who had attended an advanced HIV management course, and were working in district hospitals in the EC. The group included medical specialists and members of the district clinical support team. Clinicians posted complicated cases. The discussion that followed referenced national and international HIV guidelines and evidence-based clinical care. This provided cumulative medical expertise that assisted the clinician in the management of the case. The use of smartphone technology and MIM platforms in clinical practice is a research topic that is gaining support. Since January 2017, there are 1.2 billion active WhatsApp users worldwide.8 This service offers users the following features: the transmission of text messages, images and videos to contacts and a chat group feature that allows 256 users to share content simultaneously.9 It is important to know if this intervention is of benefit to doctors, particularly those without onsite specialist support in the South African healthcare context. It is also important to know if clinicians are aware of local occupational governing authority rules relating to patient confidentiality breaches when posting on social media. Background Many nations face problems of inequitable access to healthcare services and the shortage of suitably qualified healthcare professionals. An insufficient number of medical graduates; a scarcity of postgraduate education; the migration of healthcare professionals and a critical shortage of teaching faculty demonstrate a need for alternative approaches to improving the retention of the healthcare workforce.1 A possible contributing solution to this problem is found in continuing medical education (CME). 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. Countries must retain health professionals by providing them with opportunities for career development, CME, motivation and support.2 The evidence shows that career development and CME strongly motivate health professionals to stay in their own countries and to practise in remote areas.3 However, many health professionals struggle to access CME because of professional http://www.sajhivmed.org.za Page 2 of 9 Page 2 of 9 Original Research in the United States, or the Data Protection Directive in the European Union.14 There are currently no Health Professionals Council of South Africa (HPCSA) guidelines that address the issue of clinicians specifically posting on social media. However, their guidelines address the issue of patient confidentiality, as well as ethical concerns using telemedicine (which have been extrapolated below to the use of social media). Clinicians who wish to publish details about specific medical cases or clinical experiences online, which identify or run the risk of identifying a patient, should ensure they follow the guidelines relating to patient consent and disclosure set out by the HPCSA.15 These state that a patient’s express consent must be obtained before publishing case reports, photographs or other images in media that the public can access. WhatsApp has improved its end-to-end encryption policies and does not store chat data in a virtual cloud (like Facebook), but this form of protection has not been conclusively tested in clinical environments. Patient confidentiality is therefore still at risk. The increased use of medical social media, data and information can be very useful, but any abuse of data needs to be prevented.14 isolation, lack of locum relief and heavy workload, and this is seen particularly in rural areas.1 Much CME traditionally happens through conferences, seminars and other face-to-face meetings. Background This would raise awareness of these important ethical and legal obligations in the medical fraternity. The data obtained from this research could be used to motivate for the use of alternative platforms of learning and clinician support across different medical specialist modalities besides ID care. This intervention could then support the World Health Organization’s (WHO) recommendation that countries can aid in the retention of health professionals by providing them with opportunities for career development, CME, motivation and support.16 However, its use in the public health sector has been poorly researched with only a small number of studies published.10 The literature that is available shows that the use of this technology offers an efficient, unobtrusive and portable mode of communication for medical staff.11 Not only that, but also medical images that are captured using smartphone devices promote the delivery of medical care in a timely and resource-friendly manner.12 Kankane et al., in a study of neurosurgical communication, found that WhatsApp enabled cost-effective and quick decision-making, namely 4.06 min from image to registrar report.13 This led to earlier diagnosis and more prompt treatment. Nikolic et al. suggest that this technology has the potential to improve patient education and management, and perhaps, to impact significantly on health provision as a whole.11 Objectives non-responders possibly skewing the results.17 Forty-five per cent (74/166) of the participants did, in fact, not submit responses. The investigator attempted to minimise this threat as much as possible by regular email and WhatsApp reminders.17 The questionnaire was kept as short as possible and attempts were made to simplify access to it with an easy to use Internet link being sent to the participants – all this to minimise non-responses.17 The specific objectives are: • to analyse clinicians’ use of the WhatsApp chat group as a learning tool • to assess clinicians’ confidence in managing complicated HIV/TB patients after participating in the WhatsApp case discussion group • to describe the perceived usefulness of the chat group as a learning tool A link to the questionnaire in the Google form was initially sent to each clinician in the WhatsApp discussion chat group via WhatsApp. When the clinicians clicked on the link, they were taken to the electronic Google form. Google saved each completely filled questionnaire in the investigator’s Google drive. This form was completed by the respondent by a click on the most appropriate response. There were no open-ended or continuing questions, making the questionnaire simple and fairly quick to answer; the investigator estimated around 5 to 10 min per form. Participants were able to answer the questions within their own time frame, enabling them to have privacy or choice of space. • to understand clinicians’ knowledge and use of informed consent when sharing patient case details on a public platform such as WhatsApp. Original Research Original Research Original Research Data management and analysis The individual responses saved on Google drive were collected and transferred to an Excel spreadsheet, where data cleaning occurred. Any incompletely answered questionnaire was removed as a data source. Text responses were also allocated a numerical key for easier analysis. The data were then imported into Epi info statistical programme for analysis and were initially explored using basic frequencies for the categorical data. Research methods and design Study design An observational, descriptive cross-sectional design was used, with an anonymous Internet questionnaire, distributed to the clinicians who formed part of the WhatsApp group, as the data source. A quantitative approach was chosen for the study as the responses from the questionnaire were graded and therefore easily quantifiable. All the completed forms were available to view on the drive, which was password protected, and could be downloaded when needed for analysis. The clinicians were also emailed the link as well. Emailing helped to collect data from the clinicians that may have at any stage left the group during the period under investigation. Study population, setting and sampling The study population that was used in this study were 166 doctors from the EC province that accepted the organiser’s invitation to be part of the WhatsApp clinical discussion group from January 2016 to July 2017. The inclusion criteria for the study included doctors from the EC Department of Health, as well as clinicians from collaborating non- governmental organisations (NGOs). All the 166 doctors in the group were included to minimise any non-response, and to improve representation of the clinicians in the group.17 Aim The aim of this study is to describe the use of a WhatsApp clinical discussion group as an alternative learning tool to improve clinician access to specialised clinical management of complicated HIV/TB cases, as part of CME, and their knowledge of informed consent use when posting patient cases on social media. There are obvious concerns, however, about the transmission of confidential patient information over a social media platform. According to international guidelines, patient confidentiality and guarding their personal health data are a legal requirement under different laws, such as the Health Information Portability and Accountability Act (HIPAA) http://www.sajhivmed.org.za http://www.sajhivmed.org.za Open Access Page 3 of 9 Ethical consideration Those participants who posted cases were asked what type of cases they presented (Figure 1). There was a very similar distribution in reporting paediatric (Paeds), adult cases (including opportunistic infections [OI]) and cases of unsuppressed viral loads (Unsupp. VL) – making up the bulk of cases at 65% collectively. Other cases discussed included dermatological conditions (Derm), adverse events (Adv Ev), maternal cases and prevention of mother-to-child transmission (PMCT). Ethics approval was received from: HREC (Human Research Ethics Committee) from the University of Witwatersrand; BMREC (Biomedical Research Ethics Committee) from the University of the Western Cape. A participant information form (combined with participant consent), along with the link to this internet questionnaire was electronically available and posted on the WhatsApp group, as well as emailed to all participants who had at any stage belonged to the group within the reporting period. As the questionnaire was anonymous, no participant name was requested. There was no anticipated harm in the study, but there may have been some discomfort to the doctors in completing the online questionnaire. There was also the risk of identifying the locality of where the doctors worked (i.e. EC) but no risk of identifying individual doctors or patients/cases. Lastly, in terms of participants’ perceptions of having obtained greater clinical confidence in managing complicated HIV/TB cases (study objective two), the majority (86%) agreed that it did increase their clinical confidence. Data collection tools and collection Data were collected using a structured, anonymous Internet questionnaire. This comprised 17 statements or questions, each with a corresponding answer or choice of answers. The main themes for the questionnaire centred on access to the WhatsApp or Internet; usage of the group; aid in improving clinical confidence; usefulness as a learning tool and the confidentiality of cases posted (doctors’ perceptions). Summary statistics were presented to give a general description of the above responses using analysis tables and graphs. These categorical variables were summarised as the number and percentage of responses in each category or exposure variable. Further analysis was done by looking at other possible associations between clinical confidence to group engagement and clinical confidence because of perceived usefulness of the group as a learning tool. In the confidence variable, like–like response options were recorded for ease of analysis. Other associations included the recommendation of the group based on the perceived usefulness of the group as a learning tool. For all the above associations, frequency distributions and cross-tabulations of the above-mentioned variables were generated. Bivariate analysis was done to determine significant associations between the differing variables using p-values, odds ratios (ORs) and 95% CIs. The assessment of any significant differences was conducted using a Mid-P Exact test. This was To reduce information bias, the investigator used a standardised tool, and each doctor received the same questionnaire. The questionnaire had been reviewed by a group of three colleagues to ensure clarity and the exclusion of external bias. Once ethical approval was received and before distribution to the participants, the questionnaire in its electronic format was piloted with the same colleagues who are a part of the WhatsApp group to further improve question clarity and ease of participation. The questionnaire was self- administered, so no measurement bias was introduced by a third party.18 There was a threat to validity in terms of sampling bias when administrating the questionnaire, with the potential of http://www.sajhivmed.org.za Page 4 of 9 Original Research Page 4 of 9 Original Research computed to confirm statistical significance. Type I error rate (alpha) for statistical tests was set at 0.05 and 95% CI, and were provided when appropriate. came timeously. The participants who had posted cases felt positively about the timely case response. The majority of participants who posted cases also stated that they were satisfied with the content of the case response received. Sample description Out of the 166 belonging to the WhatsApp chat group, a total of 92 participants submitted Internet questionnaires. One form was submitted with no answers and was therefore excluded from the analysis. When participants were asked if they used the clinical guidance posted on the WhatsApp doctors group in their own patient management, 52% responded that they used the clinical guidance all the time, 44% used the guidance occasionally. Only 4% felt that the guidance given on the group was not relevant to their current patient case management. About a third of the participants reported that they actually referred back to old cases discussed all the time when a complicated clinical case presented at their clinic. Out of the remainder, 64% used the previous discussions occasionally, and 8% felt that felt that the guidance given on the group was not relevant to their current patient case management. Results The questionnaire also assessed the participants’ perceived usefulness of the group as a learning tool in managing complicated cases after taking part in the group (study objective three), and whether they would recommend this case discussion platform to other colleagues. Analysing clinicians’ use of the WhatsApp group Variable Strongly agreed Agreed Neutral Disagreed Strongly disagreed n % n % n % n % n % New clinical insights 51 56 32 35 7 8 1 1 - 0 Practical application of pre-existing knowledge 49 54 34 37 8 9 - 0 - 0 Guidance according to national or international guidelines 57 63 31 34 4 4 - 0 - 0 Recommend to colleagues 58 64 23 25 8 9 - 0 2 2 TABLE 2: Increased clinician confidence in managing patients and levels of group engagement. Variable Greater clinical confidence (%) OR 95% Confidence interval p Followed group 93 8.44 2.33–35.23 < 0.05 Posted questions 93 3.8 1.02–18.48 0.02 Posted responses 92 3.36 0.96–13.55 0.03 OR, odds ratios TABLE 2: Increased clinician confidence in managing patients and levels of group engagement. clinical insights on HIV/TB, that the information discussed in the group chat was according to national guidelines and international best practice principles and they would recommend a similar case discussion platform to other colleagues (Table 1) Clinicians’ knowledge and use of informed consent in the group (OR 48.13, 95% CI 4.99–464.49); those who referred to old chat cases were 21.42 times more confident (OR 21.42, 95% CI 4.39–104.84); there was also an increase in confidence in participants who reported that they had gained new clinical insights while participating in the group (OR 23.75, 95% CI 3.95–142.88). (OR 48.13, 95% CI 4.99–464.49); those who referred to old chat cases were 21.42 times more confident (OR 21.42, 95% CI 4.39–104.84); there was also an increase in confidence in participants who reported that they had gained new clinical insights while participating in the group (OR 23.75, 95% CI 3.95–142.88). The last objective was understanding the clinicians’ knowledge of informed consent when sharing patient information on social media. From the responses, 89% of the participants reported that they were aware that according to HPCSA regulations, they needed to obtain documented patient consent when posting a patient-related image on social media. However of those that reported posting questions, only half obtained consent (52%) versus 48% not obtaining consent, when posting patients’ photographs or other medical images on the group (even if patient identity was not revealed). When asked if they obtained documented patient consent when posting patients’ laboratory results on the group (even if patient identity was not revealed), around two-thirds (68%) of participants who had posted said they had in fact not obtained consent, less so than when posting other medical images. When looking at recommending the group to colleagues as an outcome, participants who report gaining new clinical insights were 17.33 times more likely to recommend the group (95% CI 3.13–96.01). Those who reported that the group helped them to practically apply pre-existing knowledge and felt that the guidance given was according to national or international guidelines were also, respectively, 12.82 (95% CI 2.55–64.56) and 20 (95% CI 1.63–245.63) times more likely to recommend the group to other colleagues as a case discussion platform (Table 4). In terms of group engagement and recommending the group to others, those who followed the group regularly were 4.79 times more likely to recommend it (95% CI 1.19–21.10). There was no difference in those who posted questions and responses (Table 5). Bivariate analysis Using a bivariate analysis, with cross-tabulation in Epi Info, any statistically significant associations were looked for in those clinicians who reported feeling more confident in managing their patients after group participation and whether they would recommend the group as a learning platform to other colleagues. Lastly, we looked at whether Internet access impacted clinicians’ reported ability to follow the group, but there was no clinically significant association found. Table 2 looks at any association between group engagement as an exposure variable, and increased clinician confidence as an outcome. In doctors who followed the group regularly, there was a clinically significant increase in OR (8.44, 95% CI 2.33–35.23), participants being 8.44 times more likely to have increased confidence in managing their patients. Those who posted questions also had an increase in OR, 3.8 times more likely to have an increase in their clinical confidence (95% CI 1.02–18.48). Analysing clinicians’ use of the WhatsApp group To analyse the usage of the WhatsApp group (objective one of the study), the questionnaire included questions that assessed the participant’s Internet accessibility and their engagement in the group. Satisfaction at the relevance of responses received (by content and timing) was also assessed. Lastly, participants were asked what types of cases they posted. Only 1% of participants did not have access to a form of Internet connectivity. Twenty-nine per cent of the remainder had only occasional access. Internet connectivity and access was important to permit the receipt and posting of questions on the app. Seventy-one per cent of participants had access all the time. The majority (73%) looked on the app every time a case was posted, with only 2% ignoring the group completely. Again, the majority of the participants strongly agreed that the WhatsApp group was useful in helping them gain new 1 2 3 4 5 6 7 1. Paeds (22%) 2. Adult/OI (21%) 3. Unsupp. VL (22%) 4. Derm (8%) 5. Adv Ev (12%) 6. Maternity/PMCT (12%) 7. Other (3%) FIGURE 1: Types of cases discussed (n = 81). 1 2 3 4 5 6 7 1. Paeds (22%) 2. Adult/OI (21%) 3. Unsupp. VL (22%) 4. Derm (8%) 5. Adv Ev (12%) 6. Maternity/PMCT (12%) 7. Other (3%) FIGURE 1: Types of cases discussed (n = 81). To further assess engagement in the group, participants were also asked how many times they posted cases in the group, and if they posted any responses to a case that had been posted by a colleague. Half of the participants reported to have never posted cases; 47% had posted at least 1–5 times and 3% had posted 6–10 times. In terms of posting any medical advice or responses to another colleague’s case, 52% posted occasionally, 4% all the time. To determine the satisfaction of case responses received, participants were asked if the responses to the cases posted FIGURE 1: Types of cases discussed (n = 81). FIGURE 1: Types of cases discussed (n = 81). http://www.sajhivmed.org.za Open Access Open Access TABLE 1: Usefulness of the group as a learning tool (n = 91). http://www.sajhivmed.org.za Group engagement and its usefulness as a learning tool also reported that WhatsApp facilitated, in learners, the ability to more confidently engage with peers and educators.19 This form of engagement and success can be described through the theory of cooperative learning. In cooperative learning, students who maximally engage in a group are able to extend their current knowledge base, as they are in control of the discussion construct.21 In our study’s context, participants posted a case they are most interested in and there develops a close relationship between theory, research and a practical working through the case; this underpins long-term retention of knowledge and maximises student learning.21 Group engagement and its usefulness as a learning tool Variable Recommend group (%) OR 95% Confidence interval p Used guidance to manage patients 89 1.69 0.17–16.1 0.32 Referred to old chat cases 90 2.64 0.46–14.95 0.16 New clinical insights 93 17.33 3.13–96.01 < 0.05 Practical application of pre-existing knowledge 93 12.83 2.55–64.56 < 0.05 Guidance according to National or international guidelines 91 20 1.63–245.63 < 0.05 OR, odds ratios TABLE 5: Clinician recommendation of the WhatsApp group based on their level of engagement in the group (n = 91) Variable Recommend group (%) OR 95% Confidence interval p Posted responses 88 0.83 0.19–3.28 0.4 Posted questions 88 0.97 0.24–3.89 0.46 Followed group 93 4.79 1.19–21.10 0.01 OR, odds ratios TABLE 5: Clinician recommendation of the WhatsApp group based on their level of engagement in the group (n = 91) distributed healthcare settings. Our research adds to the literature by further clarifying that the knowledge gained (whether from peers or specialists) in belonging to such a group aids in the application of new clinical insights and previous medical knowledge, as well as contributing to clinical confidence by facilitating distance learning. Lastly, our study found that participants were more likely to recommend the group to other colleagues if they had followed the group regularly (OR 4.79), and in those who reported the group as a useful learning tool. The investigators therefore surmised that the WhatsApp group seems to have promoted good group engagement which, in turn, facilitated learning, decreasing professional isolation and produced a recommendation of a similar platform to other colleagues. Such a mobile learning platform is therefore an important adjunct to current CME practices. E-learning (of which WhatsApp forms a part) can result in greater educational opportunities for participants, while at the same time enhancing student effectiveness and efficiency, as is the reported outcome in our study.22 posting of cases or responses, was regular. The results further demonstrated that there was a statistically significant association between engagement in the group and increased clinical confidence – those who followed the group regularly were 8.44 times more likely to report an increase in clinical confidence and 3.8 times more confident if they posted a case. This correlates with findings by Raiman et al., who discussed in their article that the use of mobile applications has been shown to increase student participation and therefore foster improved learning.20 Similarly, Rambe et al. Group engagement and its usefulness as a learning tool The responses from participants in this study were favourable in the reported use of the WhatsApp group and its application as a learning tool. The majority of the participants firstly cited regular Internet connectivity, which facilitated the uninterrupted use of the application and communication in real time.19 They also reported using the group discussions as a guide to further manage other patients, referred back to old chat discussions and were satisfied at the timeous response to cases (including the peer responses themselves). Group engagement, or participation, measured by following of the group and Other associations were found in increased clinician confidence in managing patients as an outcome, cross-tabulated with participant perceptions of the usefulness of the group as a learning tool (Table 3). Of statistical significance, participants who used the chat group guidance to manage their patients were 48.13 times more likely to be confident afterwards Open Access Open Access http://www.sajhivmed.org.za TABLE 3: Increased clinician confidence in managing patients based on their perceived usefulness of the group as a learning tool (n = 91). Variable Greater clinical confidence (%) OR 95% Confidence interval p Used guidance to manage patients 91 48.13 4.99–464.49 < 0.05 Referred to old chat cases 91 21.42 4.39–104.84 < 0.05 New clinical insights 90 23.75 3.95–142.88 < 0.05 Practical application of pre-existing knowledge 94 Undefined Undefined < 0.05 Guidance according to national or international guidelines 89 Undefined Undefined < 0.05 OR odds ratios BLE 3: Increased clinician confidence in managing patients based on their perceived usefulness of the group as a learning tool (n = 91). iable Greater clinical confidence (%) OR 95% Confidence interval TABLE 4: Clinician recommendation of the WhatsApp group based on their perceived usefulness of it as a learning tool (n = 91). Generalisability The results of the study show that WhatsApp is perceived as an effective means of learning and clinical support in this study group. This mobile application can then be applied to other clinical disciplines (not only IDs), from other health settings (private, district, provincial level), as a learning intervention. The target population groups that could potentially use this intervention include, for example, doctors in other clinical disciplines who need expert advice or access regarding patient management, allied health professionals (such as nurses, physiotherapists, occupational therapists) who need clinical supervision and advice from senior consultants regarding patients they are managing. As this study looks at the use of the WhatsApp group in a clinical setting for patient management and further medical learning, it would be difficult to comment if its use would be applicable outside of the medical field. Improved clinical confidence among our participants could be because of two main aspects: accessibility and case- based learning. Doctors could easily access the application, could easily access old cases in the application and could easily access new knowledge by asking for guidance on the application. Wani et al. found that doctors started management of patients quicker after using WhatsApp clinician advice because of faster access to that advice, and that they found that management to be more effective.23 It is often laborious trying to find best evidence-based management in medical literature, especially in a time-constrained clinical setting. Also, the application of that knowledge is sometimes broad, with medical theory not always correlating clearly to what is found in clinical practice. By providing input on a specific case (in a specific South African clinical setting) and supporting the clinician in managing the case in real time, a clinician’s confidence can be further bolstered. The WhatsApp group provides a form of case-based learning, which has been shown to tie theory to practice and promote deeper learning.24 Studies that use interactive techniques for CME, such as case discussions, produce a favourable change in professional practices and outcomes.25 This correlates to a similar reported outcome in our study. The second outcome of the study was to assess if patient confidentiality breaches had occurred with the posting of cases, and if doctors were aware of the legal obligations they are under when posting patient case details on a social media platform. The findings of this study could definitely be generalised to any health profession. Generalisability It would aid in raising awareness of the pitfalls of posting cases on social media, and in doing so, protect health professionals from any future litigation as well as protecting their patient’s confidentiality. Clinical confidence in managing complicated HIV and TB cases The majority of the participants in the study agreed that they had gained greater clinical confidence in managing their patients after participating in the group. The findings showed that there was also an improvement in clinical confidence among those participants who perceived the group as a useful learning tool (it has been previously mentioned how engagement in the group had a similar effect). Participants who used the chat group guidance to manage their patients were 48 times more likely to feel clinically confident. There was an increase in clinical confidence in those who referred to old chat cases (OR 21.42) and those who gained new clinical insights while participating in the group (OR 23.75). Raiman et al. reported similar findings in their study – a WhatsApp group provided a unique environment to be able to quickly access learning resources while participating in a discussion Furthermore, the participants also reported that the group gave them new clinical insights; helped them to practically apply pre-existing knowledge and felt that the guidance was aligned with international or national guidelines. In a systemic review of medical literature, Kamel Boulos et al. found collective evidence that WhatsApp has been successfully used in health and medical education and learning.9 They also concluded that apps can help to create virtual communities of enquiry and practice, and bridge distances of busy http://www.sajhivmed.org.za Open Access Original Research Page 7 of 9 Page 7 of 9 facilitated learning. Their participants also cited how useful it was to look back at old recorded learning discussions.20 facilitated learning. Their participants also cited how useful it was to look back at old recorded learning discussions.20 Limitations In our study’s WhatsApp group, group rules were posted advising clinicians to remove any patient identifiers from any medical images when posting. This helped in some ways towards preserving patient confidentiality, but further education needs to be iterated to our study group regarding obtaining actual documented consent from the patients themselves. Limitations Although WhatsApp is relatively safe in terms of hacking and leaking of confidential content because of its end-to-end encryption of data, there is still much concern about its use in medical literature and the impact on patient confidentiality.26 The majority of our study participants reported being aware that they needed to obtain documented patient consent when posting a patient-related image (photographs, case report, laboratory report) on social media. However, less than half of participants actually obtained consent. There seems to be a discrepancy in what the clinicians reported to know, and what they did to maintain patient confidentiality in this study. There are several limitations to this study. Although many attempts were made to get responses from the group, only 55% (n = 91) of participants submitted responses; the selection was therefore not random, and could introduce some bias. The small sample size and simple survey framework could affect the overall results, with resultant wide CIs. Some further bias could have been introduced by the online format of the questionnaire (with possible technical inability to fill it in correctly). A possible bias might have also occurred if any of the collaborating NGO clinicians filled in the questionnaire, although only four were active in the group at the time. We did not collect any demographic data from the respondents. The retrospective nature of the study could also affect the participants’ responses, as recall of their experiences of the chat group over a period of 1 year could vary from their original experiences. The investigators could also not determine from the study if the responses received to posted questions were from peers or specialists. A more accurate observation of the WhatsApp group would have been obtained through direct analysis of the chat contents, but this was not approved by local ethical governing bodies without written consent from each doctor (which would be beyond our scope given the study’s retrospective nature). Several authors share similar concerns that the use of patient data needs to be regulated when using social media, and that there needs to be a review of the roles and responsibilities of medical professionals when using such platforms.14 Mars and Escott found few reports of patient consent being regularly sought when sending patient information over WhatsApp.26 They concluded that doctors need to be told what steps to take to maintain confidentiality. References The investigators recommend the use of WhatsApp clinical support groups as a long-distance learning platform, based on our findings. To facilitate group success, some further recommendations include a commitment from participants in the group with active participation; a case-based method of discussion (but other learning modalities can be used); cooperative engagement led by students determining the learning construct of the group that will benefit them the most; and a range of different levels of clinical expertise in the group. There needs to be an increased awareness and education among clinicians on the legal implications of posting patient details in social media (not just WhatsApp) without proper informed consent, to protect patient confidentiality. We also suggest that further research should be conducted to obtain a more objective analysis as to whether advice given in these mobile platforms improves clinical management in patients or not. These could include auditing clinical advice given on clinician WhatsApp groups, according to best practice principles in medical literature, or by directly auditing patient outcomes in those having been managed by doctors who participate in similar mobile learning platforms in South Africa. The findings of this study will be posted on the WhatsApp group, which is still ongoing. 1. Lygidakis H, McLoughlin C, Patel K. Achieving universal health coverage: Technology for innovative primary health care education. 2016. https://doi.org/10.13140/RG. 2.2.35291.57121 1. Lygidakis H, McLoughlin C, Patel K. Achieving universal health coverage: Technology for innovative primary health care education. 2016. https://doi.org/10.13140/RG. 2.2.35291.57121 2. Buchan J, Couper I, Viraj T, et al. Early implementation of WHO recommendations for the retention of health workers in remote and rural areas. Bull World Health Organ. 2013;91(11):834–840. https://doi.org/10.2471/BLT.13.119008 2. Buchan J, Couper I, Viraj T, et al. Early implementation of WHO recommendations for the retention of health workers in remote and rural areas. Bull World Health Organ. 2013;91(11):834–840. https://doi.org/10.2471/BLT.13.119008 3. Chen L. Striking the right balance: Healthcare workfore retention in remote and rural areas. Bull World Health Organ. 2010;88(5):323. https://doi.org/10.2471/ BLT.10.078477 3. Chen L. Striking the right balance: Healthcare workfore retention in remote and rural areas. Bull World Health Organ. 2010;88(5):323. https://doi.org/10.2471/ BLT.10.078477 4. Stats SA. Mid-Year Population Estimates 2017 [homepage on the Internet]. 2017 [cited 2017 Jul 01]. Available from: https://www.ons.gov.uk/peoplepopulation andcommunity/populationandmigration/populationestimates/datasets/population estimatesanalysistool. 5. HSRC. Safe hygiene practices in Eastern Cape rural communities. 2012 6. Gray A, Vawda Y. South African health review 2017; 2017. ISSN 978-1-919839-89-9 7. SANAC. References Let our actions count: South Africa’s national strategic plan for HIV, TB and STIs 2017–2022. South African Natl AIDS Counc [serial online]. 2017 [cited 2017 Jul 01];1(March):1–132. http://sanac.org.za/wp-content/uploads/2017/05/NSP_ FullDocument_FINAL.pdf. 8. Statistica. Number of monthly active WhatsApp users worldwide from April 2013 to January 2017 (in millions) [homepage on the Internet]. Statistica; 2016. Available from: https://www.statista.com/statistics/260819/number-of-monthly- active-whatsapp-users/. [Acessed: 01 July 2017] 9. Kamel Boulos MN, Giustini DM, Wheeler S. Instagram and WhatsApp in health and healthcare: An overview. Futur Internet. 2016;8(3):1–14. https://doi.org/ 10.3390/fi8030037 10. Veroni L, Ferrari A, Acerra A. Consideration on the use of WhatsApp in physician- patient communication and relationship. Recent Prog Med. 2015;106(7):331–336. 11. Nikolic A, Anat PD, Wickramasinghe N, Claydon-Platt D. The use of communication Apps by medical staff in the Australian health care system: Survey study on prevalence and use. JMIR Med Inform. 2018;6(1):1–7. https://doi.org/10.2196/ medinform.9526 12. Kirk M, Hunter-Smith SR, Smith K, Hunter-Smith DJ,. The role of smartphones in the recording and dissemination of medical images. J Mob Technol Med. 2014;3(2):40–45. https://doi.org/10.7309/jmtm.3.2.7 Conclusion The initial aim of this study was to show that participating in a WhatsApp group was a useful adjunct learning tool http://www.sajhivmed.org.za Open Access Page 8 of 9 Original Research Disclaimer 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. Competing interests 16. WHO. Transforming and scaling up health professionals’ education and training: World Health Organization guidelines 2013. Guidelines. 2013:124. https://doi.org/ 10.1613/jair.301 The authors have declared that no competing interest exist. 17. Levin KA. Study design III: Cross-sectional studies. Evid Based Dent. 2006;7(1): 24–25. https://doi.org/10.1038/sj.ebd.6400375 Funding Information that could also clinically support doctors in geographically widespread facilities without onsite specialist support. Based on the participant responses from this research, this mobile platform does offer an alternative CME solution that can be easily and successfully implemented in various health fields. By giving healthcare professional opportunities for career development, CME, motivation and support through this novel learning platform, we can perhaps aid in their retention in the public health sector.16 Caution needs to be taken to maintain patient confidentiality when posting on social media, but that does not negate WhatsApp’s usefulness in a clinical learning setting. ACC grant, sub awarded to Wits RHI from Beyond Zero (funder-CDC). Acknowledgements 13. Kankane VK, Jaiswal GGT. Application of WhatsApp: A quick, simple, smart and cost competent method of communication in neurosurgery. Rom Neurosurg. 2016;30(12):306–312. https://doi.org/10.1515/romneu-2016-0049 The author (J.W.) would like to acknowledge the University of the Western Cape: this article was based on her recently submitted MPH thesis. She would also like to acknowledge her employer Wits RHI, for whom the article premise was also part of her KPIs. 14. Denecke K, Bamidis P, Bond C, et al. Ethical Issues of Social Media Usage in Healthcare. IMIA Yearb Med Inform. 2015;10(1):137–147. https://doi.org/ 10.15265/IY-2015-001 15. HPCSA. General ethical guidelines for the health care professions, Booklet 14 [homepage on the Internet]. HPCSA; 2008 [cited 2017 Jul 01]. Available from: www.hpcsa.co.za/Uploads/editor/UserFiles/downloads/conduct_ethics/rules/ generic_ethical_rules/booklet_10_confidentiality_protecting_and_providing_ information.pdf. Data availability statement Data sharing is not applicable to this article as no new data were created or analysed in this study. 21. Johnson DW, Johnson RT, Smith K. The state of cooperative learning in postsecondary and professional settings. Educ Psychol Rev. 2007;19(1):15–29. https://doi.org/10.1007/s10648-006-9038-8 23. Wani S, Rabah S, AlFadil S, Dewanjee N, Najmi Y. Efficacy of communication amongst staff members at plastic and reconstructive surgery section using smartphone and mobile WhatsApp. Indian J Plast Surg. 2013;46(3):502. https:// doi.org/10.4103/0970-0358.121990 22. Kinfu Y, Vovides Y, Talib Z, et al. The health worker shortage in Africa: Are enough physicians and nurses being trained? Bull World Health Organ. 2009;87(3): 225–230. https://doi.org/10.2471/BLT.08.051599 26. Mars M, Escott R. 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https://openalex.org/W2561607230	https://europepmc.org/articles/pmc5178096?pdf=render	English	null	Impact of obesity and epicardial fat on early left atrial dysfunction assessed by cardiac MRI strain analysis	Cardiovascular diabetology	2,016	cc-by	8,420	© The Author(s) 2016. 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. Cardiovascular Diabetology Cardiovascular Diabetology Evin et al. Cardiovasc Diabetol (2016) 15:164 DOI 10.1186/s12933-016-0481-7 ORIGINAL INVESTIGATION Impact of obesity and epicardial fat on early left atrial dysfunction assessed by cardiac MRI strain analysis Morgane Evin1,2,3, Kathryn M. Broadhouse1,2, Fraser M. Callaghan1,2, Rachel T. McGrath4,5, Sarah Glastras4,5, Rebecca Kozor1,2,6, Samantha L. Hocking4, Jérôme Lamy3, Alban Redheuil3, Nadjia Kachenoura3, Greg R. Fulcher4,5, Gemma A. Figtree1,2,6 and Stuart M. Grieve1,2,7* Open Access ORIGINAL INVESTIGATION Impact of obesity and epicardial fat on early left atrial dysfunction assessed by cardiac MRI strain analysis Morgane Evin1,2,3, Kathryn M. Broadhouse1,2, Fraser M. Callaghan1,2, Rachel T. McGrath4,5, Sarah Glastras4,5, Rebecca Kozor1,2,6, Samantha L. Hocking4, Jérôme Lamy3, Alban Redheuil3, Nadjia Kachenoura3, Greg R. Fulcher4,5, Gemma A. Figtree1,2,6 and Stuart M. Grieve1,2,7* Open Access Abstract Although there was no significant difference in intra-myocardial fat fraction, Dixon 3D epicardial fat volume(EFV) was significantly elevated in the obesity and type 2 diabetes versus control group (135 ± 31 vs. 90 ± 30 mL/m2, p < 0.001). There were significant correlations between LA functional indices and both BMI and EFV (p ≤ 0.007). Conclusions: LA MRI-strain may be a sensitive tool for the detection of early diastolic dysfunction in individuals with obesity and type 2 diabetes and correlated with BMI and epicardial fat supporting a possible association between adiposity and LA strain. Conclusions: LA MRI-strain may be a sensitive tool for the detection of early diastolic dysfunction in individuals with obesity and type 2 diabetes and correlated with BMI and epicardial fat supporting a possible association between adiposity and LA strain. Trials Registration Australian New Zealand Clinical Trials Registry No. ACTRN12613001069741 Trials Registration Australian New Zealand Clinical Trials Registry No. ACTRN12613001069741 Keywords: Diastolic dysfunction, Cardiac dysfunction, Magnetic resonance studies, Fat distribution Obesity and type 2 Keywords: Diastolic dysfunction, Cardiac dysfunction, Magnetic resonance studies, Fat distribution, Obesity and type 2 symptoms of heart failure [1]. Diastolic dysfunction is an asymptomatic condition leading to heart failure and is traditionally assessed by echocardiography. However, a reliable and sensitive method for the early identification of diastolic dysfunction is currently lacking [2–4]. The earliest manifestations of diastolic dysfunction are typi- cally evident in altered left atrial dynamics or pulmonary inflow patterns [5]. The features represent are good target for early diastolic dysfunction detection and improved Correspondence: stuart.grieve@sydney.edu.au 1 Sydney Translational Imaging Laboratory, Heart Research Institute, Charles Perkins Centre, University of Sydney, Camperdown, NSW 2006, Australia Full list of author information is available at the end of the article Impact of obesity and epicardial fat on early left atrial dysfunction assessed by cardiac MRI strain analysis Morgane Evin1,2,3, Kathryn M. Broadhouse1,2, Fraser M. Callaghan1,2, Rachel T. McGrath4,5, Sarah Glastras4,5, Rebecca Kozor1,2,6, Samantha L. Hocking4, Jérôme Lamy3, Alban Redheuil3, Nadjia Kachenoura3, Greg R. Fulcher4,5, Gemma A. Figtree1,2,6 and Stuart M. Grieve1,2,7 Abstract Background: Diastolic dysfunction is a major cause of morbidity in obese individuals. We aimed to assess the ability of magnetic resonance imaging (MRI) derived left atrial (LA) strain to detect early diastolic dysfunction in individuals with obesity and type 2 diabetes, and to explore the association between cardiac adipose tissue and LA function. Methods: Twenty patients with obesity and T2D (55 ± 8 years) and nineteen healthy controls (48 ± 13 years) were imaged using cine steady state free precession and 2-point Dixon cardiovascular magnetic resonance. LA function was quantified using a feature tracking technique with definition of phasic longitudinal strain and strain rates, as well as radial motion fraction and radial velocities. Results: Systolic left ventricular size and function were similar between the obesity and type 2 diabetes and control groups by MRI. All patients except four had normal diastolic assessment by echocardiography. In contrast, measures of LA function using magnetic resonance feature tracking were uniformly altered in the obesity and type 2 diabe- tes group only. Although there was no significant difference in intra-myocardial fat fraction, Dixon 3D epicardial fat volume(EFV) was significantly elevated in the obesity and type 2 diabetes versus control group (135 ± 31 vs. 90 ± 30 mL/m2, p < 0.001). There were significant correlations between LA functional indices and both BMI and EFV (p ≤ 0.007). Results: Systolic left ventricular size and function were similar between the obesity and type 2 diabetes and control groups by MRI. All patients except four had normal diastolic assessment by echocardiography. In contrast, measures of LA function using magnetic resonance feature tracking were uniformly altered in the obesity and type 2 diabe- tes group only. Although there was no significant difference in intra-myocardial fat fraction, Dixon 3D epicardial fat volume(EFV) was significantly elevated in the obesity and type 2 diabetes versus control group (135 ± 31 vs. 90 ± 30 mL/m2, p < 0.001). There were significant correlations between LA functional indices and both BMI and EFV (p ≤ 0.007). Results: Systolic left ventricular size and function were similar between the obesity and type 2 diabetes and control groups by MRI. All patients except four had normal diastolic assessment by echocardiography. In contrast, measures of LA function using magnetic resonance feature tracking were uniformly altered in the obesity and type 2 diabe- tes group only. Ventricular functional analysis Region of interest analysis was performed using the Seg- ment software (Medviso, Lund, Sweden) [16]. LV end- diastolic (EDV) and end-systolic volumes (ESV), indexed LV mass, LV stroke volume (SV) and LV ejection fraction (LVEF) were obtained from the short axis stack by manu- ally contouring end-diastolic and end-systolic endocar- dial borders and end-diastolic epicardial borders from the base to the apex. Cardiovascular magnetic resonance imaging Cardiovascular magnetic resonance imaging CMR data were acquired using a 1.5T Siemens Avanto scanner at North Shore Private Radiology, St Leonards, Australia. Cardiac volumes and left ventricle (LV) mass were quantified using 2-chamber (LVLA), 4-chamber (4CH), 3-chamber (LVOT) views and a short axis stack of cine SSFP images (TE: 1.5 ms, TR: 3.4 ms, 20 phases; flip angle: 45°, acquisition; FOV: 35 cm, slice thick- ness: 8 mm). 2-point Dixon data was also acquired (TR 6.7 ms, resolution: 1.2 mm, slice thickness: 4 mm, TE2: 2.4, 4.8 ms). One patient and one healthy volunteer were excluded from the analysis as the Dixon images were not available. LA functional and volumetric changes are amongst the first imaging changes seen in ObT2D, and therefore may be a useful marker of early disease [6–8]. Recent data shows that diabetes is an independent risk factor predictive of LA enlargement and dysfunction. Data has also linked epicardial fat to LA and ventricular func- tional changes [9], and to increased LA volume [10, 11]. A highly powered analysis using a Framingham cohort demonstrated that this association is independent of other measures of adiposity in men [11]. Although echocardiography is the generally accepted gold standard for measurement of atrial function and other diastolic functional or structural changes, in the setting of obesity the practical difficulties of imaging obese patients make routine acquisitions problematic. Cardiovascular magnetic resonance (CMR) may present a good alternative for reproducible, accurate and sensi- tive quantification of LA function. The recent develop- ment of feature tracking algorithms that can be applied in a semi-automated manner to CMR data permits myo- cardial strain measurements to be obtained from cine functional images [12–14]. CMR simultaneously offers a convenient and accurate way to quantify pericardial fat using anatomical images, or with specific fat quantifica- tion techniques such as Dixon [15]. Background Obesity and diabetes are strong risk factors for heart failure with preserved ejection fraction (HFpEF), a clini- cal syndrome that affects nearly half of patients with Full list of author information is available at the end of the article Evin et al. Cardiovasc Diabetol (2016) 15:164 Page 2 of 10 methods for capturing these physiological signs would therefore be of great clinical and scientific interest. Study populationhi The first 20 consecutive patients from the study entitled “The combined Effect of Liraglutide and Sleeve Gastrec- tomy on Metabolic, Cardiac, Neurological and Sleep Function in Obese Diabetes” (LIRASLEEVE; mean age 55 ± 8 years; 40% female) with a diagnosis of obesity and type 2 diabetes mellitus were recruited via the Depart- ment of Endocrinology, Diabetes and Metabolism at Royal North Shore Hospital, Australia. Inclusion criteria included BMI >30 kg/m2 and known T2D (HbA1c ranged from 7 to 10% at study entry). Subjects were excluded if there was a contraindication to MRI or weight in excess of 150 kg (MRI table limit). Nineteen age and gender matched controls (48 ± 13 years; 47% female) with no history of cardiac disease were recruited. The local ethics committee approved the study and all patients provided written consent. i Obesity and type 2 diabetes mellitus (ObT2D) com- monly co-exist and together they contribute to a wide range of metabolic complications; importantly, they are increasingly recognised to be major contributors to the global burden of cardiac disease. The paucity of reli- able early, non-invasive detection of diastolic changes on ObT2D population is therefore a key limitation of our current clinical and research capacity.h The changes associated with the mid and late stages of diastolic dysfunction in individuals with obesity and T2D include a complex spectrum of adipose infiltra- tion (both intra-myocardial and epicardial), fibrosis and electrophysiological alterations. Of these factors, intra-myocardial and epicardial fatty changes have received considerable attention. However, the relation- ship between cardiac fat and early dysfunction remains poorly understood. This deficit is largely attributable to the difficulties in detecting very early alterations in car- diac function. Left atrial strain and volume analysish The analysis and feature tracking algorithm has pre- viously been described in details [14]. It was quanti- fied on LVLA, 4CH, and LVOT views and averaged for a global analysis. After endocardial contour tracking, longitudinal strain was defined as the temporal varia- tion of the length of the contour, and was calculated as [Slt = (Lt − Lo)/Lo], where Lo is the initial length of the contour and Lt is the length at time t. Radial motion frac- tion corresponded to the radial relative displacement of In this study, we applied a novel MRI method for meas- uring atrial strain to detect subclinical evidence for dias- tolic dysfunction in a cohort of obese individuals with type 2 diabetes and no clinical evidence of heart failure. As a secondary aim, we assessed the association between abnormal atrial function and both intra-myocardial and epicardial fat. Evin et al. Cardiovasc Diabetol (2016) 15:164 Page 3 of 10 the considered segment towards the LA centre of mass, [Mr = (Mt − Mo)/Mo], where Mo is the initial radius, and Mt, the radius at time t. orthogonal components relative to the centre of the mitral valve (MV). The first, in the direction of the MV, was termed centric (cMr) and the other, normal to this direction (termed MV perpendicular: pMr, see Fig. 1). These two components describe the motion that broadly results from the LV translational movement (MV perpen- dicular radial motion fraction, pMr) and the LA related movements (MV centric radial motion fraction, cMr). Figure 1 summarises the LA strain functional meth- odology and analysis. Briefly, parameters are classified according to reservoir (R), conduit (C) or atrial contrac- tion (A) phases. LA phasic measurements were made for longitudinal strain (Sl) and radial motion fraction (Mr), where the phase is denoted as a suffix (SlR, SlC, SlA), (MrR, MrC, MrA) respectively. Longitudinal strain rates (SRlS′, SRlE′, SRlA′) and radial relative velocities (VrS′, VrE′, VrA′) were computed, as the time derivatives of longitudinal and radial indices, for the three atrial functional phases: reservoir (S′), conduit (E′) and LA contraction (A′). LV functional assessment Echocardiographic diastolic data for the ObT2D group are presented in Table 1-B. No echocardiographic data were obtained for the control group. Echocar- diographic measures of diastolic function were within the normal standardised range in patients except E/E′ ratio, which was elevated at >8 in four subjects within the ObT2D group, and two greater than 15 (E/E′ range 8.3–17.1). Small significant differences in indexed LV ESV (p = 0.02) and mean mid myocardial LV thickness (p = 0.003) were found in the ObT2D group compared to control (Table 1-C) as compared to healthy volunteers. Subject characteristics Table 1-A summarises the demographics, global car- diac functional measures by CMR, and echocardio- graphic measurements in the ObT2D and control groups. There were no significant differences in age or gen- der between the ObT2D and control group. The aver- age BMI in the ObT2D group classed them as morbidly obese (39 ± 3 kg/m2, range: 30–51 kg/m2); all individu- als within the control group had a BMI < 30 kg/m2. At study entry, the ObT2D group had an average duration of T2Dof 11.4 ± 6.2 years and a mean HbA1c of 8.4 ± 1.1%. Statistical analysis Statistical analyses were carried out using SPSS Version 22 (IBM, Armonk, NY). All continuous variables are expressed as mean ± standard deviation (SD). Normal- ity was checked using the Shapiro–Wilk test. Differences between groups were considered significant for p < 0.05. Intra-observer was assessed using intra class correla- tion coefficient (ICC). Groups were compared using the independent-samples t test for normally distributed con- tinuous variables and the Mann–Whitney U test for non- normally distributed variables. Echocardiography Diastolic function was assessed by echocardiography in patients using the American Society of Echocardiogra- phy guidelines [17]. The following parameters were col- lected—mitral inflow E and A waves, E′ lateral velocity of the MV annulus measured by tissue Doppler imag- ing, and aortic peak velocity and flow. One patient was excluded from the echocardiography analysis due to inadequate images secondary to the technical difficulty of the scan. Results fat images using Kmeans clustering by a semiautomatic program implemented in python and Paraview (Fig. 2). The volumes of the cluster corresponding to the adipose tissue were then calculated to define an absolute fat vol- ume, which is then divided by the whole heart volume to obtain a 3D MRI Dixon adipose fraction. In order to nor- malize the volume of epicardial fat present by body size, absolute fat volume was indexed to BSA. Epicardial and intra‑myocardial fat quantification Fat measurement was performed following segmentation of the Dixon data into fat and water images (Fig. 2) [15]. Intra-myocardial fat was quantified using a hand-drawn ROI covering the myocardial septum in the 4CH view, with care taken to avoid partial volume effects (Fig. 2a). Epicardial fat was quantified blindly between patients and controls by segmenting cardiac volumes and Dixon To further investigate the radial motion fraction modifications in term of LV related and LA standalone motions, this parameter was decomposed into two Fig. 1 Illustration of the methodology for LA function analysis. a, b Left atrial contours from a 4-chamber view and longitudinal strain and strain rate related curves of a subject from the Obese-Type 2 diabetes (ObT2D) group. Ro example of a radius used for the computation of the radial motion fraction. Decomposition of the radial motion fraction into MV centric (cMr, towards the mitral valve center) and MV corrected perpendicular (pMr, MV corrected perpendicular radial motion fraction) components. c, d SlR reservoir longitudinal strain, SlC conduit longitudinal strain and SlA La contraction longitudinal strain, SRLS′ reservoir longitudinal strain rate, SRlE′ conduit longitudinal strain rate and SRlA′ LA contraction longitudinal strain rate Fig. 1 Illustration of the methodology for LA function analysis. a, b Left atrial contours from a 4-chamber view and longitudinal strain and strain rate related curves of a subject from the Obese-Type 2 diabetes (ObT2D) group. Ro example of a radius used for the computation of the radial motion fraction. Decomposition of the radial motion fraction into MV centric (cMr, towards the mitral valve center) and MV corrected perpendicular (pMr, MV corrected perpendicular radial motion fraction) components. c, d SlR reservoir longitudinal strain, SlC conduit longitudinal strain and SlA La contraction longitudinal strain, SRLS′ reservoir longitudinal strain rate, SRlE′ conduit longitudinal strain rate and SRlA′ LA contraction longitudinal strain rate Evin et al. Cardiovasc Diabetol (2016) 15:164 Page 4 of 10 Fig. 2 2D intra-myocardial fat fraction (a), and 3D Dixon adipose tissue quantification (b), fat (a) images from DIXON sequence. K-means segmenta- tion of the heart and detection of the epicardial fat (a, b) Fig. 2 2D intra-myocardial fat fraction (a), and 3D Dixon adipose tissue quantification (b), fat (a) images from DIXON sequence. K-means segmenta- tion of the heart and detection of the epicardial fat (a, b) Left atrial size and strain measurements Table 2 summarizes the MRI-derived LA volumetric and functional strain measurements. All MRI atrial volumet- ric measures were within the normal range for both the ObT2D and control groups. There were no significant Evin et al. Cardiovasc Diabetol (2016) 15:164 Page 5 of 10 Table 1 Demographic, clinical, echocardiographic and cardiovascular MRI measurements A. subject characteristics Healthy volunteers ObT2D HV/ObT2D (n = 19) (n = 20) p value A. subject characteristics Age (years) 47.5 ± 12.5 54.6 ± 7.8 Women (%) 45 40.0 HbA1c (%) – 8.5 ± 1.0 Duration of diabetes (years) – 11.3 ± 6.1 Proportion of patients taking lipid-lowering therapy (%) – 100.0 Proportion of patients taking anti-hypertensives (%) – 80.0 History of CVD (%) – 30.0 Body surface area (m2) 1.9 ± 0.2 2.2 ± 0.2 Body mass index (kg/m2) 24.8 ± 2.6 39.7 ± 5.8 * B. echocardiographic parameters in ObT2D patients Age (years) 54.6 ± 7.8 40–60 Mitral peak E velocity (m/s) 0.75 ± 0.24 0.75 ± 0.17 Mitral peak A velocity (m/s) 0.74 ± 0.18 0.62 ± 0.15 E/A ratio 0.99 ± 0.28 1.24 ± 0.39 E′ Lateral myocardium velocity (cm/s) 10.50 ± 2.4 12.5 ± 3.0 E/E′ ratio 7.6 ± 3.7 6.3 ± 2.2 Aortic valve peak velocity (m/s) 1.43 ± 0.57 C. MRI LV function parameters Indexed LV mass (g/m2) 75.1 ± 14.9 80.8 ± 16.1 LV EDV (mL) 122.8 ± 24.0 130.8 ± 34.0 LV ESV (mL) 49.4 ± 13.4 44.8 ± 18.6 Indexed LV EDV (mL/m2) 63.8 ± 11.4 58.9 ± 13.4 Indexed LV ESV (mL/m2) 25.7 ± 7.1 20.1 ± 7.3 * LV EF (%) 60.9 ± 7.4 66.1 ± 8.8 Mean Mid. LV thickness (mm) 11.1 ± 2.0 12.9 ± 1.6 ** Table 1 Demographic, clinical, echocardiographic and cardiovascular MRI measurements Table 1 Demographic, clinical, echocardiographic and cardiovascular MRI measurements p < 0.001). The radial motion ratio was also increased by 12.7% (MrA/MrR, ObT2D vs. control, p = 0.020). differences in LA volumes or LVEF, even after indexing to BSA between the ObT2D and control groups. LA longitudinal strain (SlR), longitudinal strain rate (SRlS′), and radial motion fraction (MrR), were slightly reduced during reservoir phase in the ObT2D group com- pared to control and slightly increased for the atrial con- traction phase (SlA, SRlA′,MrA). Both longitudinal strain and radial motion fraction were significantly reduced in the conduit phase (ObT2D vs. control, p = 0.04). Left atrial size and strain measurements Expressed as an E′/A′ ratio, there was a 28.5% reduction in the lon- gitudinal strain rate ratio (SRlE′/SRlA′, ObT2D vs. control, p = 0.01). When radial motion was expressed as a rela- tive velocity, the group difference was accentuated, with a 38.2% reduction in the VrE′/VrA′ ratio (ObT2D vs. control, In the ObT2D group, the MV corrected perpendicular component of the radial motion was decreased by 20% during the reservoir phase and by 33.6% in the conduit phase (ObT2D vs. control, p = 0.04 and p = 0.09 respec- tively). During the atrial relaxation (reservoir) and con- traction phases, the MV centric radial components were higher in the ObT2D group compared to control (MV centric radial motion fraction in reservoir cMrR, p = ns; in LA contraction cMrA: p = 0.005). The increased cMrA in the ObT2D group corresponded to a magnitude change of +28% relative to controls (20 vs. 26% in the MV centric radial plane defined in Fig. 1). Evin et al. Left atrial size and strain measurements 298.9 ± 83.4 mL, p < 0.001 for absolute volume and 89.7 ± 29.6 vs. 134.6 ± 30.8 mL/m2, p < 0.001 for indexed volume). Following correction for BMI, these differences were attenuated but remained significant (p = 0.001). Correlations between left atrial function and measures of ObT2D Slight differences in intra- myocardial fat fraction were present between groups (ObT2D vs. control, 7.9 ± 3.7% vs. 6.0 ± 1.5%, p = 0.051; Table 3). In the ObT2D group, the epicardial fat measure- ment was increased by 69% for absolute volume and by 50% for indexed volume (ObT2D vs. control, 176.9 ± 76.6 Intra-observer assessment resulted in an intra-class cor- relation coefficient of 0.95. Slight differences in intra- myocardial fat fraction were present between groups (ObT2D vs. control, 7.9 ± 3.7% vs. 6.0 ± 1.5%, p = 0.051; Table 3). In the ObT2D group, the epicardial fat measure- ment was increased by 69% for absolute volume and by 50% for indexed volume (ObT2D vs. control, 176.9 ± 76.6 Left atrial size and strain measurements Cardiovasc Diabetol (2016) 15:164 Page 6 of 10 Table 2 Left atrium volumes and functional parameters derived from MRI data Results are expressed as mean ± SD * p < 0.05 *** p < 0.001 Healthy volunteers ObT2D HV/ObT2D (n = 19) (n = 20) p value LA EDV (mL) 87.1 ± 24.7 95.8 ± 22.3 LA ESV (mL) 37.4 ± 14.7 40.5 ± 12.8 Indexed LA EDV (mL/m2) 44.7 ± 9.8 43.3 ± 9.3 Indexed LA ESV (mL/m2) 19.1 ± 6.1 18.2 ± 5.2 LA EF (%) 57.7 ± 6.1 58.0 ± 7.9 Longitudinal strain (%) SlR 33.2 ± 6.8 29.4 ± 8.4 SlC 16.5 ± 4.8 13.1 ± 5.0 * SlA 16.7 ± 3.9 16.8 ± 4.8 SlA/SLR 0.5 ± 0.1 0.6 ± 0.1 Longitudinal strain rate (%/s) SRlS′ 1.4 ± 0.3 1.4 ± 0.4 SRlE′ −1.5 ± 0.4 −1.2 ± 0.6 SRlA′ −1.4 ± 0.4 −1.5 ± 0.5 SRlE′/SRlA′ 1.2 ± 0.5 0.9 ± 0.3 * Radial motion fraction (%) MrR 35.1 ± 6.7 31.5 ± 9.1 MrC 17.2 ± 4.7 13.6 ± 5.9 * MrA 17.9 ± 4.3 18.2 ± 4.3 MrA/MrR 0.5 ± 0.1 0.6 ± 0.1 * Radial relative velocity (%/s) VrS′ 1.4 ± 0.3 1.5 ± 0.6 VrE′ −1.6 ± 0.5 −1.3 ± 0.6 VrA′ −1.5 ± 0.5 −1.8 ± 0.6 VrE′/VrA′ 1.2 ± 0.5 0.7 ± 0.2 * Decomposition of the radial motion fraction (%) MV centric cMrR 34.3 ± 9.6 37.2 ± 10.6 MV centric cMrC 14.0 ± 5.7 11.3 ± 5.7 MV centric cMrA 20.3 ± 5.6 26.0 ± 6.3 MV corrected perpen- dicular pMrR 53.3 ± 17.8 42.5 ± 12.6 * MV corrected perpen- dicular pMrC 21.0 ± 15.2 14.0 ± 9.5 MV corrected perpen- dicular pMrA 32.3 ± 10.7 28.6 ± 10.7 Table 2 Left atrium volumes and functional parameters derived from MRI data vs. 298.9 ± 83.4 mL, p < 0.001 for absolute volume and 89.7 ± 29.6 vs. 134.6 ± 30.8 mL/m2, p < 0.001 for indexed volume). Following correction for BMI, these differences were attenuated but remained significant (p = 0.001). vs. 298.9 ± 83.4 mL, p < 0.001 for absolute volume and 89.7 ± 29.6 vs. 134.6 ± 30.8 mL/m2, p < 0.001 for indexed volume). Following correction for BMI, these differences were attenuated but remained significant (p = 0.001). vs. Correlations between left atrial function and measures of ObT2D Figure 3a–c illustrates the relationship between BSA and longitudinal strains and motion fractions (SlR, MrR and MrC, Panel a), VrE′/VrA′ and BMI (panel b), and VrE′/VrA′ and epicardial fat volume indexed to BSA (panel c). These demonstrated tight clustering of the VrE′/VrA′ values in the ObT2D group, with all of the subjects falling into the lowest two quartiles of VrE′/VrA′ (as defined using the normal cohort), and 70% within the lowest quartile of the normal range. There was no overlap in BMI and almost no overlap in epicardial fat. Significantly, all subjects with a BMI over 30 kg/m2 had low values of VrE′/VrA′– below or near 1 standard deviation from the control group average value. Other significant associations between Dixon 3D epicardial fat fraction and functional LA indi- ces are presented in Fig. 3d–f (SRlE/SRlA, VrE/VrA, MrA, MrA/MrR). Correlation coefficients between functional indexes and BSA or Dixon 3D epicardial fat fraction are reported Table 4. By univariate analysis, intra-myocardial fat was cor- related to VrE′/VrA′ in the control group (r = −0.602; p = 0.006). No other significant correlations were seen in either controls or in the ObT2D group (correlation VrE′/VrA′ with intra-myocardial fat in patients r = 0.18; p = ns). Figure 3 highlights that there is a lack of dynamic range for VrE′/VrA′ in the ObT2D group, with values tightly clustered together. Therefore, linear correlation between BMI or epicardial fat was not performed to fur- ther analyse for any linear relationship. The ObT2D and control groups clearly did not form a normal distribution hence a pooled analysis was not performed.h The correlations between MV centric radial motion fraction and adipose tissue quantifications and patient biology were highly significant between cMrR and BSA (r = −0.49, p = 0.002) and between cMrR and BMI (r = −0.44, p = 0.005). Significant correlations were also found for indexed and non-indexed Dixon 3D epi- cardial fat volumes with cMrR (r = −0.33, p = 0.04 and r = −0.41, p = 0.01, respectively) and pMrA (r = 0.46, p = 0.004 and r = 0.37, p = 0.02). No significant correla- tions were present between atrial parameters and dura- tion of diabetes or HbA1c. Intra‑myocardial and epicardial fat measurements ntra‑myocardial and epicardial fat measurements Intra‑myocardial and epicardial fat measurements Intra-observer assessment resulted in an intra-class cor- relation coefficient of 0.95. Discussion Our data demonstrates that MRI-derived LA strain may be sensitive to early diastolic dysfunction. Our cohort of patients with obesity and T2D had an almost normal Evin et al. Cardiovasc Diabetol (2016) 15:164 Page 7 of 10 Table 3 Fat measurements by MRI and echocardiography in healthy volunteers and obese patients Results are expressed as mean ± SD * p < 0.001 Healthy volunteers ObT2D HV/ObT2D (n = 19) (n = 20) p value Intra-myocardium dixon fat fraction (%) 7.9 ± 3.7 6.0 ± 1.5 Dixon 3D epicardial fat volume (mL) 176.4 ± 68.6 273.8 ± 64.0 * Indexed dixon 3D epicardial fat volume (mL/m2) 89.7 ± 25.2 123.6 ± 23.4 * Dixon 3D epicardial fat fraction (%) 21.0 ± 4.0 28.4 ± 5.5 * Fig. 3 Correlations between BSA and LA functional indices in the control and ObT2D groups (a). VrE′/VrA′ related to BMI (b) and indexed adipose tissue volume (c). Correlations between LA functional indices and 3D Dixon cardiac fat fraction: longitudinal strain rates and radial relative velocities ratios (d), and radial motion fraction (e) and with radial motion fraction ratio (f) Table 3 Fat measurements by MRI and echocardiography in healthy volunteers and obese patients ements by MRI and echocardiography in healthy volunteers and obese patients Fig. 3 Correlations between BSA and LA functional indices in the control and ObT2D groups (a). VrE′/VrA′ related to BMI (b) and indexed adipose tissue volume (c). Correlations between LA functional indices and 3D Dixon cardiac fat fraction: longitudinal strain rates and radial relative velocities ratios (d), and radial motion fraction (e) and with radial motion fraction ratio (f) Fig. 3 Correlations between BSA and LA functional indices in the control and ObT2D groups (a). VrE′/VrA′ related to BMI (b) and indexed adipose tissue volume (c). Correlations between LA functional indices and 3D Dixon cardiac fat fraction: longitudinal strain rates and radial relative velocities ratios (d), and radial motion fraction (e) and with radial motion fraction ratio (f) higher LV mass [8]. Consistent with this, our results demonstrated an alteration of the LA function in indi- viduals with obesity and T2D, as expressed by the ratio between conduit to LA contraction phase (E′/A′) and the LA contraction to reservoir phases. Both longitudi- nal strain rates SRlE′/SRlA′ and relative velocity VrE′/VrA′ ratios were significantly altered in the obese and diabetic group compared to matched controls, consistent with early stage diastolic dysfunction. Discussion Our data demonstrated a diminution of the conduit phase of LA function. A pos- sible explanation for this finding could be elevated LV pressure, which is well described to be an early change in diastolic dysfunction. Coronary flow reserve has also been associated with LV filling pressure [18] in ObT2D which suggest a more sophisticated pathway of diastolic dysfunction. Additionally, the longitudinal (SRlE′/SRlA′) echocardiogram and non-enlarged LA volumes by stand- ard imaging techniques [17]. Furthermore, our results show that LA strain is strongly correlated with BMI and epicardial fat supporting an association between adipos- ity and LA strain. If LA strain measures are validated, the ability to reliably detect early changes likely to be a pre- cursor to clinically apparent disease may have important applications as both a clinical and research tool. Moreo- ver, identification of individuals at risk for progression of the early diastolic dysfunction in ObT2Dmay enable (i) a more comprehensive characterisation of the patho physi- ology of early LA functional alteration in diastolic func- tion, and (ii) testing of novel disease modifying strategies. Alt ti f LA f ti i th ObT2D l ti Alterations of LA function in the ObT2D population are considered to be secondary to pressure overload and are associated with poorer LV systolic function and Evin et al. Cardiovasc Diabetol (2016) 15:164 Page 8 of 10 Table 4 Correlations between functional indexes and body surface area and Dixon 3D epicardial fat fraction * p < 0.05 p < 0.01 * p < 0.001 Body surface area (m2) Dixon 3D epicardial fat fraction (%) r p r p Longitudinal strain (%) SlR −0.561* <0.001 −0.079 0.636 SlE −0.456 0.004 −0.141 0.399 SlA −0.390* 0.014 0.090 0.591 SlA/SlR −0.373* 0.019 −0.335* 0.040 Longitudinal strain rate (%/s) SRlS′ 0.067 0.687 0.128 0.443 SRlE′ −0.302 0.062 0.043 0.798 SRlA′ 0.394* 0.013 0.096 0.566 SRlE′/SRlA′ −0.006 0.971 −0.269 0.103 Radial motion fraction (%) MrR −0.570* <0.001 −0.041 0.805 MrE −0.576* <0.001 −0.143 0.390 MrA −0.284 0.080 0.153 0.359 MrA/MrR 0.278 0.087 0.244 0.140 Radial relative velocity (%/s) VrS′ −0.064 0.698 0.252 0.127 VrE′ 0.437** 0.005 0.071 0.674 VrA′ −0.024 0.884 −0.355* 0.029 VrE′/VrA′ −0.413 0.009 −0.435 0.006 Table 4 Correlations between functional indexes and body surface area and Dixon 3D epicardial fat fraction volume. Discussion When the ObT2D and control groups were con- sidered separately, there was no significant linear rela- tionship between BMI or epicardial fat and LA function, perhaps due to our small sample size. This may suggest that LA functional alterations seen in the ObT2D group may occur over a certain threshold of epicardial adipose tissue volume or BMI. However, further data are required to test this hypothesis.h There are several proposed mechanisms by which the tissue alterations in obesity may influence the properties or mechanical load experienced by the LA. These include increased ventricular stiffness, inflammation mediated by adipokines, direct lipotoxicity or a metabolic effect mediated by insulin resistance [9]. Diastolic dysfunction has been previously shown to correlate with duration of diabetes, HbA1c and obesity [24]. Previous reports of correlations between strains and BSA or BMI were lim- ited to ventricle [25] and detailed LV strain contraction analysis highlight the rule of glycemic control [26]. In our study, we did not find an association between LA strain and duration of diabetes or HbA1c, possibly due to the early stage of diastolic dysfunction detected. Prior echo- cardiographic data support a primary atrial myopathy in diabetes [27, 28]. Our study demonstrated alterations of conduit longitudinal strain and motion fraction, changes which are supportive of primary alterations of LV relaxa- tion associated with mild diastolic dysfunction [29]. The strong correlation between MV centric motion fraction and BMI/epicardial fat might suggest MV centric motion fraction during LA contraction phase as a useful earlier predictor of diastolic dysfunction in the setting of obesity and T2D.if and radial (VrE′/VrA′) ratios depicted the change in the LA wall deformation, the passive conduit phase relative to the active phase of the LA. When radial motion was decomposed into cMr and pMr parts, the amplification of MV centric motion fraction during the LA contrac- tion phase in the ObT2D group was consistent with the anticipated response of an early decrease in ventricu- lar compliance impairing LV early filling. The observed reduction of reservoir MV corrected perpendicular motion fraction may related to LV contraction altera- tions as reported by Mochizuki et al. [19]. An increase in the functional parameters of LA contraction has been previously described in association with mild diastolic dysfunction, and has been proposed as a method of dif- ferentiating changes resulting from LV compliance as opposed to LV hypertrophy [8, 20]. Discussion and radial (VrE′/VrA′) ratios depicted the change in the LA wall deformation, the passive conduit phase relative to the active phase of the LA. When radial motion was decomposed into cMr and pMr parts, the amplification of MV centric motion fraction during the LA contrac- tion phase in the ObT2D group was consistent with the anticipated response of an early decrease in ventricu- lar compliance impairing LV early filling. The observed reduction of reservoir MV corrected perpendicular motion fraction may related to LV contraction altera- tions as reported by Mochizuki et al. [19]. An increase in the functional parameters of LA contraction has been previously described in association with mild diastolic dysfunction, and has been proposed as a method of dif- ferentiating changes resulting from LV compliance as opposed to LV hypertrophy [8, 20]. Our data demonstrated no significant differences in intra-myocardial Dixon fat between the ObT2D and control groups. A previous study reported a correlation between altered LV intra-myocardial fat fraction and LA function [10]. Our MRI data from subjects with early functional LA changes is a novel finding suggesting that intra-myocardial LV changes may not be a primary event driving very early dysfunction. The Dixon technique is only able to measure intra-myocardial fat quantity. It is insensitive to other myocardial tissue organisational alterations occurring in obesity and T2D. Further data regarding other ultra-structural analysis (e.g. from dif- fusion tensor imaging [30], T1 mapping [31], biochemi- cal changes [32], or even electrophysiology [33]) may be helpful in extending the characterisation of LV structural changes in early diastolic dysfunction. An association between BMI and LA function has been previously described [21–23]. In our study, the use of MRI enabled the quantification of LA function, LV intra- myocardial and epicardial fat during the same examina- tion. We demonstrated a clear relationship between LA relative velocity ratios and both BMI and epicardial fat MRI is an attractive alternative to the routine use of echocardiography as it overcomes the practical problems of image attainment in obese patients and simultane- ously characterises LA and LV functions as well as car- diac structure. The determination of cardiac fat volume Evin et al. Cardiovasc Diabetol (2016) 15:164 Page 9 of 10 by Dixon images has been previously reported [34]. How- ever, our study is the first to use MRI to measure cardiac size, volume atrial strain and fat in one examination. Author details 1 1 Sydney Translational Imaging Laboratory, Heart Research Institute, Charles Perkins Centre, University of Sydney, Camperdown, NSW 2006, Australia. 2 Sydney Medical School, University of Sydney, Camperdown, Australia. 3 Sorbonne Universités, UPMC Univ Paris 06, INSERM UMRS 1146, CNRSUMR 7371, Laboratoire d’Imagerie Biomédicale, ICAN Institute of Cardiometabolism and Nutrition, Paris, France. 4 Department of Endocrinology, Royal North Shore Hospital, St Leonards, Australia. 5 Kolling Institute, University of Sydney, Sydney, Australia. 6 Department of Cardiology, Royal North Shore Hospital, St Leonards, Australia. 7 Departments of Radiology, Royal North Shore Hospital and Royal Prince Alfred Hospital, Sydney, Australia. Authors’ contributions ME wrote the manuscript and researched data and KMB researched data and contributed to the manuscript, FMC reviewed/edited the manuscript, RTM researched data and contributed to the manuscript, SG reviewed/edited the manuscript, RK reviewed/edited the manuscript, SLH reviewed/edited the manuscript, JL, AR and NK contributed to the development of the feature tracking algorithm for the post-processing of the LA function data as well as reviewed/edited the manuscript. GRF reviewed/edited manuscript, GAF reviewed/edited manuscript and SMG wrote the manuscript. All authors read and approved the final manuscript. p p p This study has several limitations, including the rela- tively small sample size, slightly age range difference between patients and healthy volunteers, and uniform diagnosis of obesity and diabetes, which did not permit deconvolution of the contributions of these conditions. Additional studies are required on the decomposition of radial motion into centric and perpendicular motions. Our clinical evaluation of the control subjects was also limited and measured blood pressures were not available, a clinical diagnosis of any cardiovascular disease includ- ing hypertension was excluded for all controls. The qual- ity of the myocardial characterisation could be improved with the addition of T1 mapping or magnetic resonance spectroscopy data. In order to explore the nature of the early changes occurring in obesity and T2D, prospective studies are needed that include longitudinal imaging time points combined with objective clinical and metabolic measures (e.g. glucose tolerance test) of the progres- sive metabolic disorders over time. Although the control echocardiographic data was not available in this study, from a methodological perspective, it would be useful to directly compare diastolic assessment by echocardiogra- phy between the controls and ObT2D groups. Consent for publication Consent for publication was obtained from the patients and controls as part of the consent to participate. Discussion This approach is likely to help to understand the pathogene- sis of diastolic dysfunction in obesity and diabetes, and would enable testing of novel early therapeutic options in these patients. Further studies are needed to understand the different components of cardiac functional and struc- tural modification in the setting of obesity and diabetes in order to explore possible therapeutic routes for reversal.h mitral valve; ObT2D: obesity and type 2 diabetes; pMr: MV perpendicular radial motion fraction; SlR: reservoir longitudinal strain; SlC: conduit longitudinal strain; SlA: LA contraction longitudinal strain; SRlA′: A′ longitudinal strain rates; SRlE′: E′ longitudinal strain rates; SRlS′: S′ longitudinal strain rates; SV: stroke volume; VrA′: A′ radial relative velocities; VrE′: E′ radial relative velocities; VrS′: S′ radial relative velocities. CMR: cardiovascular magnetic resonance; cMr: MV centric radial motion fraction; EDV: LV end-diastolic; EFV: epicardial fat volume; ESV: end-systolic volumes; HFpEF: heart failure with preserved ejection fraction; ICC: intra-class correlation coefficient; LA: left atrial; LV: left ventricle; LVEF: LV ejection fraction; MrA: LA contraction radial motion fraction; MrC: conduit radial motion fraction; MRI: magnetic resonance imaging; MrR: reservoir radial motion fraction; MV: Financial support Novo Nordisk, Sydney Medical School Foundation, Heart Research Institute, Heart Research Australia, & Parker Hughes Bequest, University of Sydney, Australia. Disclosures MRI-derived strain measurements may be a useful tool to detect early abnormal LA function. Our cohort of patients with obesity and T2D had near normal echocardiograms and MRI-derived LV systolic function and volumetric measures of the LA and LV, and we demonstrated clear reductions in the conduit to LA contraction ratios (E/A) for longitudinal strain rate and radial motion fraction. Importantly these atrial measures correlated to both epi- cardial fat and to BMI. Further work in larger cohorts with a greater dynamic range of these parameters is required to understand the full implications of these findings. Novo Nordisk provided the funding for the MRI scans in this study as part of a prospective longitudinal trial—The combined Effect of Liraglutide and Sleeve Gastrectomy on Metabolic, Cardiac, Neurological and Sleep Function in Obese Diabetes: A twelve-month Randomised Study” (LIRASLEEVE). Ethics approval and consent to participate Patients were selected from the study entitled “The combined Effect of Liraglutide and Sleeve Gastrectomy on Metabolic, Cardiac, Neurological and Sleep Function in Obese Diabetes”. The Northern Sydney ethics committee approved the study. All subjects provided written consent. Competing interests h h d l h Competing interests The authors declare that they have no competing interests. Competing interests The authors declare that they have no competing interests. p g The authors declare that they have no competing interests. Acknowledgementsf We thank the staff at North Shore Private Radiology, the Departments of Cardiology (especially Drs. Chris Choong and James Mau) and Endocrinology at Royal North Shore Hospital for their extensive cooperation and enthusiastic involvement with this project. References Eur Heart J Cardiovasc Imaging. 2015;16:1191–7. 5. Borlaug BA, Paulus WJ. 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Clinical features of subclinical left ventricular systolic dysfunction in patients with diabetes mellitus. Cardiovasc Diabe- tol. 2015;14:37. Abbreviations CMR di We thank the Heart Research Institute for the funding of a Post-Doctoral Grant for ME. SMG acknowledges the support of the Heart Research Institute, Sydney Medical School Foundation and the Parker Hughes Bequest, University of Sydney. GF acknowledges Fellowship support from NHMRC, Heart Research Australia, and Heart Foundation (Australia). GRF acknowledges funding sup- port for the LIRASLEEVE study (from which these baseline data were obtained) from NOVO Nordisk. 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https://openalex.org/W2133743316	https://europepmc.org/articles/pmc3282675?pdf=render	English	null	Fluticasone furoate: once-daily evening treatment versus twice-daily treatment in moderate asthma	Respiratory research	2,011	cc-by	7,640	Abstract Background: Inhaled corticosteroids are the recommended first-line treatment for asthma but adherence to therapy is suboptimal. The objectives of this study were to compare the efficacy and safety of once-daily (OD) evening and twice-daily (BD) regimens of the novel inhaled corticosteroid fluticasone furoate (FF) in asthma patients. Methods: Patients with moderate asthma (age ≥12 years; pre-bronchodilator forced expiratory volume in 1 second (FEV1) 40-85% predicted; FEV1 reversibility of ≥12% and ≥200 ml) were randomized to FF or fluticasone propionate (FP) regimens in a double-blind, crossover study. Patients were not permitted to have used any ICS for ≥8 weeks prior to enrolment and subsequently received doses of FF or FP 200 μg OD, FF or FP 100 μg BD and matching placebo by inhalation for 28 days each. Primary endpoint was Day 28 evening pre-dose (trough) FEV1; non-inferiority of FF 200 μg OD and FF 100 μg BD was assessed, as was superiority of all active treatment relative to placebo. Adverse events (AEs) and 24-hour urinary cortisol excretion were assessed. Results: The intent-to-treat population comprised 147 (FF) and 43 (FP) patients. On Day 28, pre-dose FEV1 showed FF 200 μg OD to be non-inferior (pre-defined limit -110 ml) to FF 100 μg BD (mean treatment difference 11 ml; 95% CI: -35 to +56 ml); all FF and FP regimens were significantly superior to placebo (p ≤0.02). AEs were similar to placebo; no serious AEs were reported. Urinary cortisol excretion at Day 28 for FF was lower than placebo (ratios: 200 μg OD, 0.75; 100 μg BD, 0.84; p ≤0.02). Conclusions: FF 200 μg OD in the evening is an efficacious and well tolerated treatment for asthma patients and is not inferior to the same total BD dose. Trial registration: Clinicaltrials.gov; NCT00766090. Keywords: Asthma, fluticasone furoate, inhaled corticosteroid, once daily, efficacy, safety Inhaled corticosteroids (ICS) are the most effective controller medications for the first-line treatment of asthma in adults and children and are also used at later stages in combination with other medications, specifically long-acting beta2 agonists (LABA) [6]. ICS are typically developed for twice-daily dosing but once-daily evening dosing of an ICS has been reported to significantly improve adherence to therapy compared with twice-daily dosing (93.3% vs. 89.3% [p < 0.001] as measured by auto- matic dose counter) in an open-label 12 week study of mometasone furoate [7]. RESEARCH Open Access © 2011 Woodcock 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 This is a benefit that has the potential to improve patient outcomes, given the associa- tion between poor adherence rates (particularly for con- troller medications) and uncontrolled asthma in children Fluticasone furoate: once-daily evening treatment versus twice-daily treatment in moderate asthma Ashley Woodcock1, Eugene R Bleecker2, William W Busse3, Jan Lötvall4, Neil G Snowise5, Lucy Frith5, Loretta Jacques5, Brett Haumann5 and Eric D Bateman6 Woodcock et al. Respiratory Research 2011, 12:160 http://respiratory-research.com/content/12/1/160 Woodcock et al. Respiratory Research 2011, 12:160 http://respiratory-research.com/content/12/1/160 Correspondence: ashley.woodcock@manchester.ac.uk 1School of Translational Medicine, University of Manchester, Manchester, UK Full list of author information is available at the end of the article Background A variety of treatments are available for asthma but there remains potential to improve the level of disease control in adults and children [1-4]. Failure to achieve asthma control affects patients’ daily lives, for example through persistent symptoms, more frequent exacerba- tions and missed work and school time, placing demands on emergency care facilities [2,5]. Further improvements to the range of therapeutic options for asthma are needed so that patients can achieve better disease control. * Correspondence: ashley.woodcock@manchester.ac.uk 1School of Translational Medicine, University of Manchester, Manchester, UK Full list of author information is available at the end of the article Woodcock et al. Respiratory Research 2011, 12:160 http://respiratory-research.com/content/12/1/160 Woodcock et al. Respiratory Research 2011, 12:160 http://respiratory-research.com/content/12/1/160 Page 2 of 8 Page 2 of 8 SABA and had not taken ICS for ≥8 weeks, but could have taken LABAs, xanthines, cromones, or leukotriene modifiers provided they had been stopped at screening. Patients had to refrain from using oral, parenteral, and depot forms of corticosteroids in the 8 weeks before screening and anti-IgE therapy in the 12 weeks before screening. We excluded patients who had smoked in the year before the study, those with a smoking history of > 10 pack-years, and individuals with a respiratory infec- tion, life-threatening asthma, or asthma exacerbations requiring either hospitalization in the 6 months before screening or oral corticosteroids in the 8 weeks before screening. Drug therapy was withheld for baseline spiro- metry, treatment with LABAs and leukotriene modifiers was ceased the day before assessment, and patients could not take salbutamol during the 6-hour period before the clinic visit. and adults [8,9], and the reported correlation between falling rates of adherence to ICS and higher rates of asthma-related hospitalization in adults [10]. Animal and human pharmacology studies show that the novel ICS fluticasone furoate (FF) has a long dura- tion of action and prolonged retention in the lung, sug- gesting that it is suitable for once-daily dosing [11]. FF and fluticasone propionate (FP) are structurally different. At the C-17a position, FF contains an ester derived from 2-furoic acid which replaces the simpler propio- nate ester. These differences mean that FF has more complete occupancy of the 17a pocket in the glucocor- ticoid receptor [12] and higher glucocorticoid receptor binding affinity than FP [13]. Patients Patients with moderate persistent asthma, aged 12 years or more with a pre-bronchodilator FEV1 of 40-85% of predicted normal value and reversibility of FEV1 to inhaled salbutamol of at least 12% and at least 200 ml were eligible for inclusion [17]. Patients were taking Study design This was a randomized, placebo-controlled crossover study designed to compare the efficacy and safety of 28 days’ treatment of FF given as a once-daily and twice- daily regimen in adolescents and adults with asthma. The study was approved by local ethics review commit- tees and was conducted in accordance with the Declara- tion of Helsinki and Good Clinical Practice guidelines at 16 investigative sites in the USA between October 2008 and March 2009. All patients gave written informed consent. The trial is registered as NCT00766090 on the Clinicaltrials.gov registry and the sponsors’ study num- ber is FFA112202. Background As part of the overall phase II development plan investigating FF, dose-ran- ging studies in asthma patients have demonstrated that FF has a favourable efficacy and safety profile when administered once-daily in the evening [14-16]. The aims of the current study were to compare the efficacy and safety of once-daily versus twice-daily FF regimens with each other and with placebo in patients uncontrolled on a non-corticosteroid controller or short-acting beta2 agonist (SABA) alone. The study spe- cifically tested the hypothesis that a once-daily regimen is not inferior to a twice-daily regimen with respect to lung function (evening pre-dose forced expiratory volume in 1 second (FEV1)) after 4 weeks’ treatment. FP once-daily and twice-daily regimens were included as active controls to confirm that the primary efficacy vari- able of trough FEV1 (measured pre-dose in the evening) on Day 28 was sensitive enough to detect differences between active treatments and placebo. Randomization Eli ibl i Eligible patients entered a run-in period of at least 7 days during which safety evaluations were conducted including a 24-hour urine collection for determination of cortisol excretion (see below). Patients were randomly assigned to either an FF group or an FP group, in a 7:2 ratio, respectively. To be eligible to enter the treatment period patients were required, at the end of the run-in period to exhibit the following; (I) evening FEV1 between 40% and < 80% and at least one of a daily symptom score of ≥1, rescue medication use on 4 of the last 7 days or PEF variability of ≥20% on 4 of the last 7 days; (2) evening FEV1 between ≥80% and 85% and at least one of a daily symptom score of ≥1, rescue medication use on all of the last 7 days or PEF variabil- ity of ≥20% on all of the last 7 days. Additionally patients were required to have a 24-hour urine cortisol sample available at the end of the run-in period. In the FF group all patients received drug via the NDPI and in the FP group via the Diskus™; thus although patients and investigators were blinded to which treatment they were receiving within a group, they were not blinded to whether they were allocated to an FF or an FP group. The central randomization schedule was generated by the sponsor using a validated computerized system (RandAll). The Registration and Medication Ordering System (RAMOS), an automated, interactive telephone based system, was used by the investigator or designee to register and randomize the patient and receive medi- cation assignment information. Treatment assignment could be unblinded only in an emergency, through a call to the interactive telephone system. Outcome measurements Assuming an average within-patient standard devia- tion in pre-bronchodilator evening trough FEV1 of 210 ml and a non-inferiority limit of -110 ml, 84 completed patients would be required in the FF patient set to demonstrate non-inferiority of FF 200 μg once daily relative to FF 100 μg twice daily with 92% statistical power and a 2.5% one-sided significance level. For the superiority comparisons with placebo, this number of patients would enable detection of a difference of 200 ml between each of the FF groups and placebo with > 99% power. For the FP patient set, the target number of completed patients (n = 24) would enable detection of a 200 ml difference between FP dosed once daily or twice daily and placebo with 91% power, based on a 2-sided 5% significance level and a within-subject standard deviation of 210 ml. The primary endpoint was the pre-dose, pre-rescue bronchodilator FEV1 on the evening of Day 28 of the treatment period. The protocol required that spirometry was performed on Days 0 and 28 at 8.00 pm +/- 3 hours, at least 6 hours after the last administration of salbutamol and within 1 hour of the time of the Day 0 measurement. Statistical analysis h The intent-to-treat (ITT) population comprised all patients who received at least one dose of study medica- tion; the per-protocol (PP) population was the subset of patients in the ITT population who completed at least one treatment period without a protocol deviation. Both populations were used to assess the primary comparison of FF once daily versus FF twice daily. For the assess- ment of differences between active and placebo groups, the ITT population was used. The PP population was considered to be a supportive analysis. The UC popula- tion consisted of patients who had urine samples with no confounding factors that would limit the analysis of UC. From the screening visit onwards, no additional asthma medications were allowed except for rescue sal- butamol. Intranasal and topical corticosteroids, and oral, ocular, and intra-nasal antihistamines were permitted. Treatments Patients were assigned to 1 of 12 possible treatment sequences (table 1), each sequence comprising three 28- Woodcock et al. Respiratory Research 2011, 12:160 http://respiratory-research.com/content/12/1/160 Page 3 of 8 Page 3 of 8 Table 1 Distribution of patients between treatment sequences Sequence Allocation ratio Treatments Delivery device Period 1 Period 2 Period 3 1 7 Placebo FF 200 μg OD FF 100 μg BD Novel dry powder inhaler 2 7 Placebo FF 100 μg BD FF 200 μg OD 3 7 FF 100 μg BD Placebo FF 200 μg OD 4 7 FF 100 μg BD FF 200 μg OD Placebo 5 7 FF 200 μg OD Placebo FF 100 μg BD 6 7 FF 200 μg OD FF 100 μg BD Placebo 7 2 Placebo FP 200 μg OD FP 100 μg BD Diskus™inhaler 8 2 Placebo FP 100 μg BD FP 200 μg OD 9 2 FP 100 μg BD Placebo FP 200 μg OD 10 2 FP 100 μg BD FP 200 μg OD Placebo 11 2 FP 200 μg OD Placebo FP 100 μg BD 12 2 FP 200 μg OD FP 100 μg BD Placebo FF = fluticasone furoate; FP = fluticasone propionate; BD = twice daily; OD = once daily. Table 1 Distribution of patients between treatment sequences as any worsening of asthma that required emergency intervention, hospitalization, or treatment with an asthma medication not allowed by the study protocol) were withdrawn from the study. day treatment periods separated by two 2-week washout periods. Six sequences contained FF 200 μg once daily in the evening (with placebo in the morning), FF 100 μg twice daily and matching placebo twice daily dosed from a novel dry powder inhaler. Six sequences contained FP 200 μg once daily in the evening (with placebo in the morning), FP 100 μg twice daily and matching placebo twice daily dosed from a Diskus™inhaler. The differ- ence between the delivery devices used to deliver FF and FP meant that investigators could distinguish whether patients were assigned to an FF or FP sequence, but were double-blind to whether placebo or either of the two active regimens were being administered. Safety evaluation Adverse events (AEs) were coded using Medical Dic- tionary for Regulatory Activities (MedDRA, version 11). Safety assessments included routine laboratory tests, vital signs and oropharyngeal examination, and change in 24-hour urinary cortisol (UC) excretion between study baseline and the end of each 28-day treatment period. Patients who had asthma exacerbations (defined Woodcock et al. Respiratory Research 2011, 12:160 http://respiratory-research.com/content/12/1/160 Woodcock et al. Respiratory Research 2011, 12:160 http://respiratory-research.com/content/12/1/160 Page 4 of 8 Page 4 of 8 Table 2 Baseline characteristics of patients in each set of treatment sequences FF sequences (n = 147) FP sequences (n = 43) Total (n = 190) Age (years) 31.4 (15.30) 35.2 (16.03) 32.3 (15.51) Range 12-68 12-76 12-76 Females, n (%) 87 (59) 21 (49) 108 (57) Race (%) White 90 (61) 22 (51) 112 (59) African American/ African heritage 50 (34) 20 (47) 70 (37) Other 7 (5) 1 (2) 8 (4) History of atopy, n (%) 93 (63) 27 (63) 120 (63) Lung function at screening Reversibility of FEV1 (%) 27.20 (13.667) 27.52 (16.449) 27.27 (14.298) Reversibility of FEV1 (ml) 608.2 (304.64) 591.4 (367.47) 604.4 (318.98) Lung function at study baseline Pre-bronchodilator FEV1 (L) 2.296 (0.6176) 2.293 (0.6990) 2.296 (0.6350) Pre-bronchodilator FEV1 (% predicted) 69.85 (9.704) 67.73 (11.204) 69.37 (10.071) Values are mean and standard deviation unless stated. Data shown are for the ITT population. FEV1 = forced expiratory volume in 1 second; FF = fluticasone furoate; FP = fluticasone propionate Table 2 Baseline characteristics of patients in each set of treatment sequences For the primary efficacy analysis, comparison of treat- ment differences was performed using mixed model analysis of covariance (ANCOVA) with fixed effects for treatment, period, sex, and age and including period baseline as part of a bivariate response. In this analysis, ANCOVA was also used to compare treatment differ- ences in UC excretion, with treatment, period, age, sex, and study baseline as fixed effects and patient as a ran- dom effect. For each treatment group, least square (LS) mean values were calculated for absolute pre-dose FEV1 and change in pre-dose FEV1 from period baseline. All analyses were pre-planned before the study blind was broken. No subgroup analyses were performed. Study population Of 190 patients randomly assigned to study treatment, 147 were assigned to one of the six FF sequences and 43 to one of the six FP sequences; 134 and 41 patients, respectively, completed the study. Reasons for failure at the screening stage and reasons for withdrawal after the randomization stage are shown in Figure 1. The ITT population consists of all 190 patients who were rando- mized and 177 patients met the criteria to be included in the PP population. Baseline demographic and clinical characteristics of patients assigned to the two sets of treatment sequences (FF and FP) are shown in table 2. Asthma was generally long-standing with 164 patients (86%) having asthma for at least 5 years. ITT and PP populations. Pre-dose FEV1 increased in all groups, but the mean increases in the four active treat- ment groups were approximately twice those in the pla- cebo group. The differences versus placebo were statistically significant in all four active treatment groups, as assessed in the ITT population. For FF 200 μg once daily, FF 100 μg twice daily and FP 100 μg twice daily, the p value for the difference was < 0.001, while for FP 200 μg once daily the p value for the differ- ence was 0.02. Efficacy The mean values of pre-dose FEV1 on Day 28 in each treatment group and the mean changes compared with period baseline (Day 0) are shown in table 3 for both 327 Screened 190 Randomly allocated to one of two treatment sequences 137 Not randomized 110 Failed inclusion/exclusion criteria 13 Failed randomization criteria 6 Study closed 5 Withdrew consent 2 Investigator decision 1 Lost to follow-up 147 FF 200 µg OD, 100 µg BD and matching placebo 134 Completed 43 FP 200 µg OD, 100 µg BD and matching placebo 2 Withdrawn 1 Lack of efficacy 1 Lost to follow-up 13 Withdrawn 5 Lack of efficacy 3 Lost to follow-up 2 Withdrew consent 2 Protocol deviation 1 Investigator decision 41 Completed Figure 1 Disposition of patients. BD = twice daily; OD = twice daily. In the ITT population, the lower 95% confidence interval (CI) for the mean difference between FF 200 μg once daily and FF 100 μg twice daily in pre-dose FEV1 on Day 28 was -35 ml (LS mean difference of 11 ml) (table 3; Figure 2). This difference was within the pre- defined limit of -110 ml, thus demonstrating non-infer- iority of the FF 200 μg once-daily regimen. Similar results were obtained from the non-inferiority analysis in the PP population: the lower 95% CI for the treat- ment difference was -49 ml (LS mean difference 0 ml). Data from patients treated with FP indicated numeri- cally reduced improvement in pre-dose FEV1 with the 200 μg once-daily dose in comparison with 100 μg twice daily, although no statistical comparison of these groups was performed. Figure 1 Disposition of patients. BD = twice daily; OD = twice daily. Woodcock et al. Safety No serious AEs were reported and no AEs led to perma- nent discontinuation of drug or to patient withdrawal. The frequency of on-treatment AEs was higher in the FF 200 μg once-daily, FF 100 μg twice-daily and placebo NDPI groups (16%, 18%, and 14%, respectively) than in the FP 200 μg once-daily, FP 100 μg twice-daily and Diskus™placebo groups (5%, 7% and 12% respectively). Upper respiratory tract infections (URTI) were the most commonly reported AEs, occurring in 5% of patients in each of the FF groups and 1% in the placebo group; no other AEs were reported by more than 1% of patients in either of the FF groups or the placebo group during the Asthma exacerbations occurred in five (3%) patients on placebo, and one (< 1%) patient on FF 200 μg once daily. None of the exacerbations were severe enough to require hospitalization. UC excretion data were analyzed in 170 patients with adequate 24-hour collections at study baseline and Day 28. The Day 28 ratio to placebo was statistically signifi- cantly lower in the FF 200 μg once-daily and FF 100 μg twice-daily arms (ratio 0.75, p < 0.001 and 0.84, p = 0.020, respectively), but the ratios with FP were not sta- tistically significant for the FP 200 μg once-daily and FP 100 μg twice-daily arms (ratio 1.03, p = 0.808 and 0.89, p = 0.338, respectively) (Figure 3). No AEs associated with abnormal urinary-free cortisol were reported. There were no clinically important changes in any laboratory test parameter or vital signs with any study treatment during any of the treatment periods. FP 100 µg BD – placebo FP 200 µg OD – placebo FF 100 µg BD – placebo Treatment comparison –200 FF 200 µg OD – placebo –100 0 100 Treatment difference and 95% CI (ml) 200 300 FF 200 µg OD – 100 µg BD Figure 2 Mean treatment difference (and 95% CI) adjusted for treatment, period, sex and age, for comparisons between active treatments and placebo and between the two FF dosage regimens (ITT population). Dotted line at 0 shows the point at which the two interventions would have an equal effect on pre-dose FEV1. The lower dotted line (for the FF 200 μg OD vs. FF 100 μg BD comparison) shows the predefined -110 ml threshold for non-inferiority of FF 200 μg OD versus FF 100 μg BD. Safety BD = twice daily; CI = confidence interval; OD = twice daily; FF = fluticasone furoate; FP = fluticasone propionate FP 100 µg BD – placebo FP 200 µg OD – placebo FF 100 µg BD – placebo Treatment comparison –200 FF 200 µg OD – placebo –100 0 100 Treatment difference and 95% CI (ml) 200 300 FF 200 µg OD – 100 µg BD Efficacy Respiratory Research 2011, 12:160 http://respiratory-research.com/content/12/1/160 Page 5 of 8 Page 5 of 8 Table 3 Evening pre-dose FEV1 on Day 28 of treatment and improvement from period baseline for each treatment regimen (all placebo treatments were pooled for these analyses) Placebo FF 200 μg OD FF 100 μg BD FP 200 μg OD FP 100 μg BD Number of patients 187 140 142 42 43 LS mean, ml (SE) 2605 (43.4) 2714 (44.4) 2703 (44.3) 2693 (53.5) 2737 (53.3) LS mean change from period baseline, ml (SE) 112 (18.6) 221 (20.9) 210 (20.7) 199 (36.5) 244 (36.1) LS mean difference (active-placebo), ml (95% CI) NA 108 (64-153); p < 0.001 98 (54-142); p < 0.001 87 (14-161); p = 0.020 132 (59-205); p < 0.001 LS mean difference (FF 200 μg OD-FF 100 μg BD), ml (95% CI) NA 11 (-35-56); p = 0.641 NA NA NA Absolute values and all differences are LS means, with 95% CI for non-inferiority and p values for superiority analysis for all comparisons between treatments. Data shown are for the ITT population. BD = twice daily; CI = confidence interval; FF = fluticasone furoate; FP = fluticasone propionate; LS = least square, NA = not applicable; OD = once daily; SE = standard error Table 3 Evening pre-dose FEV1 on Day 28 of treatment and improveme regimen (all placebo treatments were pooled for these analyses) Table 3 Evening pre-dose FEV1 on Day 28 of treatment and improvement from period baseline for each treatment regimen (all placebo treatments were pooled for these analyses) V1 on Day 28 of treatment and improvement from period baseline for each treatment ents were pooled for these analyses) ovement from period baseline for each treatmen treatment period (table 4). However, only three of the AEs reported (headache/dry throat, FF 100 μg twice daily; tachycardia, FP 200 μg once daily) were consid- ered to be potentially drug-related. One patient reported dysphonia (FP 200 μg once daily), but there were no cases of oral candidiasis. Discussion In this crossover study in adolescents and adults with moderate asthma, the same daily dose of a novel ICS, FF, administered once daily in the evening or as a twice- daily regimen was compared over a 28-day treatment period. For the primary efficacy variable of evening pre- dose FEV1, FF 200 μg once daily in the evening was non-inferior to FF 100 μg twice daily. All four active treatment arms were associated with significantly higher pre-dose FEV1 values than placebo. With FP, a numeri- cally higher increase in pre-dose FEV1 with twice-daily dosing than with once-daily dosing was observed. The Figure 2 Mean treatment difference (and 95% CI) adjusted for treatment, period, sex and age, for comparisons between active treatments and placebo and between the two FF dosage regimens (ITT population). Dotted line at 0 shows the point at which the two interventions would have an equal effect on pre-dose FEV1. The lower dotted line (for the FF 200 μg OD vs. FF 100 μg BD comparison) shows the predefined -110 ml threshold for non-inferiority of FF 200 μg OD versus FF 100 μg BD. BD = twice daily; CI = confidence interval; OD = twice daily; FF = fluticasone furoate; FP = fluticasone propionate Figure 2 Mean treatment difference (and 95% CI) adjusted for treatment, period, sex and age, for comparisons between active treatments and placebo and between the two FF dosage regimens (ITT population). Dotted line at 0 shows the point at which the two interventions would have an equal effect on pre-dose FEV1. The lower dotted line (for the FF 200 μg OD vs. FF 100 μg BD comparison) shows the predefined -110 ml threshold for non-inferiority of FF 200 μg OD versus FF 100 μg BD. BD = twice daily; CI = confidence interval; OD = twice daily; FF = fluticasone furoate; FP = fluticasone propionate Woodcock et al. Discussion difference between once and twice daily FP was in line with differences previously reported for FP once versus twice daily [18,19], although this supports the current indication for twice-daily dosing of FP in asthma the study was not powered nor designed to assess differ- ences between once-daily and twice-daily dosing of FP, only to assess differences between either FP dosing regi- men and placebo. FF appears to be suitable for once- daily dosing as both once-daily and twice-daily dosing (same total daily dose) produced similar improvements in lung function,. The efficacy results for FF in the cur- rent study are consistent with the results of three dose- ranging studies in patients with different levels of asthma severity, in which 8 weeks of FF administered once daily in the evening produced superior Both regimens of FF were well tolerated in this study and AE reporting rates were similar to placebo, espe- cially when considering the AEs reported for placebo and FF using the NDPI, and placebo and FP using the Diskus™. It is possibly the case that the higher inci- dence of AEs reported with the NDPI regardless of treatment (placebo or FF) resulted from a lack of famil- iarity with the device. There was only one asthma exacerbation among patients receiving FF and no dys- phonia or oropharyngeal candidiasis. URTIs were the only event to be reported more often in the FF groups than with placebo, and were not associated with loss of asthma control. A reduction in UC of 16% and 25% (relative to placebo) was observed with FF 100 μg BD and FF 200 μg OD, respectively, and this finding con- trasts those of other FF dose-ranging studies in which patients with asthma did not show any UC suppression relative to placebo after 8 weeks’ treatment at doses up to 600 μg once daily [14-16]. There was a numerical reduction in UC of the same magnitude (19%) in the FP 100 μg twice-daily regimen. No adverse safety events were recorded in the current study that could be attrib- uted to cortisol suppression. Further studies are needed to assess the magnitude of any potential effect of corti- sol suppression in susceptible patients. Discussion Respiratory Research 2011, 12:160 http://respiratory-research.com/content/12/1/160 Page 6 of 8 Page 6 of 8 Table 4 Number and proportion of patients reporting AEs during treatment, for events reported by at least 1% of patients in the FF or placebo arms Number of patients reporting event, n (%) Placebo nDPI (n = 145) Placebo DISKUS (n = 42) FF 200 μg OD (n = 140) FF 100 μg BD (n = 142) FP 200 μg OD (n = 42) FP 100 μg BD (n = 43) Any on-treatment AE 21 (14) 5 (12) 22 (16) 26 (18) 2 (5) 3 (7) URTI 2 (1) 0 7 (5) 7 (5) 0 0 Sinusitis 0 1 (2) 1 (< 1) 2 (1) 0 0 Pharyngitis 2 (1) 0 1 (< 1) 0 0 0 Cellulitis 2 (1) 0 0 0 0 0 Tooth infection 0 0 2 (1) 0 0 0 Cough 0 0 0 2 (1) 0 0 Headache 2 (1) 0 2 (1) 0 0 0 Tension headache 0 0 2 (1) 0 0 0 Data shown are for the ITT population. AE = adverse events; BD = twice daily; FF = fluticasone furoate; OD = once daily; URTI = upper respiratory tract infections. Table 4 Number and proportion of patients reporting AEs during treatment, for events reported by at least 1% of patients in the FF or placebo arms Number of patients reporting event, n (%) Placebo nDPI Placebo DISKUS FF 200 μg OD FF 100 μg FP 200 μg OD (n = 42) FP 100 μg BD (n = 43) nd proportion of patients reporting AEs during treatment, for events reported by at least 1% of or placebo arms Table 4 Number and proportion of patients reporting AEs during treatment, for events reported patients in the FF or placebo arms Data shown are for the ITT population. Data shown are for the ITT population. AE = adverse events; BD = twice daily; FF = fluticasone furoate; OD = once daily; URTI = upper respiratory tract infections. improvements in lung function and symptoms relative to placebo at doses of 50-800 μg once daily [14-16]. Discussion Treatment Adjusted treatment ratio and 95% CI 2.00 1.00 0.50 FP 100 µg BD FP 200 µg OD FF 100 µg BD FF 200 µg OD Figure 3 Treatment differences for 24-hour urinary cortisol excretion on Day 28 of treatment, expressed as the adjusted ratio (active:placebo) of the absolute excretion values (UC population). BD = twice daily; CI = confidence interval; OD = twice daily; FF = fluticasone furoate; FP = fluticasone propionate Treatment Adjusted treatment ratio and 95% CI 2.00 1.00 0.50 FP 100 µg BD FP 200 µg OD FF 100 µg BD FF 200 µg OD The crossover design used for the current study had the advantage of reducing potential variability compared with a parallel-group design. The study was not comple- tely double-blind, as the differences in the inhaler devices used to deliver FF and FP enabled investigators to distinguish between those two groups. Patients may also have known whether they received FF or FP, but they had no involvement or choice in their treatment sequence or choice of active drug. However, patients Figure 3 Treatment differences for 24-hour urinary cortisol excretion on Day 28 of treatment, expressed as the adjusted ratio (active:placebo) of the absolute excretion values (UC population). BD = twice daily; CI = confidence interval; OD = twice daily; FF = fluticasone furoate; FP = fluticasone propionate Figure 3 Treatment differences for 24-hour urinary cortisol excretion on Day 28 of treatment, expressed as the adjusted ratio (active:placebo) of the absolute excretion values (UC population). BD = twice daily; CI = confidence interval; OD = twice daily; FF = fluticasone furoate; FP = fluticasone propionate Page 7 of 8 Woodcock et al. Respiratory Research 2011, 12:160 http://respiratory-research.com/content/12/1/160 Page 7 of 8 received only one active treatment (FF or FP) throughout the study and no formal statistical comparisons were made between FF and FP. Given the considerations of trial practicality, we believe that the 2-week washout per- iod between treatments was adequate for lung function and UC to return to baseline; this is consistent with the recommended minimum time reported for studies on ICS treatments [20]. The numbers of patients in the ITT and PP populations exceeded the numbers stipulated by the sample size calculation, as a higher than expected proportion of screened patients met the eligibility criteria for study treatment. List of abbreviations AE Ad t ANC AE: Adverse event; ANCOVA: Analysis of covariance; CI: Confidence interval; FEV1: Forced expiratory volume in 1 second; FF: Fluticasone furoate; FP: Fluticasone propionate; ICS: Inhaled corticosteroid; ITT: Intent-to-treat; LABA: Long-acting beta2 agonist; LS: Least square; NDPI: Novel Dry Powder Inhaler; PEFR: Peak expiratory flow rate; PP: Per protocol; UC: Urinary cortisol; URTI: Upper respiratory tract infection; SABA: Short-acting beta2 agonist Discussion We do not believe the use of differ- ent devices for FF and FP should be considered as a con- founder for the main study outcomes, although it could be considered as a limitation of the study. Furthermore, trough FEV1 was the sole efficacy endpoint of this study and as such the non-inferiority of FF 200 μg once-daily dosing to FF100 μg twice-daily dosing cannot be inferred for other measures of treatment responsiveness such as PEFR, symptoms and exacerbations. evening compared with morning dosing [26]. Mometasone furoate 200 μg taken in the evening appeared superior to morning dosing as measured by change in FEV1, forced vital capacity, and PEFR from baseline after 12 weeks (although formal statistics were not applied) [27]. In con- trast, there appeared to be no difference between morning and evening once-daily dosing for budesonide compared with twice-daily dosing [28]. Author details 1 1School of Translational Medicine, University of Manchester, Manchester, UK. 2Center for Genomics and Personalized Medicine, Wake Forest University Health Sciences, Winston-Salem, NC, USA. 3Department of Medicine, University of Wisconsin, Madison, USA. 4Krefting Research Centre, University of Gothenburg, Gothenburg, Sweden. 5Respiratory Medicines Development Centre, GlaxoSmithKline, Uxbridge, UK. 6Department of Medicine, University of Cape Town, Cape Town, South Africa. Acknowledgements We thank all patients who took part in the study and all of the investigators at the 16 centres. The authors wish to acknowledge the contribution of Richard Forth, GlaxoSmithKline, in the statistical analysis of the data and review of the manuscript at the outline stage. The independent steering committee (AW, ERB, EDB, WWB, JL) together with authors employed by the sponsor (NGS, LF, LJ, BH) had full access to the data and were responsible for the decision to publish the paper. All listed authors meet the criteria for authorship set forth by the International Committee for Medical Journal Editors. The sponsor did not place any restriction on authors about the statements made in the final paper. Editorial support in the form of development of draft outline, development of manuscript first draft, editorial suggestions to draft versions of this paper, assembling tables and figures, collating author comments, copyediting, fact checking, referencing, and graphic services was provided by Geoffrey Weller at Gardiner-Caldwell Communications and was funded by GlaxoSmithKline. The manuscript processing fee was paid by GlaxoSmithKline. The current study used a once-daily evening dose regi- men. Previous studies have compared evening with morn- ing dosing for once-daily ICS regimens. In a previous study on FF, a 400 μg once-daily evening dose regimen had similar efficacy to a 200 μg twice-daily regimen, but the 400 μg once-daily morning dose, although effective, was less effective than FF 200 μg twice daily [24]. Data on other ICS also suggest improved efficacy for evening dos- ing. Triamcinolone once daily was more effective when the dose was given in the afternoon than in the morning [25]. Ciclesonide 200 μg once daily had a significantly greater improvement from baseline in morning PEFR with Conclusions l In conclusion, four weeks’ treatment with FF given as a 200 μg dose once daily in the evening has superior effi- cacy and similar tolerability compared with placebo in patients with moderate asthma, and is non-inferior to a FF 100 μg twice-daily regimen as measured by pre-dose FEV1 response. Some cortisol suppression was noted with FF, although this was not observed in previous stu- dies that used higher doses of FF and for longer treat- ment durations. Although confirmatory studies are required, the data support the use of FF as a once-daily, evening dosed, treatment in asthma. A once-daily ICS regimen has the potential to improve adherence by offering greater convenience while ensur- ing continuous 24-hour control of inflammation and symptoms. Lack of adherence to ICS treatment in asthma patients is a predictor of suboptimal disease control and poor outcome in children and adults [8,10,21]. A retrospective study in children and adults showed that in patients who needed asthma-related emergency care, persistence with ICS use in the pre- vious 12 months was low (fewer than three prescriptions filled), and that despite an increase in the number of prescriptions dispensed in the month of the emergency event, dispensing rate returned to the level observed previously in the second month after the event [22]. In another retrospective analysis, adolescents/young adults with mild asthma receiving mometasone furoate once daily showed better adherence and asthma control than those receiving other twice-daily ICS treatments [23]. However, while a once-daily regimen is approved for some agents for maintenance treatment of mild asthma [6], the recommended dosing frequency for the majority of ICS and for most patients is twice daily. Competing interests p g AW has served as consultant to Almirall, AstraZeneca, Chiesi, GlaxoSmithKline, Merck Sharpe and Dohme and Novartis; has received lecture fees and travel expenses for attendance at ATS and ERS meetings from GlaxoSmithKline; has been PI on clinical trials conducted by University Hospital of South Manchester. ERB has served as a consultant to and received lecture fees from GlaxoSmithKline; and has performed clinical trials for GlaxoSmithKline, which have been administered by his employer Wake Forest University Health Sciences. EDB has served as a consultant to and received lecture fees from GlaxoSmithKline; and his institution has received remuneration for participation in clinical trials sponsored by GlaxoSmithKline. WWB has served as a consultant to AstraZeneca, Boehringer Ingelheim, Novartis and TEVA; served on advisory boards for Altair, Amgen, Centocor, GlaxoSmithKline, Johnson & Johnson, Merck Sharpe and Dohme, Pfizer and Wyeth; received lecture fees from Merck Sharpe and Dohme; and received research funding from AstraZeneca, Ception, GlaxoSmithKline, MedImmune and Novartis. JL has served as a consultant to and received lecture fees from AstraZeneca, GlaxoSmithKline, Merck Sharpe and Dohme, Novartis and UCB Pharma; has been partly covered by some of these companies to attend previous scientific meetings including the ERS and the AAAAI; and has participated in clinical research studies sponsored by AstraZeneca, GlaxoSmithKline, Merck Sharpe and Dohme, and Novartis. JL is also editor of Respiratory Research and recused himself fully from the editorial process of h d l f d h ld k 12. Biggadike K, Bledsoe RK, Hassell AM, Kirk BE, McLay IM, Shewchuk LM, Stewart EL: X-ray crystal structure of the novel enhanced-affinity glucocorticoid agonist fluticasone furoate in the glucocorticoid receptor- ligand binding domain. J Med Chem 2008, 51:3349-3352. 13. Salter M, Biggadike K, Matthews JL, West MR, Haase MV, Farrow SN, Uings IJ, Gray DW: Pharmacological properties of the enhanced-affinity glucocorticoid fluticasone furoate in vitro and in an in vivo model of respiratory inflammatory disease. Am J Physiol Lung Cell Mol Physiol 2007, 293:L660-L667. 14. Bateman ED, Bleecker ER, Busse W, Lötvall J, Woodcock A, Forth R, Medley H, Jacques L, Haumann B: Fluticasone furoate (FF), a once-daily inhaled corticosteroid (ICS), demonstrates dose-response efficacy in patients symptomatic on non-steroidal asthma therapy. Eur Respir J 2010, 36(Suppl 54):204s. 15. References 1. Rabe KF, Vermeire PA, Soriano JB, Maier WC: Clinical management of asthma in 1999: the Asthma Insights and Reality in Europe (AIRE) study. Eur Respir J 2000, 16:802-807. 20. Phillips K, Oborne J, Lewis S, Harrison TW, Tattersfield AE: Time course of action of two inhaled corticosteroids, fluticasone propionate and budesonide. Thorax 2004, 59:26-30. 2. Rabe KF, Adachi M, Lai CK, Soriano JB, Vermeire PA, Weiss KB, Weiss ST: Worldwide severity and control of asthma in children and adults: the global asthma insights and reality surveys. J Allergy Clin Immunol 2004, 114:40-47. 21. Suissa S, Ernst P, Benayoun S, Baltzan M, Cai B: Low-dose inhaled corticosteroids and the prevention of death from asthma. N Engl J Med 2000, 343:332-336. 3. Haughney J, Barnes G, Partridge M, Cleland J: The Living and Breathing Study: a study of patients’ views of asthma and its treatment. Prim Care Respir J 2004, 13:28-35. 22. Stempel DA, Roberts CS, Stanford RH: Treatment patterns in the months prior to and after asthma-related emergency department visit. Chest 2004, 126:75-80. p 4. Mintz M, Gilsenan AW, Bui CL, Ziemiecki R, Stanford RH, Lincourt W, Ortega H: Assessment of asthma control in primary care. Curr Med Res Opin 2009, 25:2523-2531. 4. Mintz M, Gilsenan AW, Bui CL, Ziemiecki R, Stanford RH, Lincourt W, Ortega H: Assessment of asthma control in primary care. Curr Med Res Opin 2009, 25:2523-2531. 23. McLaughlin J, Navaratnam P, Urdaneta E, Friedman H: Patient adherence in adolescent/young adult mild asthma patients treated with inhaled corticosteroids. Proceedings of the Paediatric Academic Society Annual Meeting 1-4 May 2010 , E-PAS20101476.251. 5. Haselkorn T, Fish JE, Zeiger RS, Szefler SJ, Miller DP, Chipps BE, Simons FE, Weiss ST, Wenzel SE, Borish L, Bleecker ER, TENOR Study Group: Consistently very poorly controlled asthma, as defined by the impairment domain of the Expert Panel Report 3 guidelines, increases risk for future severe asthma exacerbations in The Epidemiology and Natural History of Asthma: Outcomes and Treatment Regimens (TENOR) study. J Allergy Clin Immunol 2009, 124:895-902. 24. Woodcock A, Bleecker ER, Bateman ED, Bleecker ER, Lötvall J, Snowise NG, Forth R, Jacques L, Haumann B: Fluticasone furoate, a novel inhaled corticosteroid, demonstrates once-daily efficacy in asthma when dosed in the evening. Eur Respir J 2010, 36(Suppl 54):205s. 25. Authors’ contributions ll h d l d h All authors developed the design and concept of the study. NGS, LF, LJ, and BH approved the statistical plan. NGS served as the clinical investigation lead and in that role coordinated generation of the protocol and data gathering. LF led the statistical analysis. All authors vouch for the accuracy and Page 8 of 8 Page 8 of 8 Woodcock et al. Respiratory Research 2011, 12:160 http://respiratory-research.com/content/12/1/160 Woodcock et al. Respiratory Research 2011, 12:160 http://respiratory-research.com/content/12/1/160 11. van den Berge M, Luijk B, Bareille P, Dallow N, Postma DS, Lammers J-WJ: Prolonged protection of the new inhaled corticosteroid fluticasone furoate against AMP hyperresponsiveness in patients with asthma. Allergy 2010, 65:1531-1535. completeness of the data and the data analysis. All authors read and approved the final manuscript. completeness of the data and the data analysis. All authors read and approved the final manuscript. Received: 19 July 2011 Accepted: 21 December 2011 Published: 21 December 2011 Received: 19 July 2011 Accepted: 21 December 2011 Published: 21 December 2011 18. Masoli M, Weatherall M, Beasley R: Fluticasone given once versus twice a day: meta-analysis. Respirology 2005, 10:183-8. 19. Purucker ME, Rosebraugh CJ, Zhou F, Meyer RJ: Inhaled fluticasone propionate by diskus in the treatment of asthma: a comparison of the efficacy of the same nominal dose given either once or twice a day. Chest 2003, 124:1584-93. Competing interests Bleecker ER, Bateman ED, Busse W, Lötvall J, Woodcock A, Tomkins S, House K, Jacques L, Haumann B: Fluticasone furoate (FF), an inhaled corticosteroid (ICS), is efficacious in asthma patients symptomatic on low doses of ICS therapy. Eur Respir J 2010, 36(Suppl 54):204s. 16. Busse WW, Bleecker ER, Bateman ED, Lötvall J, Forth R, Davis AM, Jacques L, Haumann B, Woodcock A: Fluticasone furoate demonstrates efficacy in patients with asthma symptomatic on medium doses of inhaled corticosteroid therapy: an 8-week, randomised, placebo-controlled trial. Thorax 2011, doi:10.1136/thoraxjnl-2011-200308. Respiratory Research and recused himself fully from the editorial process of this manuscript. NGS, LF, LJ and BH are employees of and hold stock in Respiratory Research and recused himself fully from the editorial process of this manuscript. NGS, LF, LJ and BH are employees of and hold stock in GlaxoSmithKline. 17. National Institutes of Health (NIH): Guidelines for the diagnosis and management of asthma - Expert panel report 3. 2007, U.S. Department of health and human services, Bethesda, MD. NIH publication No. 07-4051. doi:10.1186/1465-9921-12-160 Cite this article as: Woodcock et al.: Fluticasone furoate: once-daily evening treatment versus twice-daily treatment in moderate asthma. Respiratory Research 2011 12:160. References Pincus DJ, Humeston TR, Martin RJ: Further studies on the chronotherapy of asthma with inhaled steroids: the effect of dosage timing on drug efficacy. J Allergy Clin Immunol 1997, 100:771-774. 6. Global Initiative for Asthma (GINA). Global strategy for asthma management and prevention. [http://www.ginasthma.com]. 7. Price D, Robertson A, Bullen K, Rand C, Horne R, Staudinger H: Improved adherence with once-daily versus twice-daily dosing of mometasone furoate administered via a dry powder inhaler: a randomized open-label study. BMC Pulm Med 2010, 10:1. 26. Postma DS, Sevette C, Martinat Y, Schlösser N, Aumann J, Kafé H: Treatment of asthma by the inhaled corticosteroid ciclesonide given either in the morning or evening. Eur Respir J 2001, 17:1083-88. 27. Noonan M, Karpel JP, Bensch GW, Ramsdell JW, Webb DR, Nolop KB, Lutsky BN: Comparison of once-daily to twice-daily treatment with mometasone furoate dry powder inhaler. Ann Allergy Asthma Immunol 2001, 86:36-43. 8. Smith LA, Bokhour B, Hohman KH, Miroshnik I, Kleinman KP, Cohn E, Cortés DE, Galbraith A, Rand C, Lieu TA: Modifiable risk factors for suboptimal control and controller medication underuse among children with asthma. Pediatrics 2008, 122:760-769. 28. Jones AH, Langdon CG, Lee PS, Lingham SA, Nankani JP, Follows RM, Tollemar U, Richardson PD: Pulmicort Turbohaler once daily as initial prophylactic therapy for asthma. Respir Med 1994, 88:293-299. 9. Stanford RH, Gilsenan AW, Ziemiecki R, Zhou X, Lincourt WR, Ortega H: Predictors of uncontrolled asthma in adult and pediatric patients: analysis of the Asthma Control Characteristics and Prevalence Survey Studies (ACCESS). J Asthma 2010, 47:257-262. Tollemar U, Richardson PD: Pulmicort Turbohaler once daily as initial prophylactic therapy for asthma. Respir Med 1994, 88:293-299. doi:10.1186/1465-9921-12-160 Cite this article as: Woodcock et al.: Fluticasone furoate: once-daily evening treatment versus twice-daily treatment in moderate asthma. Respiratory Research 2011 12:160. 10. Williams LK, Pladevall M, Xi H, Peterson EL, Joseph C, Lafata JE, Ownby DR, Johnson CC: Relationship between adherence to inhaled corticosteroids and poor outcomes among adults with asthma. J Allergy Clin Immunol 2004, 114:1288-1293. 10. Williams LK, Pladevall M, Xi H, Peterson EL, Joseph C, Lafata JE, Ownby DR, Johnson CC: Relationship between adherence to inhaled corticosteroids and poor outcomes among adults with asthma. J Allergy Clin Immunol 2004, 114:1288-1293.
https://openalex.org/W4318475094	https://www.e3s-conferences.org/10.1051/e3sconf/202336502023/pdf	English	null	Comparison of microcrack formation boundaries determined by complex of physical methods with long-term strength of expanded clay concrete under different types of stress state	E3S web of conferences	2,023	cc-by	7,240	E3S Web of Conferences 365, 02023 (2023) CONMECHYDRO - 2022 E3S Web of Conferences 365, 02023 (2023) CONMECHYDRO - 2022 https://doi.org/10.1051/e3sconf/202336502023 Corresponding author: raupovch@gmail.com Comparison of microcrack formation boundaries determined by complex of physical methods with long-term strength of expanded clay concrete under different types of stress state Chorikul Raupov and Ganisher Malikov Tashkent State Transport University, Tashkent, 100067, Uzbekistan Tashkent State Transport University, Tashkent, 100067, Uzbekistan Abstract. This work aims to experimentally study the strength and strain of expanded clay concrete during short-term and long-term compression and tension under various loading modes. A technique for testing expanded clay concrete under short-term and long- term compression and tension, including the boundaries of microcrack formation by a complex of physical methods (tensometric, ultrasonic pulsed, and acoustic emission), is given. p ) g The results of tests of expanded clay concrete under short-term and long- term monotonic loading under compression and tension and low-cycle loading under compression, as well as the boundaries of microcrack formation by a complex of physical methods, are obtained. The boundaries of microcrack formation are compared with the long-term strength of expanded clay concrete under various types of stressed states. Strain diagrams of expanded clay concrete under axial compression and tension, and "ultrasound transmission speed - stress level" and "number of acoustic pulses - stress level" diagrams are also obtained. Empirical formulas are proposed for determining the boundaries of microcracking in expanded clay concrete and the relationship between the level of long-term strength and the time of staying specimens under load in compression and tension. The results allow the formulation of several proposals and clarifications for normative documents to calculate and design lightweight concrete elements and structures. © 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/). 1 Introduction The widespread use of local artificial porous aggregates instead of natural heavy aggregates in seismic regions of the Republic of Uzbekistan with a dry, hot climate is a governing condition for increasing the efficiency of capital investments in the construction of transport and other important structures. A distinctive feature of expanded clay, which in terms of producing lightweight concrete from porous aggregates, ranks first in comparison with other porous aggregates, is its relatively high strength at a relatively lower bulk E3S Web of Conferences 365, 02023 (2023) CONMECHYDRO - 2022 E3S Web of Conferences 365, 02023 (2023) CONMECHYDRO - 2022 https://doi.org/10.1051/e3sconf/202336502023 density. Expanded clay concrete in bridge building instead of traditional concrete can significantly reduce the weight of structures, material consumption, transport and installation costs, and labor costs while maintaining the necessary strength, reliability, and durability of structures. It can improve performance, reduce loads on foundations, reduce the cost of bridge construction and, at the same time, speed up their construction [1 – 7]. Theories of strength and fracture under compression and tension are of great importance for predicting the physical and mechanical characteristics of concrete, in particular, the concepts of the boundaries of microcracking ( and ) [3, 8 – 15] and long-term strength (Rbl and Rbtl) [2, 3, 16, 17]. With these theories, it is possible to assess the kinetics of the process of microcracking in concrete and the safety margins of structures. The boundaries of microcracking and long-term compressive and tensile strength should be considered important characteristics of concrete to ensure reliable operation of the structure. The values of the boundaries of microcrack formation are quite important for describing the features of concrete behavior since they allow concluding the stage of the stress-strain state (SSS) [9]. An analysis of the results of studies on the boundaries of microcrack formation shows that these characteristics are ambiguously related to the compressive strength Rb and can vary significantly depending on the proportion of expanded clay concrete mix, the type and consumption of coarse aggregate, and other factors [3, 8–14]. Particular attention is paid to the upper limit of microcrack formation since reaching this boundary indicates the transition to the third stage of the SSS, i.e., this indicates that microcracks merge into macrocracks and divide the concrete structure into blocks. The blocks under load are displaced relative to each other, which causes the concrete matrix destruction [9-12]. 1 Introduction If the load level is close to the upper limit of microcracking but does not exceed it, plastic strains under static loading stabilize over time (even under cyclic changes of loading [18, 19]). At present, the derivation of new formulas (more universal ones) applicable to concretes of different types and classes is relevant. The opinions of researchers in determining the relative limit of long-term strength differ, as evidenced by the data given in [2, 3, 15, 16]. In recent years, several formulas have been proposed that allow a more differentiated approach to assessing the relative limit of the long-term strength of concrete. Therefore, for heavy concretes of ordinary classes, used earlier, good results are given by the following formula [16] / = 0.92 −0.4 lg( − ) (1) (1) age of concrete at the time of loading. where is the age of concrete at the time of loading. where is the age of concrete at the time of loading. For heavy concrete of class B30 and higher at an old age, the following dependence can be used: / = 0.35lg + 0.175) (2) (2) If the concrete of the same classes is loaded at middle age, when the hardening processes continue to influence parameter R, then the long-term strength can be determined by the following formula = + 0.4. (3) (3) Since the parameters depend mainly on the class of concrete, its age at the time of loading, the gain in strength, and the conditions of moisture exchange with the surrounding medium, it can be assumed that the ultimate strength depends mainly on the same factors. 2 2 E3S Web of Conferences 365, 02023 (2023) CONMECHYDRO - 2022 E3S Web of Conferences 365, 02023 (2023) CONMECHYDRO - 2022 https://doi.org/10.1051/e3sconf/202336502023 However, several issues related to the study of lightweight concrete elements and structures, including their operation under various stress states and loading modes, have not yet been studied or are insufficiently studied. The proposed article presents the main results of these studies, which allow us to formulate several proposals and specifications for regulatory documents on the calculation and design of lightweight concrete elements and structures. Table 2. Characteristics of expanded clay concrete Note: Specimens were tested at the age of 28 days. Prisms dimensions - 150x150x600 mm, cub ribs - 150 mm. The cone slump of concrete mix - 1...2 cm. Note: Specimens were tested at the age of 28 days. Prisms dimensions - 150x150x600 mm, cubes ribs - 150 mm. The cone slump of concrete mix - 1...2 cm. 2 Methods The composition and main characteristics of concrete are given in Tables 1 and 2. Expanded clay gravel of two fractions, 5–10 and 10–20 mm in a ratio of 40:60, was taken as a coarse aggregate produced in the Tashkent cement plant. As Portland cement, the cement of the Navoi cement plant was used, and the sand of the Tashkent quarry was used as quartz sand. Table 1. Composition of expanded clay concrete (EC) Actual consumption of materials per m3 of concrete Cement, kg 427 sand, kg 629 expanded clay, kg (l) 414 (727) W/C (water/cement ratio) 0.49 Table 1. Composition of expanded clay concrete (EC) Table 2. Characteristics of expanded clay concrete Bulk density of dry expanded clay concrete, kg/m3 1760 Cubic strength, R, MPa 33.0 Prism strength, Rb, MPa 28.4 Prism strength factor, Rb/R 0.86 Initial modulus of elasticity, Eb, Pa 15.4 Note: Specimens were tested at the age of 28 days. Prisms dimensions - 150x150x600 mm, cubes Table 2. Characteristics of expanded clay concrete 2.1 The procedure for testing expanded clay concrete during short-term testing; measuring instruments Monotonic loading under compression and tension. Loading of specimens (prisms) of 70x70x280 mm and 150x150x600 mm dimensions and cylinders of 70 mm in diameter and 235 mm in height, made of expanded clay concrete under short-term compression and tension, was performed according to the standard procedure on testing machines UMM-20 and P-250 with a maximum capacity of 200 and 2500 kN, respectively. Specimen loading was conducted in steps of no more than 0.1 of the expected breaking load with staying on the steps until the increase in short-term creep deformations ceased. The duration of staying on the steps under stepwise compression loading of specimens did not exceed 5 min. Low-cycle loading under compression. Some specimens were tested under cyclic loading. Each step contained 1 cycle of loading and destruction. The duration of load staying at the upper and lower stress levels of a given step was determined by reaching such a shape of the deformation diagram when, within the measurement accuracy, the hysteresis loop ceased to increase. This loading pattern was realized up to a stress level not exceeding the upper limit of microcrack formation [20, 21]. 3 3 E3S Web of Conferences 365, 02023 (2023) CONMECHYDRO - 2022 E3S Web of Conferences 365, 02023 (2023) CONMECHYDRO - 2022 https://doi.org/10.1051/e3sconf/202336502023 During short-term tests, longitudinal and transverse strains were measured with wiring strain paper gauges of the CNIISK experimental mechanical plant with their bases of 20 and 50 mm. To obtain information about the integral value of strains, longitudinal and transverse strains were measured in several sections along the length of the specimen, covering a certain volume of material [5]. The adopted pattern of strain gauge gluing is shown in Figures. 1 a, b, c. The dimensions of the tested prisms and the base of strain gauges determined the use of each pattern. Strain gauges were glued according to the standard method - three months before the start of the test. The scheme of prisms and cylinders installation in a testing machine is shown in Figure 2. Fig. 1. Scheme of ultrasonic and tensometric measurements Fig. 1. Scheme of ultrasonic and tensometric measurements Fig. 1. Scheme of ultrasonic and tensometric measurements Under compression tests, the specimens were centered along the physical axis by trial loading of no more than 0.20Rb (Figure 2, a). 2.1 The procedure for testing expanded clay concrete during short-term testing; measuring instruments Under tensile testing, the load on the specimen was transferred through Hooke's hinges using collet grippers, ensuring uniform strain distribution along the specimen's length and reliable transfer of axial tensile force to the specimen. (Figure 2, b). а) b) Fig. 2. Testing specimens under short-term compression (a) and tension (b) b) а) Fig. 2. Testing specimens under short-term compression (a) and tension (b) E3S Web of Conferences 365, 02023 (2023) CONMECHYDRO - 2022 https://doi.org/10.1051/e3sconf/202336502023 2.2 The procedure for testing expanded clay concrete during long-term testing; measuring instruments Long-term loading of specimens to a given level of stresses under compression and tension was conducted in spring and lever installations with a maximum force of 210 kN and 30 kN, respectively. One or two specimens were placed in the installation. The first scheme was used under high loading levels (Figure 3, a), and the second was used under low loading levels (Figure 3, b). Metal plates 20 mm thick were laid between them when installing two prisms, and when installing two cylinders, a Hooke hinge was laid (Figure 3, c). The value of the load was set according to the range of springs, pre-calibrated on the press, on which the short-term loading was performed. Under tensile tests, the cylinders were placed in the installation using collets and Hooke's hinges, and under compression tests, the prisms were installed with metal base plates 30 cm thick with ball joints glued to the ends. а) b) c) Fig. 3. Testing of specimens under long-term compression (a, b) and tension (c). b) b) а) Fig. 3. Testing of specimens under long-term compression (a, b) and tension (c). The range of springs during calibration was measured with PAO-6 deflection meters with a division value of 0.01 mm, installed on two opposite sides (generatrices) of springs. The range of springs during calibration was measured with PAO-6 deflection meters with a division value of 0.01 mm, installed on two opposite sides (generatrices) of springs. To eliminate the error related to non-additivity of shrinkage and creep, the specimens before loading were waterproofed from the sides with a layer of paraffin 2–3 mm thick and two layers of polyethylene film with seams glued by insulating tape. To eliminate the error related to non-additivity of shrinkage and creep, the specimens before loading were waterproofed from the sides with a layer of paraffin 2–3 mm thick and two layers of polyethylene film with seams glued by insulating tape. 2.3 Short-term strength of expanded clay concrete under axial compression and tension Monotonic compression and tension loading. The strength and strain characteristics of expanded clay concrete under compression and tension are summarized in Tables 3 and 4. 5 5 https://doi.org/10.1051/e3sconf/202336502023 E3S Web of Conferences 365, 02023 (2023) CONMECHYDRO - 2022 Table 3. Results of short-term axial compression tests Characteristics Values Cubic strength of expanded clay concrete, R, at the age of 220, 530, and 800 days, respectively, MPa 36.5, 43.5 and 48.2 Prism strength, Rb, at the age of 220, 530, and 800 days, respectively, МPа 34.3, 37.4 and 35.7 Prism strength factor, Rb/R 0.94, 0.86 and 0.74 Rb/ R, at the age of 220, 530, and 800 days, respectively 0.94, 0.96 and 0.74 Modulus of elasticity, Eb, at the age of 220, 530, and 800 days, respectively, GPa 18.7, 22.0 and 22.9 Poisson's ratio, e, at the age of 220, 530, and 800 days, respectively 0.20, 0.19 and 0.20 Compressibility at 0.95Rb x(=0.95Rb)10-5, at the age of 220, 530 and 800 days, respectively 192, 181 and 168 Note: Results for expanded clay concrete at the age of 220 and 530 days were obtained for specimens 70x70x280 mm, at the age of 800 days - for specimens 150x150x600 mm. Table 3. Results of short-term axial compression tests Table 4. Results of short-term axial tensile tests Table 4. Results of short-term axial tensile tests Characteristics Values Tensile strength, Rbt, at the age of 220 and 530 days, respectively, MPa 2.06 and 2.30 Modulus of elasticity, Eb, at the age of 220 and 530 days, respectively, GPa; 23.3 and 23.5 Poisson's ratio, e, at the age of 220 and 530 days, respectively 0.20 and 0.20 Tensile strength at 0.95Rbt x(=0.95Rb)10-5, at the age of 220 and 530 days, respectively 10 and 10 The data obtained (Table 3) on the increase in time of cubic R and prism Rb strength of expanded clay concrete confirmed that the measures we took to protect specimens from drying out during storage were sufficient, and they provided a normal hardening condition for 800 days, which is very important when comparing the results of short-term and long- term tests. The increase in the elasticity modulus of expanded clay concrete under compression over time by the end of the experiment (1= 800 days) was 30-50% higher than at the age of 1=28 days (Tables 2 and 3). The increase in the tensile modulus was less significant. 2.3 Short-term strength of expanded clay concrete under axial compression and tension So, by 1= 530 days, it increased only by 5% compared to 1= 220 days, while under compression over this time interval, the value of Eb increased by 15%. For expanded clay concrete, the values of Rb/R exceed the ones specified in the existing standards for traditional concrete. Other researchers noted the increased prismatic strength of expanded clay concrete as well [1-5, 9-13]. They explained that porous aggregates have a more developed surface and better adhesion with the cement-sand component than dense aggregates. This largely restrains the transverse deformations of the prisms. However, with an increase in age, there was a noticeable decrease in the prism strength coefficient of expanded clay concrete (Table 3), and at the age of 800 days, Rb/R was 88% of Rb/R at 28 days of age (Table 2). According to our experimental data, the coefficient of variation for the strength and strain properties of expanded clay concrete under compression and tension was 1–6% and 3–14%, respectively. High values of the coefficients of variation under tension obtained in the experiments are due to two factors associated with the different nature of concrete destruction under compression and tension, respectively, and with different relative accuracy of 6 E3S Web of Conferences 365, 02023 (2023) CONMECHYDRO - 2022 E3S Web of Conferences 365, 02023 (2023) CONMECHYDRO - 2022 https://doi.org/10.1051/e3sconf/202336502023 measurements. Under axial tension, the destruction of the sample passes along one section due to a local defect in the macrostructure. As experiments have shown [5], there is practically no redistribution of stresses and strains between sections in concrete, and a weak section determines the strength of the entire sample. measurements. Under axial tension, the destruction of the sample passes along one section due to a local defect in the macrostructure. As experiments have shown [5], there is practically no redistribution of stresses and strains between sections in concrete, and a weak section determines the strength of the entire sample. Under compression, the effect of strain and stress localization is less pronounced due to their partial distribution over the entire volume of the specimen. The relative accuracy of measurements under compression and tension is because the values of stress and strain measured during tensile testing are an order of magnitude less than under compression, while the resolution of force gauges and especially strain gauges is the same. 2.3 Short-term strength of expanded clay concrete under axial compression and tension This factor is especially pronounced when measuring tensile strains since their measurement accuracy, which does not exceed 1(10–5, is commensurate with the measured strains, especially at low loading levels. The ratio of compressive and tensile strengths. The results of determining the axial tensile strength Rbt were compared with the results obtained for the cubic strength R and prism strength Rb (Tables 1–3). In our experiments, the Rbt/R ratio was 0.05...0.06, and Rbt/Rb was 0.06...0.07. The high ratio of Rbt/R for expanded clay concrete in our experiments is explained by the test procedure, where practical axial tension was ensured under the testing of cylinders. In addition, tensile tests of cylinders show better results than prisms due to the smaller effect of friction in the corners of the specimen. The ratio of Rbt/R for expanded clay concrete can be approximately taken equal to 0.05. Low-cycle compressive loading. The test results under low-cycle compressive loading are summarized in Table 5. A comparison of the data obtained with the data given in Tables 2 and 3 showed that low-cycle loading under compression did not affect the strength of expanded clay concrete and mixes (the difference between the values of Rb did not exceed 6%). Table 5. Test results under low-cycle compressive loading Prism strength Rb, MPa 35.1 Modulus of elasticity Eb, GPa at the loading cycle The first 19.7 The last 19.0 Initial Poisson's ratio e at the loading cycle The first 0.21 The last 0.22 Table 5. Test results under low-cycle compressive loading The results indicate the best resistance of expanded clay concrete under low-cycle compressive loading and the expediency of their use in transport engineering, where the structure is subject to multiple low-cycle loads. The values of Poisson's ratio e practically did not change and did not differ from those obtained under monotonic loading. The elasticity modulus Eb of expanded clay concrete decreased by the last cycle by approximately 4%. 3.1 Determination of the boundaries of micro-crack formation in expanded clay concrete by a complex of physical methods nsometric method. Determination of o crc R and crc R by tensometric methods based on the analysis of experimental "–" diagrams was performed according to the method described in [15] by measuring longitudinal and transverse strains under loading. Figures 4 and 5 show averaged diagrams of expanded clay concrete deformation 7 E3S Web of Conferences 365, 02023 (2023) CONMECHYDRO - 2022 https://doi.org/10.1051/e3sconf/202336502023 obtained when testing specimens under compression and tension. Visually, general patterns of the deformation diagrams under axial compression and tension are similar. However, in the case of compression, the nonlinearity of the diagrams manifested itself earlier, and the degree of its curvature was greater than in the case of tension. A characteristic feature of all strain diagrams was an almost linear dependence between stresses and strains over a large part of the stress range. Inelastic strains were noted only at loading levels greater than 0.70. obtained when testing specimens under compression and tension. Visually, general patterns of the deformation diagrams under axial compression and tension are similar. However, in the case of compression, the nonlinearity of the diagrams manifested itself earlier, and the degree of its curvature was greater than in the case of tension. A characteristic feature of all strain diagrams was an almost linear dependence between stresses and strains over a large part of the stress range. Inelastic strains were noted only at loading levels greater than 0.70. Based on the measurement results, the average values of the longitudinal х and transverse у strains of the specimen were calculated, and, on their basis, volumetric strain v, its increment v, and the differential coefficient of transverse strain were determined at each stage of loading. The graphs of changes in these characteristics (Figure 4) were plotted depending on the relative level of loading. Five prisms were tested for expanded clay concrete. The values of mechanical characteristics (prism strength, modulus of elasticity, Poisson's ratio) were determined as an average of data for five specimens. Fig. 4. Strain of expanded clay concrete under axial compression (prism dimensions 70x70x280 mm) Fig. 4. Strain of expanded clay concrete under axial compression (prism dimensions 70x70x280 mm A comparison of the strains determined by individual strain gauges on the specimen showed that the spread of their values was relatively small and changed little as the load increased. 3.1 Determination of the boundaries of micro-crack formation in expanded clay concrete by a complex of physical methods The coefficient of variation of readings from longitudinal strain gauges glued on different faces of the prism did not exceed 10%. This scatter was mainly because the actual axis of load application did not coincide with the physical axis of the specimen. In comparing the readings from strain gauges located in different sections along the height on the same face, their coefficient of variation did not exceed 5%. Considering these results, the strains were determined as the arithmetic average values according to the readings of all strain gauges. g g A comparison of the values of transverse strains, determined by individual strain gauges on the specimen, showed that they were characterized not only by a higher spread (the coefficient of variation of their values was up to 40%) but by a sharp difference in the readings of individual strain gauges at individual points on the surface of the specimen (in the zone of potential destructions) almost from the beginning of loading. From experimental diagrams, strain values at 0.95 of breaking load were determined since data on the compressibility and extensibility of concrete are used to solve several issues in the design and evaluation of research results. For expanded clay concrete and heavy concrete, compressibility depends on several factors, of which the most important are the strength of concrete, the material of the aggregate, and the stress level of concrete [2, 6, 17]. 8 8 E3S Web of Conferences 365, 02023 (2023) https://doi.org/10.1051/e3sconf/202336502023 CONMECHYDRO - 2022 E3S Web of Conferences 365, 02023 (2023) CONMECHYDRO - 2022 https://doi.org/10.1051/e3sconf/202336502023 Fig. 5. Strain of expanded clay concrete under axial tension Fig. 5. Strain of expanded clay concrete under axial tension Ultrasonic pulse method. The average diagrams of the change in the velocity of transmission of the generated ultrasonic pulses through the specimen are shown in Figures 6 and 7, and the results of determining are given in Table 6. Here, as well as in the case of the tensometric measurement method, the values of , determined from different scanning traces, can differ from each other by 20%. Fig. 6. Diagram "velocity of ultrasound transmission vs. stress level in expanded clay concrete specimens" Fig. 6. Diagram "velocity of ultrasound transmission vs. stress level in expanded clay concrete specimens" 9 E3S Web of Conferences 365, 02023 (2023) CONMECHYDRO - 2022 https://doi.org/10.1051/e3sconf/202336502023 CONMECHYDRO - 2022 Fig. 7. Diagram "velocity of ultrasound transmission vs. 3.1 Determination of the boundaries of micro-crack formation in expanded clay concrete by a complex of physical methods stress level in expanded clay concrete specimens" at perpendicular (1) and diagonal (2) scanning Fig. 7. Diagram "velocity of ultrasound transmission vs. stress level in expanded clay concrete specimens" at perpendicular (1) and diagonal (2) scanning Acoustic emission method. The experiments show (Figure 8) that the acoustic pulses accompanying the destruction of concrete structures are recorded almost from the moment of application of the compressive load and have several intensity peaks during the loading process. A point on the " – N" diagram before the beginning of the first peak of the intensity of the growth of impulses N is taken as the lower boundary of microcracks, and a point before the last peak is taken as the upper boundary of microcracks. The first peak corresponds to the microcrack development scheme proposed in [3, 15], to the formation of a system of microcracks originating from the initial cracks on the grains of the coarse aggregate. The next possible peaks should correspond to the formation of local systems of intergrown microcracks, and the subsequent peak should correspond to the formation of main macrocracks of destruction. The values of , obtained by the acoustic emission method (Table 6) are in good agreement with the values of , obtained by the tensometric method in the fracture zone (the discrepancy is no more than 8%). Fig. 8. Diagram "number of acoustic pulses vs. stress level in expanded clay concrete specimens" Fig. 8. Diagram "number of acoustic pulses vs. stress level in expanded clay concrete specimens 10 E3S Web of Conferences 365, 02023 (2023) CONMECHYDRO - 2022 https://doi.org/10.1051/e3sconf/202336502023 The results of determining the boundaries of microcrack formation / and / by a complex of physical methods (strain gauge, ultrasonic pulse, and acoustic emission method) are given in Table 6. Table 6. 3.1 Determination of the boundaries of micro-crack formation in expanded clay concrete by a complex of physical methods Results of determining the boundaries of microcrack formation under compression by a complex of physical methods Name of loading methods and types Boundaries of microcrack formation Values Under compression by the tensometric method (ТМ) under cyclic loading By average strains R /R 0.81 By strains in the fracture zone R /R 0.75 By maximum transverse and middle longitudinal strains R /R 0.35 under monotonous loading By average strains R /R 0.46 R /R 0.94 By deformations in the fracture zone R /R 0.46 R /R 0.90 By maximum transverse and average longitudinal strains R /R 0.23 R /R 0.75 By ultrasonic pulse method (UPM) R /R 0.43 By acoustic emission method (AEM) R /R 0.43 R /R 0.73 By tensometric method for tension on average strains R /R 0.75 Note: 150x150x600 mm prism specimens were tested at the age of 800 days. Table 6. Results of determining the boundaries of microcrack formation under compression by a complex of physical methods te: 150x150x600 mm prism specimens were tested at the age of 800 days. Note: 150x150x600 mm prism specimens were tested at the age of 800 days. The lower limit of microcrack formation of expanded clay concrete according to the tensometric method is weakly pronounced. It can be determined with a sufficiently large approximation only at the beginning of the increase in longitudinal and transverse strains (Figure 4) or with sufficient accuracy by acoustic emission and ultrasound [15]. ( g ) y y [ ] The authors of the reference [9] conducted experimental studies of the operation of various types of concretes; as a result, new empirical dependencies were proposed to determine the boundaries of microcrack formation: = / = 0.33 × −0.15 (4) = / = 0.33 × + 0.1 (5) (5) where is the average strength of concrete, MPa; kcrc is the empirical coefficient. Experimental data determined that there is a linear relationship between the values of relative loads for the upper and lower limits of microcrack formation [22]. The ratio of the values of the load level corresponding to the lower boundary of microcracking to the value of the load level corresponding to the upper boundary remains constant regardless of the class of concrete, i.e. / = const. 3.1 Determination of the boundaries of micro-crack formation in expanded clay concrete by a complex of physical methods The value of ratio / can be taken: The value of ratio / can be taken: ̶ / ≈0.67 – for normal concrete [23]; ̶ / ≈0.67 – for normal concrete [23]; ̶ / ≈0.60 – for expanded clay concrete [23]; ̶ / ≈0.60 – for expanded clay concrete [23]; The empirical coefficient kcrc, taken based on value , can be used when performing calculations for concrete and reinforced concrete structures: The empirical coefficient kcrc, taken based on value , can be used when performing calculations for concrete and reinforced concrete structures: 11 11 E3S Web of Conferences 365, 02023 (2023) CONMECHYDRO - 2022 https://doi.org/10.1051/e3sconf/202336502023 = × / (6) = × / (6) (6) (6) where kc1 ≈ 1.2 – for expanded clay concrete with concrete density ρ < 2200 kg/m3, and for other types of concretes kc1 = 1.0 with ρ > 2200 kg/m3. where kc1 ≈ 1.2 – for expanded clay concrete with concrete density ρ < 2200 kg/m3, and for other types of concretes kc1 = 1.0 with ρ > 2200 kg/m3. To determine the boundaries of microcrack formation in high-strength expanded clay concrete of a dense structure according to formulas (4 and 5), the values of numerical coefficients are determined by the least squares method based on the experimental data obtained by the authors = / = 0.21 × −0.11, (7) = / = 0.21 × + 0.21. (8) (8) The average values of and obtained by the authors in the experiment for expanded clay concrete were compared with the values calculated using empirical formulas (proposed by Semenyuk S.D., Moskalkova Yu.G. (4, 5) [9, 24] and the authors of (7, 8)) (Table 7). The average values of and obtained by the authors in the experiment for expanded clay concrete were compared with the values calculated using empirical formulas (proposed by Semenyuk S.D., Moskalkova Yu.G. (4, 5) [9, 24] and the authors of (7, 8)) (Table 7). Table 7. Calculation results of relative values of microcrack boundaries Table 7. Calculation results of relative values of microcrack boundaries Concrete type Average prism strength, . MPa Values of ratio / Relative values of microcrack formation boundaries Deviations of calculated values from experimental ones, % Test Adopted Test values Values calculated according to formulas (4), (5) According to the method of Semenyuk S.D. and Moskalkova Yu.G. 11 Normal concrete [9, 24] 22.3 0.667 0.67 0.54 0.81 0.536 0.786 0.4 2.4 28.1 0.639 0.67 0.53 0.83 0.588 0.838 -5.8 -0.8 28.2 0.679 0.67 0.57 0.84 0.588 0.838 -1.8 0.2 29 0.674 0.67 0.58 0.86 0.595 0.845 -1.5 1.5 Expanded clay concrete [23] 15.9 0.667 0.60 0.50 0.75 0.507 0.757 -0.7 -0.7 17.7 0.600 0.60 0.45 0.75 0.533 0.783 -8.3 -3.3 Authors' data according to formulas (7, 8) 35.7 0.57 0.60 0.43 0.75 0.431 0.751 -0.1 -0.1 The data given in Table 4 clearly demonstrate the adequacy of the application of the empirical formulas proposed by the authors in (7, 8) to determine the relative values of the boundaries of microcracking in high-strength expanded clay concrete of a dense structure. 3.2 Long-term strength of expanded clay concrete On the graphs of Figure 6, the values of the level of long-term strength in semilogarithmic coordinates are plotted depending on the staying time of the specimens under load (t–1). At the same time, curves corresponding to dependence (3) were plotted. Figure 3 shows that for expanded clay concrete, there is a good agreement between the experimental data and the approximating dependence. The deviations of the experimental values of the level of 12 E3S Web of Conferences 365, 02023 (2023) CONMECHYDRO - 2022 https://doi.org/10.1051/e3sconf/202336502023 long-term strength from the values calculated by dependence (3) do not exceed 10%. long-term strength from the values calculated by dependence (3) do not exceed 10%. The relationship between the level of long-term strength and the time of staying of specimens under loading was taken as: long-term strength from the values calculated by dependence (3) do not exceed 10%. The relationship between the level of long-term strength and the time of staying of specimens under loading was taken as: The relationship between the level of long-term strength and the time of staying of specimens under loading was taken as: = ( , ) = − log ( − ), (9) (9) where a = 0.868 and b = 0.032 – the values under axial compression, and a = 0.915 and b = 0.028 are the values under axial tension, determined by the least squares method based on the experimental data obtained by the authors. where a = 0.868 and b = 0.032 – the values under axial compression, and a = 0.915 and b = 0.028 are the values under axial tension, determined by the least squares method based on the experimental data obtained by the authors. Fig. 9. Change in the long-term strength of expanded clay concrete over time under axial tension (a) and compression (b) by dependence (3) Fig. 9. Change in the long-term strength of expanded clay concrete over time under axial tension (a) and compression (b) by dependence (3) 3.3 Comparison of the boundaries of microcrack formation with the long-term strength of expanded clay concrete A comparison of values of , determined by the tensometric method (TM) under cyclic and monotonic loading and by the acoustic emission method (AEM) with the limit of long- term compressive and tensile strength is given in Table 8. Table 8. Comparison of the boundaries of microcrack formation of expanded clay concrete under compression and tension (R /R and R /R ) with the long-term strength (/ и /) Type of load Value Compression under cyclic loading ТМ by average strains 0.81 by strains in the zone of destruction 0.74 under monotonous loading by average strains 0.94 by strains in the zone of destruction 0.90 AEM 0.75 / 0.74 R under cyclic loading ТМ by average strains 1.09 by strains in the zone of destruction 1.00 under monotonous loading by average strains 1.27 by strains in the zone of destruction 1.22 AEM 0.99 Tension R under monotonous loading ТМ 0.75 / 0.79 R / 0.95 13 E3S Web of Conferences 365, 02023 (2023) CONMECHYDRO - 2022 E3S Web of Conferences 365, 02023 (2023) CONMECHYDRO - 2022 https://doi.org/10.1051/e3sconf/202336502023 https://doi.org/10.1051/e3sconf/202336502023 Analysis of the comparison results shows that for expanded clay concrete, the best comparison of the values of the limit of long-term strength Rbl and the upper limit of microcracking under compression [22] gives the value of The acoustic emission method and the tensometric method in the fracture zone under cyclic loading are determined. From this, it follows that to determine the long-term strength based on the results of short-term tests, it is recommended to test specimens with both monotonically increasing and cyclic loads with the unloading at each stage, measuring strains on loading and unloading in several sections (more than three) along the height of the specimen in the zone of a homogeneous stressed state. As the limit of the long-term strength of expanded clay concrete, we take the stress corresponding to the extremum on the diagram of the inelastic component of volumetric strain, determined from the measurement data of strains in the destruction zone as the difference between the total diagram according to the results of monotonic loading and the elastic component according to the results of cyclic loading. Or else, it is recommended to determine the value of by the method of acoustic emission under monotonic loading. 3.3 Comparison of the boundaries of microcrack formation with the long-term strength of expanded clay concrete A comparison of the values of the long-term strength and the upper limit of microcracking under axial tension (Table 8) shows that for expanded clay concrete, the values of and Rbtl correspond to each other (the discrepancy is no more than 13%). 4 Conclusions 1. It was determined that the ratio of Rbt/R was 0.05...0.06, and Rbt/Rb - 0.06...0.07, and the values of the initial Poisson's ratio of expanded clay concrete under compression and tension correspond to the values regulated by building standards in SNiP. 1. It was determined that the ratio of Rbt/R was 0.05...0.06, and Rbt/Rb - 0.06...0.07, and the values of the initial Poisson's ratio of expanded clay concrete under compression and tension correspond to the values regulated by building standards in SNiP. p g y g 2. The results obtained indicate the best resistance of expanded clay concrete under low- cycle compression loading and the expediency of their use in the structures of transport facilities, where the structures are subject to multiple low-cycle loads. 2. The results obtained indicate the best resistance of expanded clay concrete under low- cycle compression loading and the expediency of their use in the structures of transport facilities, where the structures are subject to multiple low-cycle loads. 3. It was determined that the values of , calculated by different methods are in good agreement with each other, which cannot be said about R . The values of , obtained by the acoustic emission method are in good agreement with the values of , obtained by the tensometric method in the fracture zone (the discrepancy is no more than 8%). 4. The statement confirmed that for expanded clay concrete, the values of relative levels of crack formation R /R and R /R are higher than traditional heavy concrete of the same strength. 5. An empirical formula appropriate for practical application was obtained for determining the boundaries of microcrack formation and describing the pattern of change in the long-term strength of expanded clay concrete under axial compression and tension; this formula allows (at the stage of projecting) considering the long-term strength for any time interval. 6. It was determined that for expanded clay concrete, the best comparison of the values of the long-term strength Rbl and the upper limit of microcrack formation under compression gives the value of , determined by the acoustic emission method and the tensometric method in the fracture zone under cyclic loading. References 1. Raupov Ch., Shermukhamedov U., and Karimova A. Assessment of strength and deformation of lightweight concrete and its components under triaxial compression, taking into account the macrostructure of the material. In E3S Web of Conferences, 264, p. 02015 (2021) 14 E3S Web of Conferences 365, 02023 (2023) CONMECHYDRO - 2022 https://doi.org/10.1051/e3sconf/202336502023 2. Raupov Ch., Karimova A., Zokirov F., and Khakimova Y. Experimental and theoretical assessment of the long-term strength of lightweight concrete and its components under compression and tension, taking into account the macrostructure of the material. In E3S Web of Conferences, 264, p. 02024 (2021) 3. Raupov Ch.S. Expanded clay concrete for transport construction: Monograph, Tashkent: Tamaddun, p. 356 (2020) 4. Raupov Ch.S., Umarov Kh.K. Recommended areas of application of expanded clay concrete in bridge building and its effectiveness. Bulletin of TashIIT. 1, pp. 6-9 (2010) 5. Ashrabov A.A., Raupov Ch.S. Work of lightweight concrete beams in view of a descending branch of the diagram. International Conference held in Malaysia, the collection of scientific researches, pp 139–142 (2002) 6. Ashrabov A.A., Raupov Ch.S. The normalization of long-lived durability of lightweight concrete at monoaxial stressing. International Conference held in Malaysia. The collection of scientific researches, pp 134–138 (2002) 7. Raupov Ch.S. Technology for the manufacture of bridge structures from high-strength expanded clay concrete. Bulletin of TashIIT. 2, pp. 11–15 (2008) 8. Farhad Ansari. Mechanism of microcrack formation in concrete. ACI Materials Journal, 86(5), pp. 459–464 (1989) 9. Semenyuk S.D., Moskalkova Yu.G. Methods for determining the boundaries of microcrack formation. Construction of unique buildings and structures, 7(70). pp. 22- 30 (2018) doi: 10.18720/CUBS.70.2 10. Chini A.R., Villavicencio E. J. Detection of Microcracks in Concrete Cured at Elevated Temperature. University of Florida, p. 86. Gainesville, (2006) 11. Thomas T. C. Hsu. Fatigue and microcracking of concrete. Materials and Structures. 17(1). pp. 51–54 (1984) 12. Camacho, E. J. V. Analysis of Microcrack Behavior in Mass Concrete. Doctoral dissertation, University of Florida (2006). 13. A. Ashrabov, C. S. Raupov, A. A. A. Samad, J. Jayaprakash. Stady on force transfer mechanizm in cracked reinforced concrete elements. The International Conference on problems of mechanics and seismodynamics of structures, pp. 28–31 (2004) 14. A. A. Ashrabov, Y.V. Zaitsev, S. Spotar, C. S. Raupov. Modelling and strength simulation for concrete materials containing cracks. Journal of Problems of Mechanics. № 4, pp. 11–17, Tashkent (2005) 15. Guchkin I.S. References Study of the process of micro-destruction of expanded clay concrete under uniaxial compression by a complex of physical methods. p. 150, Penza (1973) 16. Golyshev A.B., Bachinsky V.Ya., Polishchuk V.P. Reinforced concrete structures, Strength of concrete. Kyiv (2001) 17. A. A. Ashrabov, M. S. Jaafar, W.A.M. Thanoon and C. S. Raupov. Static Fatigue Strength of Lightweight Concrete at Uniaxial Loading. Proceedings of the 2nd World Engineering Congress Sarawak. Structural engineering and construction anagement. Ingineering Innovation and Sustainability: Global Challenges and Issues. pp. 98–101 Malaysia (2002) 18. Moskalkova Yu. G. Strength and deformability of bent reinforced concrete elements, reinforced by the build-up of a compressed zone, under static and low-cycle loading: Belarusian-Russian University Mogilev, p. 199 (2013) 19. Semenyuk S.D., Moskalkova Yu.G. 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https://openalex.org/W2904536696	https://europepmc.org/articles/pmc6311832?pdf=render	English	null	Malignant Hypertension Causing a Pulmonary-Renal Syndrome	Case reports in nephrology	2,018	cc-by	1,841	Hindawi Case Reports in Nephrology Volume 2018, Article ID 3273695, 4 pages https://doi.org/10.1155/2018/3273695 Hindawi Case Reports in Nephrology Volume 2018, Article ID 3273695, 4 pages https://doi.org/10.1155/2018/3273695 Hindawi Case Reports in Nephrology Volume 2018, Article ID 3273695, 4 pages https://doi.org/10.1155/2018/3273695 Bryan Yong1 and David A. Power 2 1Clinical School, University of Melbourne, Parkville, Victoria, Australia 2Department of Nephrology, Austin Health, Studley Road, Heidelberg 3084, Victoria, Australia Correspondence should be addressed to David A. Power; david.power@austin.org.au Received 9 August 2018; Accepted 2 December 2018; Published 17 December 2018 Academic Editor: Theodore I. Steinman Copyright © 2018 Bryan Yong and David A. Power. 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. Background. Pulmonary-renal syndrome is characterised by acute kidney injury, haematuria, and haemoptysis and is a well- recognised presentation of diseases such as ANCA vasculitis that require urgent immunosuppression. Case Presentation. A patient presented with a brief history of haemoptysis, acute renal failure, microscopic haematuria, and severe hypertension. The diagnosis was initially not clear so he was treated with antihypertensives, renal replacement therapy, and immunosuppression. Renal biopsy subsequently showed evidence of malignant hypertension. Autoantibodies were uniformly negative. Conclusions. This case demonstrates that malignant hypertension can present as pulmonary-renal syndrome. 1. Background status. There were bilateral mid and lower zone inspiratory crackles and he had small volume haemoptysis. There were no focal neurological signs. Optic fundoscopy revealed flame haemorrhages and cotton wool spots, consistent with grade 3 hypertensive retinopathy (Figure 1). An electrocardiogram revealed high amplitude QRS complexes in V1 and V6 con- sistent with left ventricular hypertrophy. The echocardiogram showed normal left ventricular size but moderate to severe left ventricular wall thickness. Pulmonary haemorrhage with acute kidney injury and Refer- ences is classically caused by immunologically mediated dis- eases such as ANCA-associated vasculitis, anti-GBM disease, which is also known as Goodpasture’s syndrome, or systemic lupus erythematosus [1]. In the acute clinical setting, it is often necessary to treat patients with immunosuppressive agents and plasma exchange prior to accurate diagnosis. We present an unusual case of haemoptysis and acute kidney injury, treated with initial immunosuppression, where hypertension was found to be the causative problem. t On admission, he had a normocytic anaemia (Hb 75 g/L, RR = 130-180 g/L) and thrombocytopenia (platelets 98 × 109/L, RR = 150-400 × 109/L). The serum creatinine was elevated at 810 𝜇mol/L (RR = 62-106 𝜇mol/L), with an esti- mated glomerular filtration rate (eGFR) of 7 mL/min (RR = ≥90mL/min) (Table 1). The haemolysis screen revealed a bilirubin of 46 𝜇mol/L (RR = <18 𝜇mol/L), reticulocyte count of 333 × 109/L (RR = 20-100 × 109/L), lactate dehydrogenase (LDH) of 1177 IU/L (RR = 135-225IU/L), and a haptoglobin of <0.1 mg/L (RR = 0.3-2.0mg/L). The peripheral blood smear showed moderate schistocytosis and moderate polychroma- sia, in keeping with haemolysis (Table 1). A dipstick was pos- itive for proteinuria (2+) and haematuria (2+). Urine mı- croscopy showed 40 × 106 erythrocytes (RR < 40 × 106). Ultra- sound revealed normal sized kidneys without hydronephro- sis. Corticomedullary differentiation was normal. The chest 2. Case Summary A thirty-three-year-old Indian gentleman presented with a two-week history of haemoptysis and a three-day history of fever, epigastric discomfort, nausea, and headache. This occurred on a background of hypertension, diagnosed 8 years previously. He was a smoker with a 5 pack-year smoking history. He took no regular or over the counter medications. At presentation, he was markedly hypertensive (225/ 145 mmHg) and tachycardic (110 bpm). He was afebrile with normal oxygen saturations and had dual heart sounds with a loud P2. Clinical examination indicated euvolemic fluid Case Reports in Nephrology 2 Figure 1: Right and left funduscopic images showing flame haemorrhages and cotton wool spots, consistent with grade 3 hypertensive retinopathy. Figure 1: Right and left funduscopic images showing flame haemorrhages and cotton wool spots, consistent with grade 3 hypertensive retinopathy. Figure 1: Right and left funduscopic images showing flame haemorrhages and cotton wool spots, consistent with grade 3 hypertensive retinopathy. Table 1: Laboratory results. FBC Hb MCV WCC Platelets 75 87 11.3 98 Ref. range 130-180 g/L 80-96 FL 4-11 x 109/L 150-400 x 109/L UEC Na K Cr eGFR 135 2.5 810 7 Ref. range 136-145 mmol/L 3.5-5.1 mmol/L 62-106 𝜇mol/L ≥90 mL/min Haemolysis screen Bilirubin Reticulocytes LDH Haptoglobin 46 333 1177 <0.1 Ref. range <18 𝜇mol/L 20-100 x 109/L 135-225 IU/L 0.3-2.0 mg/L Blood smear Moderate schistocytosis and polychromasia Serology ANA ANCA Anti-GBM ENA panel ADAMTS13 Negative Negative <5 Negative 52 Ref. range <20 U/mL 40-130% X-ray showed cardiomegaly, patchy bilateral airspace opaci- ties that were worse on the right side.h X-ray showed cardiomegaly, patchy bilateral airspace opaci- ties that were worse on the right side.h of the acute kidney injury. Therapy with steroids, cyclophos- phamide, and plasma exchange was ceased once the renal biopsy results became available. Furthermore, the autoim- mune screening panel which included antinuclear antibodies (ANA), antinuclear cytoplasmic antibodies (ANCA), anti- GBM antibodies, extractable nuclear antibodies (ENA), and complement levels was normal. The ADAMTS13 levels in addition were later found to be normal and did not suggest complement mediated HUS. The clinical suspicion was that of an ANCA vasculitis or anti-GBM disease, with a differential diagnosis that included complement mediated haemolytic uraemic syndrome (HUS), thrombotic thrombocytopaenic purpura (TTP), and malig- nant hypertension.h yp The hypertension was treated with metoprolol, methyl- dopa, and hydralazine to achieve a BP of 150/100. Pulse methylprednisolone therapy, followed by cyclophosphamide, and plasma exchange were commenced. 3. Discussion that of aHUS when thrombotic microangiopathy (TMA) is present. In TMA, the appearances on renal biopsy include intravascular and glomerular thrombi as well as mesangial changes. In malignant hypertension, however, more severe vascular changes in small arterioles would be expected.h Pulmonary haemorrhage with acute kidney injury is clas- sically caused by immunologically mediated diseases such as ANCA associated vasculitis, Goodpasture’s syndrome, or systemic lupus erythematosus [1]. The combination of hypertension, hypertensive changes on fundoscopy, labora- tory evidence of microangiopathic haemolytic anaemia, and a renal biopsy consistent with thrombotic microangiopa- thy suggested that the unifying diagnosis in this case was malignant hypertension. Renal biopsy findings and negative ANCA, anti-GBM, and ANA serologies were supportive of a nonimmunological cause. Clinical and biochemical improve- ments with strict hypertension control also supported the diagnosis of malignant hypertension. The mechanism of haemoptysis in malignant hyper- tension is unclear. Malignant hypertension may cause pul- monary haemorrhage via direct vascular endothelial injury from mechanical stress. Elevated left ventricular pressure and pulmonary oedema causing pulmonary haemorrhage may be an alternate mechanism. It was reported that 63% of patients with malignant hypertension had renal impairment on pre- sentation [8]. Renal survival was notably found to be 84% and 72% at five and ten years, respectively. i While immunological causes of pulmonary-renal syn- drome are more common, malignant hypertension should be considered in the differential diagnosis in the correct clini- cal setting. Hypertension management alone may result in improvement in haemolysis markers and pulmonary symp- toms; however long term renal outcome remains poor. Malignant hypertension typically presents with non- specific symptoms such as agitation, an altered conscious state, chest discomfort, and headache. It is a rare cause of pulmonary-renal syndrome [2–5]. As we were unable to perform a kidney biopsy due to hypertension, it was difficult to exclude an immunological cause for the pulmonary- renal syndrome. The patient presented on the weekend and it proved impossible to obtain emergency serology, which might have helped to exclude several of the diagnoses enter- tained. This would likely have spared the patient unnecessary immunosuppression. It is also worth noting that severe hypertension is unusual in patients with anti-GBM disease and ANCA vasculitis, which is described in a few case series [6, 7]. 2. Case Summary The headache, haemoptysis, and evidence of haemolysis rapidly resolved following this treatment. Renal biopsy was initially delayed due to hypertension and was subsequently performed on day 3 (Figure 2). The biopsy contained 18 glomeruli. Most glomeruli showed a consolidated, bloodless appearance with obscuration of capillary loops by endothelial cell swelling and subendothelial zone expansion. Staining for IgA, IgM, IgG, fibrinogen, C3c, and C1q was negative. The biopsy suggested malignant hypertensive nephropathy as the underlying cause Control of hypertension was associated with resolution of the biochemical evidence of haemolysis. CT angiogram showed no evidence of renal artery stenosis. The patient remained dialysis dependent for 7 months and then regained sufficient renal function to cease dialysis. Fourteen months after presentation, the BP is 127/87 with serum creatinine 337 𝜇mol/L and eGFR 19 ml/min. Current medications are perindopril, indapamide, carvedilol, frusemide, calcitriol, and rosuvastatin. A recent echocardiogram shows left ven- tricular hypertrophy and low normal systolic function. He remains under regular review by cardiology and nephrology. Case Reports in Nephrology 3 Figure 2: Renal biopsy. Left: arteriolar thickening and myxoid changes. Top right: negative immunofluorescence staining. Bottom right: onion skinning of arterioles on silver staining. Figure 2: Renal biopsy. Left: arteriolar thickening and myxoid changes. Top right: negative immunofluorescence staining. Bottom right: onion skinning of arterioles on silver staining. The authors declare that they have no conflicts of interest. The authors declare that they have no conflicts of interest. References [1] C. McCabe, Q. Jones, A. Nikolopoulou, C. Wathen, and R. Luq- mani, “Pulmonary-renal syndromes: An update for respiratory physicians,” Respiratory Medicine, vol. 105, no. 10, pp. 1413–1421, 2011. This patient had clear evidence of intravascular haemoly- sis. There were schistocytes observed in the peripheral blood smear, which would support a diagnosis of atypical HUS. Pul- monary haemorrhage is considered quite unusual in aHUS. The diagnosis was considered possible, however, until the prompt resolution of haemolysis with control of blood pres- sure suggested that aHUS was unlikely. The appearances of malignant hypertension on renal biopsy are similar to [2] H. S. Park, Y. A. Hong, B. H. Chung et al., “Malignant hyper- tension with an unusual presentation mimicking the immune mediated pulmonary renal syndrome,” Yonsei Medical Journal, vol. 53, no. 6, pp. 1224–1227, 2012. [3] S. Aithal, N. Marley, and G. Venkat-Raman, “An unusual non- immunological cause of renal pulmonary syndrome,” Clinical Nephrology, vol. 72, no. 4, pp. 322–325, 2009. Case Reports in Nephrology 4 [4] P. Dalal, G. Phadke, A. Gill et al., “A patient with hemoptysis and renal failure,” International Journal of Hypertension, vol. 2011, Article ID 268370, 3 pages, 2011. [5] K. Hida, J. Wada, M. Odawara et al., “Malignant hypertension with a rare complication of pulmonary alveolar hemorrhage,” American Journal of Nephrology, vol. 20, no. 1, pp. 64–67, 2000. [6] M. J. Guti´errez-S´anchez, V. Petkov-Stoyanov, and J. A. Mart´ın- Navarro, “Reversible posterior leukoencephalopathy syndrome in Goodpasture’s syndrome,” Nefrolog´ıa, vol. 32, no. 4, pp. 540- 541, 2012. [7] J. Odaka, T. Kanai, T. Ito et al., “Renal-limited necrotizing granulomatous vasculitis in a pediatric patient,” Pediatrics International, vol. 57, no. 4, pp. 777–780, 2015. [8] R. Gonz´alez, E. Morales, J. Segura, L. M. Ruilope, and M. Praga, “Long-term renal survival in malignant hypertension,” Nephrol- ogy Dialysis Transplantation , vol. 25, no. 10, pp. 3266–3272, 2010.
https://openalex.org/W2619273508	https://www.econstor.eu/bitstream/10419/194684/1/1028127936.pdf	English	null	The impact of trade openness on economic growth: The case of Cote d’Ivoire	Cogent economics & finance	2,017	cc-by	9,598	Article The impact of trade openness on economic growth: The case of Cote d'Ivoire Cogent Economics & Finance Provided in Cooperation with: Taylor & Francis Group Provided in Cooperation with: Taylor & Francis Group Suggested Citation: Keho, Yaya (2017) : The impact of trade openness on economic growth: The case of Cote d'Ivoire, Cogent Economics & Finance, ISSN 2332-2039, Taylor & Francis, Abingdon, Vol. 5, Iss. 1, pp. 1-14, https://doi.org/10.1080/23322039.2017.1332820 Terms of use: Die Dokumente auf EconStor dürfen zu eigenen wissenschaftlichen Zwecken und zum Privatgebrauch gespeichert und kopiert werden. Documents in EconStor may be saved and copied for your personal and scholarly purposes. Sie dürfen die Dokumente nicht für öffentliche oder kommerzielle Zwecke vervielfältigen, öffentlich ausstellen, öffentlich zugänglich machen, vertreiben oder anderweitig nutzen. You are not to copy documents for public or commercial purposes, to exhibit the documents publicly, to make them publicly available on the internet, or to distribute or otherwise use the documents in public. If the documents have been made available under an Open Content Licence (especially Creative Commons Licences), you may exercise further usage rights as specified in the indicated licence. Sofern die Verfasser die Dokumente unter Open-Content-Lizenzen (insbesondere CC-Lizenzen) zur Verfügung gestellt haben sollten, gelten abweichend von diesen Nutzungsbedingungen die in der dort genannten Lizenz gewährten Nutzungsrechte. https://creativecommons.org/licenses/by/4.0/ https://creativecommons Keho, Yaya 1. Introduction Since the works by Grossman and Helpman (1990), Romer (1990) and Young (1991), the role of trade in promoting economic growth has stimulated a growing body of economic studies. The question is whether trade acts as an engine for economic growth, as stated by the trade-led growth hypothesis. Yaya Keho1* Corresponding author: Yaya Keho, Department of Applied Economics, Ecole Nationale Supérieure de Statistique et d’Economie Appliquée (ENSEA), 08 BP 03 Abidjan 08, Abidjan, Côte d’Ivoire E-mail: yayakeho@yahoo.fr Reviewing editor: Miao Grace Wang, Marquette University, USA Additional information is available at the end of the article Received: 07 March 2017 Accepted: 15 May 2017 Published: 31 May 2017 Abstract: The relationship between trade openness and economic growth has been extensively investigated yielding to mixed and inconclusive results. This might be attributed to the omission of the role of capital stock and labor in the trade-growth nexus. This paper examines the impact of trade openness on economic growth for Cote d’Ivoire over the period 1965–2014 in a multivariate framework including capital stock, labor and trade openness as regressors. It uses the Autoregressive Distributed Lag bounds test to cointegration and the Toda and Yamamoto Granger causality tests. The results show that trade openness has positive effects on eco- nomic growth both in the short and long run. Furthermore, they reveal a positive and strong complementary relationship between trade openness and capital forma- tion in promoting economic growth. Additional information is available at the end of the article Subjects: Econometrics; International Trade (incl. trade agreements & tariffs); Development Economics Keywords: economic growth; trade openness; cointegration; Cote d’Ivoire Keho, Cogent Economics & Finance (2017), 5: 1332820 https://doi.org/10.1080/23322039.2017.1332820 ABOUT THE AUTHOR These policies were fueled by the failure of import-substitution industrializa- tion strategy and also by findings from empirical studies showing that more outward-oriented econ- omies record higher economic growth rates. Furthermore, the spectacular success of East Asian economies was partly attributed to their early openness to trade (Stiglitz, 1996; World Bank, 1993). It is not surprising that in the late 1970s, many developing countries have adopted trade liberaliza- tion reforms involving the reduction of import and export tariffs and non-tariff barriers. However, another strand of research argues that increase in trade openness may be detrimental to economic growth by increasing inflation and lowering exchange rates (Cooke, 2010; Jafari Samimi, Ghaderi, Hosseinzadeh, & Nademi, 2012). Trade openness may impact economic growth negatively for coun- tries which specialize in production of low-quality products (Haussmann, Hwang, & Rodrik, 2007). For instance, countries exporting primary products are vulnerable to terms of trade shocks. Despite these conflicting views, the general belief is that openness to international trade is beneficial to economic development, especially for developing countries. A number of studies point to positive growth effects of trade openness (e.g. Chang, Kaltani, & Loayza, 2009; Dollar & Kraay, 2004; Frankel & Romer, 1999; Freund & Bolaky, 2008). Other studies contradict the existence of a positive link between trade and economic growth (e.g. Musila & Yiheyis, 2015; Polat, Shahbaz, Rehman, & Satti, 2015; Ulaşan, 2015; Vlastou, 2010). The mixed results from the empirical literature might be attributed to the econometric techniques, the sample of countries, and the indicator used as proxy for trade openness. Most of existing studies employ panel data re- gression approaches that impose cross-sectional homogeneity on coefficients, with the hope that the results could be applied to all countries. The cross-sectional homogeneity assumption is likely to be violated given the heterogeneity of economies with respect to trade policy, economic conditions and technological and institutional developments. What do Burundi, Kenya, Mali, India, and France have in common to be included into a same panel data analysis? The objective of this study is to examine the link between trade openness and economic growth in Cote d’Ivoire using a multivariate framework. Cote d’Ivoire recorded a remarkable economic success from 1960 to 1979, with a growth rate averaging 6.5% and trade openness accounting for 70.2% of GDP. This impressive economic performance was attributed mainly to political stability, favorable terms of trade, and massive public investment. ABOUT THE AUTHOR Yaya Keho, PhD is currently Professor of Econometrics and Statistics at the National School of Statistics and Applied Economics (ENSEA) of Abidjan, Côte d’Ivoire. He earned his PhD in Economics from the University of Saint Quentin-en-Yveline, at France. He has taught at many Universities in Africa (Cote d’Ivoire, Benin, Burundi, Cameroon, Senegal). Prof KEHO has published in many international revues like South African Journal of Economics, Energy Policy, Energy, International Economic Journal, Empirical Economic Letters, Economics Bulletin, International Journal of Energy Economics and Policy, International Journal of Statistics and Economics, Journal of Statistical and Econometric Methods, International Journal of Economics and Finance, International Journal of Business and Economics, Asian Economic and Financial Review, Asian-African Journal of Economics and Econometrics. His research interests focus on public finance, energy economics, international economics and applied econometrics. Openness to international trade has influences on economic growth. However, there are studies that support both negative and positive impact, with very scanty literature from Sub-Saharan Africa most especially Cote d’Ivoire. The findings of this study indicate a positive and strong complementary relationship between trade openness and capital formation in promoting economic growth. This result can be useful for analyzing trade policies and economic growth in other African economies with similar characteristics as Cote d’Ivoire. © 2017 The Author(s). This open access article is distributed under a Creative Commons Attribution (CC-BY) 4.0 license. © 2017 The Author(s). This open access article is distributed under a Creative Commons Attribution (CC-BY) 4.0 license. Page 1 of 14 Page 1 of 14 Keho, Cogent Economics & Finance (2017), 5: 1332820 https://doi.org/10.1080/23322039.2017.1332820 It has been shown that in the long-run, trade openness can potentially enhance economic growth by providing access to goods and services, achieving efficiency in the allocation of resources and improving total factor productivity through technology diffusion and knowledge dissemination (Barro & Sala-i-Martin, 1997; Rivera-Batiz & Romer, 1991). It is therefore expected that countries with more trade openness will relatively outperform those with less openness. From this perspective, developing countries have much to gain by trading with advanced countries. It is mainly in view of these expected gains that international institutions and donor governments routinely recommend trade liberalization policies to developing countries in the hope of opening up and integrating them into the global market. 2. Literature review The relationship between trade openness and economic growth has received a great deal of atten- tion both in the theoretical and empirical literature during the last three decades. However, there is no consensus on whether greater openness to trade stimulates economic growth. According to the theory of comparative advantage, if a country wants to trade with another country the latter will produce goods in which it has a comparative advantage. It specializes in the sector for which it has better factor endowments and produces goods on a larger scale. As a result, productivity and ex- ports of this sector will go up and this will boost the overall economic growth. This theory has been further extended by other economists. Krueger (1978) and Bhagwati (1978) argue that trade liberali- zation encourages specialization in sectors which have economies of scale that contribute to im- prove the efficiency and productivity in long-run. New endogenous growth models explain a positive relationship between trade openness and economic growth as the result of the international diffu- sion of advanced technologies (Coe & Helpman, 1995; Grossman & Helpman, 1991a; Romer, 1994). A country with a higher degree of openness has a greater ability to use technologies generated in advanced economies, and this capability leads them to grow more rapidly than a country with a lower degree of openness. Edwards (1998) argues that the cost of imitation also matters in the trade-growth relationship. If the imitation cost of innovation in the poorer countries is lower than that in advanced economies, the poorer countries will grow faster than the advanced ones and there will be a tendency toward convergence. All these arguments suggest that developing economies have much to gain from international trade with technologically advanced nations. However, some opposite arguments point out that trade openness may be detrimental to economic growth. This is the case when the country specializes in sectors where research and development activities are not the core ones (Almeida & Fernandes, 2008). Moreover, trade composition in terms of goods also matters regarding its growth effect (Haussmann et al., 2007; Kali, Méndez, & Reyes, 2007). Whether or not a country gains from international trade also depends on the ease with which foreign tech- nologies are mastered and adapted to the local environment (Grossman & Helpman, 1991b). On the empirical front, a growing literature has examined the relationship between trade and economic growth. ABOUT THE AUTHOR The 1980s brought with a decline in economic growth which became negative in many years. Over the period 1980–1993, the economic growth rate aver- aged −0.3% and the share of exports plus imports in GDP accounted for about 67.3%. Structural transformation of the economy also slowed down during this period. The weak growth that charac- terized the Ivorian economy from 1980 to 1993 has been blamed largely on external shocks and structural weaknesses in the economy. From 1999, the country experienced a period of political uncertainty leading to political tension that lasted from 1999 to 2011. The economy suffered from this situation. With the end of conflict in April 2011 and the return of peace, the country is experienc- ing an impressive economic revival and a rebuilding of its capital stock through public investment in infrastructures. The economic growth rate and trade performance have reached 9 and 87%, respec- tively, over the period 2012–2014. The recent performance in economic growth and trade spark some questions: is a significant part of economic growth trade-led? If yes, is trade-led growth a long-run or short-run phenomenon? The study will try to address these questions. The hypothesis to be tested in this study is that trade has a positive impact on economic growth in Cote d’Ivoire. The study employs the Autoregressive Distributed Lag (ARDL) bounds test of Pesaran, Shin, and Smith (2001) to depict the long-run relationship between trade and economic growth. Further, it applies Page 2 of 14 Page 2 of 14 Keho, Cogent Economics & Finance (2017), 5: 1332820 https://doi.org/10.1080/23322039.2017.1332820 the Granger-causality test suggested by Toda and Yamamoto (1995) to unravel the causal relation- ships among the variables. These approaches are more reliable in studies involving variables inte- grated of different orders. the Granger-causality test suggested by Toda and Yamamoto (1995) to unravel the causal relation- ships among the variables. These approaches are more reliable in studies involving variables inte- grated of different orders. The remainder of the paper is organized as follows. Section 2 provides a review of the literature regarding the trade-growth nexus. Section 3 outlines the model, data and econometric methodology. Section 4 discusses the empirical results. Finally, Section 5 summarizes the main findings of the study and provides some policy recommendations. 2. Literature review Trade openness is conducive to economic growth in low-inflation countries but has insignificant impact on growth in high-inflation countries. Kim, Lin, and Suen (2012) provide evidence that trade promotes economic growth in high-income, low-inflation, and non-agricultural countries but has a negative impact in countries with the opposite attributes. For a panel of 46 countries, Huang and Chang (2014) find that the growth effect of trade depends on the extent of stock market development. Trade enhances economic growth only when the country reaches a threshold level of stock market development. Sakyi, Villaverde, and Maza (2015) provide evidence of positive bi-directional causal relationship be- tween trade and economic growth for a sample of 115 developing countries. Were (2015) finds that trade exerts a positive and significant effect on economic growth rate in developed and developing countries, but its effect is not significant for least developed countries which largely include African countries. In a study of China, Hye, Wizarat, and Lau (2016) show that trade openness is positively related to growth in the long and short run. Regarding the Sub-Saharan African countries the evidence is also mixed. Deme (2002) validates the trade-led growth hypothesis for Nigeria. Chang and ying (2008) confirm the positive growth effects of trade and air freight for a sample of Economic Commission for Africa (ECA) countries. Gries, Kraft, and Meierrieks (2009) investigate the case of 16 Sub-Saharan African countries and do not find significant long-run relationships among the variables for most of the sample. They also provide evidence that economic growth causes trade openness in Ethiopia, Gabon, Kenya, Mauritius, Senegal, Sierra Leone, and Togo, whereas a feedback causal relationship exists for Cameroon, Cote d’Ivoire, Nigeria and Rwanda. On the contrary, no causal relationship between trade and growth was found for Burundi, Ghana, Madagascar, South Africa, and Gambia. For a sample of 34 African countries, Vlastou (2010) finds that openness to trade has a negative impact on economic growth. He also reports a causal re- lationship running from openness to growth. In a study of 27 African least developed countries, Tekin (2012) finds no significant causality between foreign aid, trade openness and real per capita GDP. Asfaw (2014) analyses the impact of trade liberalization on economic growth in a sample of 47 Sub- Saharan African countries. The results reveal that openness to trade stimulates both economic growth and investment. 2. Literature review The evidence from this literature is mixed and conflicting across methodologies and countries. The studies by Bahmani-Oskooee and Niroomand (1999), Frankel and Romer (1999), Karras (2003), Yanikkaya (2003), Dollar and Kraay (2004), Wang, Liu, and Wei (2004), Freund and Bolaky (2008), Das and Paul (2011), Marelli and Signorelli (2011), Nowbutsing (2014) and Zarra- Nezhad, Hosseinpour, and Arman (2014) confirm the positive impact of trade on economic growth. In contrast, Vamvakidis (2002) and Ulaşan (2015) find no support for the trade-led growth hypoth- esis. Rigobon and Rodrik (2005) find a significant negative impact of trade on income levels. Fenira (2015) finds a weak relationship between trade openness and economic growth. Rassekh (2007) investigates the trade-growth nexus for 150 countries and finds that lower income countries benefit more from international trade as compared to higher income economies. In a study of 82 countries, Chang et al. (2009) report a positive relationship between trade openness and economic growth. Kim and Lin (2009) apply the instrument-variable threshold regression approach to 61 countries and find an income threshold level above which greater trade enhances economic growth. Below the threshold level, however, trade openness has detrimental effects on growth. Afzal and Hussain (2010) find no causal relationship between exports and economic growth as well as between im- ports and economic growth in Pakistan. This finding has been challenged by Klasra (2011) and Shahbaz (2012) who confirm the trade-led growth hypothesis for Pakistan. Dufrenot, Mignon, and Page 3 of 14 Page 3 of 14 Keho, Cogent Economics & Finance (2017), 5: 1332820 https://doi.org/10.1080/23322039.2017.1332820 Tsangarides (2010) apply the quantile regression approach to explore the trade-growth nexus for 75 developing countries. Their results indicate that the effect of openness on economic growth is higher in low-growth countries relative to high-growth countries. The low-growth economies include coun- tries from all the continents, but a majority is in Africa (Benin, Cote d’Ivoire, Madagascar, and Zambia) and Latin America. Kim, Lin, and Suen (2011) use instrumental variable threshold regressions to examine whether the trade-income relationship varies with the level of economic development. Their results show that trade openness has positive effects on financial development, capital accu- mulation, and economic development in high-income countries. In low-income countries, however, the effect is negative and significant. Kim (2011) shows that openness to trade has positive effects on economic growth and real income in developed countries but negative effects in developing countries. 2. Literature review Furthermore, the real effect of trade also depends on the level of financial development and inflation. Openness to trade has negative effect on growth in countries with low financial devel- opment, but has insignificant impact in countries with high financial development. Trade openness is conducive to economic growth in low-inflation countries but has insignificant impact on growth in high-inflation countries. Kim, Lin, and Suen (2012) provide evidence that trade promotes economic growth in high-income, low-inflation, and non-agricultural countries but has a negative impact in countries with the opposite attributes. For a panel of 46 countries, Huang and Chang (2014) find that the growth effect of trade depends on the extent of stock market development. Trade enhances economic growth only when the country reaches a threshold level of stock market development. Sakyi, Villaverde, and Maza (2015) provide evidence of positive bi-directional causal relationship be- tween trade and economic growth for a sample of 115 developing countries. Were (2015) finds that trade exerts a positive and significant effect on economic growth rate in developed and developing countries, but its effect is not significant for least developed countries which largely include African countries. In a study of China, Hye, Wizarat, and Lau (2016) show that trade openness is positively related to growth in the long and short run. Tsangarides (2010) apply the quantile regression approach to explore the trade-growth nexus for 75 developing countries. Their results indicate that the effect of openness on economic growth is higher in low-growth countries relative to high-growth countries. The low-growth economies include coun- tries from all the continents, but a majority is in Africa (Benin, Cote d’Ivoire, Madagascar, and Zambia) and Latin America. Kim, Lin, and Suen (2011) use instrumental variable threshold regressions to examine whether the trade-income relationship varies with the level of economic development. Their results show that trade openness has positive effects on financial development, capital accu- mulation, and economic development in high-income countries. In low-income countries, however, the effect is negative and significant. Kim (2011) shows that openness to trade has positive effects on economic growth and real income in developed countries but negative effects in developing countries. Furthermore, the real effect of trade also depends on the level of financial development and inflation. Openness to trade has negative effect on growth in countries with low financial devel- opment, but has insignificant impact in countries with high financial development. 3.1. Model and data The hypothesis to be tested in this study is that trade openness stimulates economic growth in Cote d’Ivoire. To test this hypothesis, we start with Cobb–Douglas production function combining capital and labor as follows: (1) Qt = AtK훼 t L1−훼 t (1) Qt = AtK훼 t L1−훼 t Qt = AtK훼 t L1−훼 t (1) where Q is real economic output, K is capital stock, L is labor force, and A is technological progress. We extend this production function by assuming that technological progress can be influenced by trade openness. This leads us to specify A as follows: (2) At = 휙OP 훿1 t Z휌 t (2) At = 휙OP 훿1 t Z휌 t where OP stands for trade openness and Z represents other factors that may influence the state of technology. Substituting Equation (2) into Equation (1), gives: where OP stands for trade openness and Z represents other factors that may influence the state of technology. Substituting Equation (2) into Equation (1), gives: (3) Qt = 휙OP 훿1 t K훼 t L1−훼 t Z휌 t Qt = 휙OP 훿1 t K훼 t L1−훼 t Z휌 t (3) Diving both sides by labor and taking logs, Equation (3) can be modeled as follows: (4) yit = 휃0 + 휃1Kit + 휃2Lit + 휃3OPit + 휇it (4) yit = 휃0 + 휃1Kit + 휃2Lit + 휃3OPit + 휇it where y, K, L, OP represent the log of real GDP per capita, log of real capital stock per capita, log of labor force, and log of real trade per capita, respectively. where y, K, L, OP represent the log of real GDP per capita, log of real capital stock per capita, log of labor force, and log of real trade per capita, respectively. The capital stock series is computed from the gross fixed capital formation figures using the per- petual inventory model which is: Kt = It + (1 − δ)Kt−1 with an annual rate of depreciation of δ = 6%. The average growth rate (ρ) of investment over the sub-period 1965–1980 was used to generate the ini- tial level of capital stock as K0 = I0/(ρ + δ). Earlier studies put too much emphasis on exports as meas- ure for trade openness ignoring the role of imports. 3.1. Model and data According to the theory of comparative advantage, trade leads to a more efficient use of domestic resources through the imports of capital goods and intermediate inputs that otherwise are too costly to produce locally (Yanikkaya, 2003). These goods are necessary for the production of exports in less developed countries. Thus, imports are as important as exports for economic growth in developing countries. Hence, in this study trade openness is measured as the sum of real exports per capita and real imports per capita. Real exports and imports have been computed on the basis of their respective shares in GDP. Population is used as proxy for labor force and to convert data in per capita terms. All data are in constant local cur- rency and converted into natural logarithms. The data-set comes from the World Development Indicators and covers the period 1965–2014. 2. Literature review Besides, trade policies such as average weighted tariff rate and real effective ex- change rate affect economic performance through trade. Menyah, Nazlioglu, and Wolde-Rufael (2014) investigate the causal nexus among financial development, trade openness and economic growth for 21 Sub-Saharan African countries. They find limited support for the trade-led growth hy- pothesis. The trade-led growth hypothesis holds only for Benin, Sierra Leone, and South Africa. In a more recent work, Brueckner and Lederman (2015) employ the instrumental variable ap- proach to a panel of 41 Sub-Saharan African countries. They find that trade openness increases economic growth both in the short and long run. Musila and Yiheyis (2015) investigate the case of Kenya and find that trade openness has positive effect on investment ratio but not on the rate of economic growth. Polat et al. (2015) find that trade openness impedes economic growth in South Africa. Finally, Lawal, Nwanji, Asaleye, and Ahmed (2016) apply the ARDL methodology to Nigeria and find a negative long-run impact of trade openness on economic growth but a positive growth effect in the short run. Further, a two-way causality was found between the two variables. Page 4 of 14 Keho, Cogent Economics & Finance (2017), 5: 1332820 https://doi.org/10.1080/23322039.2017.1332820 3. Model, data, and methodology Figure 1. Real GDP and trade openness over time, 1965–2014. Keho, Cogent Economics & Finance (2017), 5: 1332820 https://doi.org/10.1080/23322039.2017.1332820 lagged level variables equal to zero. That is, the null hypothesis of no long-run relationship is: 휙1 = 휙2 = 휙3 = 0. This hypothesis is tested through an F-test. The asymptotic critical values are provided by Pesaran et al. (2001) for large sample sizes. However, these critical values may not be appropriate for our case which has 50 observations. Therefore, we use the simulation procedure sug- gested by Pesaran et al. (2001) to generate exact critical values. Furthermore, the ARDL bounds testing procedure is sensitive to the selection of the lag structure (m, n, p). In this study, maximum lag length on each variable was set to five and the optimal lag structure was selected using the in- formation criteria. The model has been tested by the diagnostic tests that are serial correlation, normality, and heteroskedasticity tests. Stability tests have also been used to test the goodness of fit of the ARDL model. The ARDL approach tests whether or not a long-run relationship exists between the variables, but not the direction of causality. To provide information on the direction of causal relationships among the variables, we apply the Granger-causality approach suggested by Toda and Yamamoto (1995). This approach has the advantage of not requiring pre-testing for cointegration among the variables. It makes inference valid even when the variables are integrated of different orders. The basic idea of this approach is to artificially augment the correct VAR order, p, with d extra lags, where d is the maximum order of integration of the variables. Thus, the model VAR to be estimated is as follows: (6) ⎡ ⎢ ⎢⎣ yt OPt Zt ⎤ ⎥ ⎥⎦ = ⎡ ⎢ ⎢⎣ 훼1 훼2 훼3 ⎤ ⎥ ⎥⎦ + p i=1 ⎡ ⎢ ⎢⎣ 훽1i 훾1i 훿1i 훽2i 훾2i 훿2i 훽3i 훾3i 훿3i ⎤ ⎥ ⎥⎦ × ⎡ ⎢ ⎢⎣ yt−i OPt−i Zt−i ⎤ ⎥ ⎥⎦ + p+d i=p+1 ⎡ ⎢ ⎢⎣ 훽1i 훾1i 훿1i 훽2i 훾2i 훿2i 훽3i 훾3i 훿3i ⎤ ⎥ ⎥⎦ × ⎡ ⎢ ⎢⎣ yt−i OPt−i Zt−i ⎤ ⎥ ⎥⎦ + ⎡ ⎢ ⎢⎣ e1t e2t e3t ⎤ ⎥ ⎥⎦ (6) ⎡ ⎢ ⎢⎣ yt OPt Zt ⎤ ⎥ ⎥⎦ = ⎡ ⎢ ⎢⎣ 훼1 훼2 훼3 ⎤ ⎥ ⎥⎦ + p i=1 ⎡ ⎢ ⎢⎣ 훽1i 훾1i 훿1i 훽2i 훾2i 훿2i 훽3i 훾3i 훿3i ⎤ ⎥ ⎥⎦ × ⎡ ⎢ ⎢⎣ yt−i OPt−i Zt−i ⎤ ⎥ ⎥⎦ + p+d i=p+1 ⎡ ⎢ ⎢⎣ 훽1i 훾1i 훿1i 훽2i 훾2i 훿2i 훽3i 훾3i 훿3i ⎤ ⎥ ⎥⎦ × ⎡ ⎢ ⎢⎣ yt−i OPt−i Zt−i ⎤ ⎥ ⎥⎦ + ⎡ ⎢ ⎢⎣ e1t e2t e3t ⎤ ⎥ ⎥⎦ (6) Once this augmented level VAR is estimated, a standard Wald test is applied to the first lagged p explanatory variables to make causal inference. The null hypothesis that trade openness does not cause GDP is γ11 = γ12 = … = γ1p = 0. Similarly, GDP does not cause trade openness if β21 = β22 = … = β2p = 0. The computed Wald-statistic has an asymptotic chi-square distribution with the degree of freedom equal to the number of constraints. Once this augmented level VAR is estimated, a standard Wald test is applied to the first lagged p explanatory variables to make causal inference. The null hypothesis that trade openness does not cause GDP is γ11 = γ12 = … = γ1p = 0. Similarly, GDP does not cause trade openness if β21 = β22 = … = β2p = 0. The computed Wald-statistic has an asymptotic chi-square distribution with the degree of freedom equal to the number of constraints. 3.2. Econometric methodology The empirical investigation involves three steps. The first step examines the stationarity of the vari- ables using unit root tests. The second step tests the presence of long-run relationships between the variables. The third step is to carry out causal relationships among the variables using Granger cau- sality tests. The ARDL approach to cointegration developed by Pesaran et al. (2001) is used to depict the long-run relationship among the variables. The advantages of this approach over other tradi- tional methods are well documented in the econometric literature. The ARDL bounds testing ap- proach to cointegration is based on the following error-correction model: (5) Δyt = 휙0 + 휙1yt−1 + 휙2OPt−1 + 휙3Zt−1 + m ∑ i=1 훾1iΔyt−i + n ∑ i=0 훾2iΔOPt−i + p ∑ i=0 훾3iΔZt−i + et (5) where Δ is the difference operator and Z = (K, L). Equation (5) is estimated using each variable as the dependent variable. The presence of long-run relationship is tested by restricting coefficients of Page 5 of 14 Page 5 of 14 Page 5 of 14 Page 5 of 14 4.1. Descriptive statistics of the data Figure 1 presents the trend of real GDP and trade openness during the sample period. We can see that the two variables present an upward trend until 1980 where economic crisis starts thus they decline in the following years. We also observe a decline in trade openness in 1994 the year of the devaluation of the CFA Franc currency. Page 6 of 14 12.6 12.8 13.0 13.2 13.4 13.6 13.8 14.0 1965 1970 1975 1980 1985 1990 1995 2000 2005 2010 Real GDP per capita Real trade per capita 12.6 12.8 13.0 13.2 13.4 13.6 13.8 14.0 1965 1970 1975 1980 1985 1990 1995 2000 2005 2010 Real GDP per capita Real trade per capita Real GDP per capita Real trade per capita Page 6 of 14 Page 6 of 14 Keho, Cogent Economics & Finance (2017), 5: 1332820 https://doi.org/10.1080/23322039.2017.1332820 Table 1. Descriptive statistics and correlation matrix Note: Figures in parentheses are p-values. Indicates statistical significance at the 5% level. Variables ln GDP ln K ln L ln OP Panel A: summary statistics Mean 13.498 14.059 16.212 13.194 Median 13.444 13.982 16.296 13.180 Maximum 13.934 14.528 16.913 13.639 Minimum 13.204 13.698 15.255 12.739 Std. dev. 0.2030 0.2580 0.5020 0.2090 Interquartile range 0.3643 0.4027 0.8647 0.2251 Skewness 0.5160 0.3900 −0.3770 0.3990 Kurtosis 2.0770 1.9480 1.8680 2.8660 Jarque–Bera 4.0010 3.5710 3.8560 1.3650 Probability 0.1350 0.1670 0.1450 0.5050 Panel B: correlation matrix ln GDP 1.000 ln K 0.793* (0.000) 1.000* ln L −0.773* (0.000) −0.512* (0.000) 1.000* ln OP 0.759* (0.000) 0.511* (0.000) −0.389* (0.005) 1.000* Table 1 provides descriptive statistics and correlations of the variables. It can be observed that log of real GDP per capita has an average level of 13.5 and was at its highest peak in 1978 at 13.9. Trade openness in log averaged 13.2 and reached its maximum in 1977. It can also be observed that the probability values of the Jarque–Bera statistic suggest that our variables are normally distributed. The correlation matrix indicates a positive relationship between trade and GDP. However, correlation does not imply causality. A positive correlation between trade and GDP can be compatible with the trade-led growth hypothesis, the growth-led trade hypothesis or a two-way causality between trade and GDP. Does any causality exist between trade and GDP after controlling for capital and labor? Note: 5% critical values for PP and KPSS tests are −3.504 and 0.146, respectively. 4.2. Unit root and cointegration testsi As a first step of our empirical analysis, we test for the order of integration of the series using the PP test of Phillips and Perron (1988) and the KPSS test of Kwiatkowski, Phillips, Schmidt, and Shin (1992). This step is necessary because the ARDL bounds test requires the dependent variable to be integrated of order one and the explanatory variables to be I(0) or I(1). If any variable is I(2) then the F-test will provide biased results. The results displayed in Table 2 suggest that the variables are non-stationary in their levels but achieve stationary status after taking the first differences. This implies the possibility of long-run relationship among the variables. Page 7 of 14 Table 2. Results of unit root tests Note: 5% critical values for PP and KPSS tests are −3.504 and 0.146, respectively. Series Level First difference PP KPSS PP KPSS GDP −2.541 0.109 −4.418 0.179 K −2.293 0.185 −1.181 0.164 L 0.083 0.242 −2.119 0.110 OP −2.026 0.104 −6.560 0.132 Table 2. Results of unit root tests N t 5% iti l l f PP d KPSS t t 3 504 d 0 146 ti l Series Level First difference PP KPSS PP KPSS GDP −2.541 0.109 −4.418 0.179 K −2.293 0.185 −1.181 0.164 L 0.083 0.242 −2.119 0.110 OP −2.026 0.104 −6.560 0.132 Keho, Cogent Economics & Finance (2017), 5: 1332820 https://doi.org/10.1080/23322039.2017.1332820 Table 3. Results of the ARDL cointegration test Notes: Lag length on each variable is selected using the AIC criterion with maximum lag set to 5. Critical values are generated under the model with unrestricted intercept and no trend. Indicates the rejection of the null hypothesis of no cointegration at 5% level of significance. Model ARDL F-stat. Diagnostic tests x2 (Normality) x2 (Heteroscedasticity) x2 (Correlation) GDP = f(K, L, OP) ARDL(1,3,0,0) 7.969 0.822 0.729 0.454 OP = f(GDP, K, L) ARDL(1,5,5,0) 4.922* 0.656 0.634 0.247 K = f(GDP, L, OP) ARDL(2,1,0,0) 4.878* 0.743 0.292 0.313 Level Critical values (T = 50) Lower bounds I(0) Upper bounds I(1) 5% 3.495 4.689 10% 2.891 3.984 Table 3. Results of the ARDL cointegration test Notes: Lag length on each variable is selected using the AIC criterion with maximum lag set to 5. Critical values are generated under the model with unrestricted intercept and no trend. p Indicates the rejection of the null hypothesis of no cointegration at 5% level of significance. Statistical significance at the 10% levels. Statistical significance at the 10% levels. 4.2. Unit root and cointegration testsi The results of the ARDL bounds test are displayed in Table 3. From this table, we see that a com- pelling long-run relationship exists among the variables when regression is normalized in GDP, trade, and capital stock. In each case, the computed F-statistic exceeds the upper critical value at 5% level of significance. At the 5% significance level, all diagnostic tests do not exhibit any evidence of viola- tion of the classical linear regression model assumptions. After finding the existence of cointegration between the variables, we further estimate the long- run effects of capital, labor, and trade openness on economic growth. We estimate the long-run relationship using ARDL, Fully Modified OLS, and Dynamic OLS methods. The results are disclosed in Table 4. All estimated coefficients are statistically significant and have correct signs as expected. The results indicate that capital contributes positively to economic growth in the long run. Other things remain the same, a 1% increase in capital stock leads to about 0.30% increase in real GDP per capita. Furthermore, trade openness is positively related to economic output and this relation is statistically significant at 5% level of significance. Keeping all else the same, a 1% rise in trade open- ness increases output by 0.15%. This finding validates the trade-led growth hypothesis that is ex- pansion of trade leads to higher level of economic output. The short-run dynamics results are reported in Table 5. The coefficient on the lagged error correc- tion term is significant with the correct sign, supporting the evidence of a stable long-run relation- ship among the variables. This coefficient suggests that a deviation from the long run equilibrium level of output in one year is corrected by 70% over the following year. The elasticity of output with respect to capital or trade openness in the short run is positive and statistically significant. In the short-run, capital and trade openness contribute to economic growth. Table 4. Long run estimates St ti ti l i ifi t th 5% l l Regressor Dependent variable: Log(GDP) ARDL FMOLS DOLS Coefficient t-stat. Coefficient t-stat. Coefficient t-stat. Capital (K) 0.308* 4.481 0.301* 5.527 0.289* 4.845 Labor (L) −0.190* −4.990 −0.166* −5.993 −0.173* −6.051 Trade (OP) 0.155** 1.775 0.350* 5.670 0.394* 5.728 Constant 10.20* 9.842 7.343* 6.027 7.033* 5.553 Page 8 of 14 Keho, Cogent Economics & Finance (2017), 5: 1332820 https://doi.org/10.1080/23322039.2017.1332820 Table 5. 4.2. Unit root and cointegration testsi Short run estimation results Statistical significance at the 5% level. Regressor Dependent variable: ΔLog(GDP) Coefficient t-stat. Prob. ΔK 1.804 8.276 0.000 ΔL −1.692 −1.044 0.302 ΔOP 0.117* 3.575 0.000 Constant 7.256* 6.966 0.000 ECM (−1) −0.706* −6.820 0.000 The results of this study show that international trade plays a significant role in the economic growth of Cote d’Ivoire, validating the trade-led growth hypothesis both in the short and long run. This finding accords with Asfaw (2014), Zarra-Nezhad et al. (2014), and Brueckner and Lederman (2015), but contradicts with Vlastou (2010), Polat et al. (2015), Ulaşan (2015), Were (2015) and Lawal et al. (2016) who reported a negative or insignificant impact of trade openness on economic growth. Some of these studies do not include into the analysis capital or labor as additional explana- tory variables. It is well-known that econometric tests are sensitive to omitted variables and hence studies relying on a bivariate framework may be subject to misspecification bias (Lütkepohl, 1982). On the other hand, differences in economic structure and trade policy may explain why the trade- growth nexus is country-specific. The fact is that Cote d’Ivoire mainly relies on exports of agricultural products such as cocoa, coffee, and cashews that account for 47% of total exports. It also imports raw materials, machines and productive technology that are used as capital for production of goods. Note: Figures reported are p-values of Wald statistics. Indicates significance at the 5% level. Note: Figures reported are p-values of Wald statistics. Indicates significance at the 5% level. 4.3. Granger causality tests and variance decomposition analysis g y p y Before testing for causality, it is necessary to determine the lag length of the level VAR model. The optimal lag length is determined using four statistics: Akaike Information Criterion (AIC), Schwarz Information Criterion (SC), Hannan–Quinn Information Criterion (HQ), and Final Prediction Error (FPE). The optimal lag selected is p = 5. As the maximal integrated order of the series is 1, we esti- mate a level VAR of order K = 6 in the Toda-Yamamoto procedure. The results of the Granger- causality tests are presented in Table 6. Clearly, there exists a strong unidirectional causality from capital, labor, and trade openness to GDP. The results also reveal bidirectional causality between trade openness and capital stock, indicating that international trade contributes to increase the capital stock of the economy, which in turn increases output. The finding of economic growth being caused by trade openness supports the trade-led growth hypothesis in the case of Cote d’Ivoire. The Granger-causality test does not determine the relative strength of causal relations between the variables beyond the selected time period. This weakens the reliability of causality results. To examine the strength of the causal link from one variable to another and to check the relative ef- fectiveness of causality effects ahead of sample period, we apply variance decomposition method. This method explains how much of the predicted error variance for any variable is explained by in- novations generated throughout each independent variable over various time horizons. The results displayed in Table 7 show that economic growth is explained predominantly by its own innovative Table 6. Results of Granger causality tests Dep. var. Causal variable GDP Capital Labor Trade GDP – 0.006* 0.000* 0.000* Capital 0.302 – 0.000* 0.035* Openness 0.250 0.008* 0.246 – Note: Figures reported are p-values of Wald statistics. Keho, Cogent Economics & Finance (2017), 5: 1332820 https://doi.org/10.1080/23322039.2017.1332820 Table 7. Variance decomposition Period S.E. shocks (38.2%) while innovative shocks of capital, labor, and trade contribute to GDP by 20.5, 16.9, and 24.2%, respectively. This clearly shows that a 38.2% portion of economic growth is explained by factors outside the empirical model such as financial, political, and institutional factors. Further, the contribution of trade openness is larger as compared to capital and labor. This shows that interna- tional trade is a major driver of economic growth in Cote d’Ivoire. 5. Conclusion and policy implications The impact of trade openness on economic growth is a subject of debate in the existing literature. The impact was found to be positive in some studies and nonsignificant or even negative in others. The mixed results might be attributed to analytical framework and country specific characteristics. This study examines the impact of trade openness on economic growth in Cote d’Ivoire over the period 1965–2014. The empirical analysis has used a multivariate framework with capital and labor as controlling variables. The ARDL bounds testing approach to cointegration has been applied to test the long-run relationship among the variables. Further, the Toda and Yamamoto Granger-causality approach is used to unravel the direction of causality between trade openness and growth. The re- sults confirm the existence of a long-run relationship between economic growth, capital stock, labor, and trade openness. It was found that capital and openness to trade have positive impacts on eco- nomic growth both in the short and long run. Furthermore, we found positive and strong comple- mentarity between trade openness and capital formation in promoting economic growth. Therefore, the results of the study validate the trade-led growth hypothesis in the case of Cote d’Ivoire. This implies that a substantial portion of the economic expansion of Cote d’Ivoire is external. Therefore, Cote d’Ivoire needs to further reduce trade barriers and promote international trade by reducing and simplifying procedures and controls. However, the heavily dependence on international trade may be detrimental to fiscal sustainability and economic growth under the Prebisch–Singer law of decline in the terms of trade. Cote d’Ivoire exports mainly primary products, which prices are unstable and determined on the international market. For outward-oriented strategy to have much larger impact on economic growth, the country should modify the composition of trade by switching from exports of raw materials and semi-manufactured goods to high valued-added goods. Furthermore, trade policy should promote investments in capital intensive sectors and develop human capital that can absorb technologies coming from advanced countries. Despite the promising results, this study suffers from some limitations. First, the empirical analysis has been conducted using trade at the aggregate level. An area of fruitful future re- search would be to analyze the trade composition in terms of goods and its impact on economic growth. Such an analysis provides useful information about what underpins the positive impact of trade on economic growth. 4.3. Granger causality tests and variance decomposition analysis GDP K L OP Variance decomposition of log(GDP) 1 0.028270 100.0000 0.000000 0.000000 0.000000 3 0.035729 77.64844 2.724370 0.240319 19.38687 5 0.045975 56.19793 11.25348 2.238418 30.31018 7 0.051533 48.48627 16.01268 6.665601 28.83544 8 0.052068 47.56086 15.68864 6.704608 30.04589 9 0.053037 47.04607 15.12396 8.150519 29.67945 10 0.054101 45.47760 14.65369 11.23233 28.63639 11 0.055241 43.80483 14.19886 14.31596 27.68034 12 0.055395 43.58881 14.13734 14.74695 27.52690 13 0.056310 42.80612 14.29173 15.37085 27.53130 14 0.058534 40.34656 17.44010 16.26982 25.94351 15 0.060548 38.27093 20.58389 16.89917 24.24601 Variance decomposition of log(K) 1 0.010288 51.34748 48.65252 0.000000 0.000000 3 0.020250 29.75501 61.38695 0.713085 8.144957 5 0.026251 18.23969 43.76284 0.955979 37.04149 7 0.032313 14.16981 33.28088 1.827843 50.72147 8 0.033602 14.34860 33.84939 1.883073 49.91894 9 0.034424 14.09440 35.21607 1.880525 48.80900 10 0.035634 13.45668 37.39790 2.856560 46.28886 11 0.037493 12.80026 39.80089 5.204063 42.19478 12 0.038975 12.48126 41.04775 7.200671 39.27032 13 0.040149 13.05982 42.17079 7.278688 37.49070 14 0.041530 13.96427 43.66750 6.802938 35.56529 15 0.043064 14.74589 45.41643 6.509529 33.32815 Variance decomposition of log(OP) 1 0.058160 4.414221 1.738856 14.19921 79.64772 3 0.070971 17.87181 1.504599 11.88647 68.73713 5 0.080574 18.50804 4.569071 17.71544 59.20744 7 0.092263 19.16977 14.64157 16.80543 49.38323 8 0.093174 19.13460 15.87941 16.52216 48.46383 9 0.094553 18.91261 17.74884 16.15847 47.18008 10 0.096258 18.27924 19.08557 16.90301 45.73218 11 0.098557 19.54410 20.12771 16.49501 43.83317 12 0.100810 19.28760 21.60437 16.50621 42.60182 13 0.104314 19.81820 23.45972 16.87295 39.84913 14 0.106439 19.72065 24.78345 16.85021 38.64569 15 0.107640 19.28397 24.47340 16.53918 39.70346 Page 10 of 14 Page 10 of 14 Keho, Cogent Economics & Finance (2017), 5: 1332820 https://doi.org/10.1080/23322039.2017.1332820 On the other hand, trade openness explains a 33.3% portion of capital stock by its innovative shocks while 45.4% is due to its own shocks and 14.7% by economic growth. This finding implies a causal relationship running from trade to capital formation and is consistent with the Granger causality analysis. The variance decomposition of trade openness reveals that economic growth and capital explain trade growth by 19.3 and 24.5%, respectively. This suggests that both economic growth and capital formation cause trade openness. The causality from economic growth to trade openness was not found in the Granger-causality analysis. Thus, the variance decomposition approach suggests bidirectional causality between trade openness and capital formation, and between economic growth and trade openness. There is a positive complemen- tarity between trade openness and capital formation in promoting economic growth in Cote d’Ivoire. 5. Conclusion and policy implications It will throw light on whether the trade-led growth in Cote d’Ivoire is due to agricultural exports or non-agricultural imports. Second, the estimation method used here may be subject to the problem of potential omitted variable bias and endogeneity of some regressors. Therefore, another useful extension of this research would be to include other rele- vant variables in a system of equations where trade and capital are also determined by other economic variables. This helps disentangle the channels through which trade affects economic growth. Page 11 of 14 Page 11 of 14 Keho, Cogent Economics & Finance (2017), 5: 1332820 https://doi.org/10.1080/23322039.2017.1332820 https://doi.org/10.18488/journal.aefr https://doi.org/10.18488/journal.aefr Frankel, J. A., & Romer, D. (1999). Does trade cause growth? American Economic Review, 89, 379–399. https://doi.org/10.1257/aer.89.3.379 Citation information Freund, C., & Bolaky, B. (2008). Trade, regulations, and income. Journal of Development Economics, 87, 309–321. https://doi.org/10.1016/j.jdeveco.2007.11.003 Cite this article as: The impact of trade openness on economic growth: The case of Cote d’Ivoire, Yaya Keho, Cite this article as: The impact of trade openness on economic growth: The case of Cote d’Ivoire, Yaya Keho, economic growth: The case of Cote d’Ivoire, Yaya Keho, Cogent Economics & Finance (2017), 5: 1332820. Cogent Economics & Finance (2017), 5: 1332820. Gries, T., Kraft, M., & Meierrieks, D. (2009). Linkages between financial deepening, trade openness, and economic development: Causality evidence from Sub-Saharan Africa. World Development, 37, 1849–1860. https://doi.org/10.1016/j.worlddev.2009.05.008 Acknowledgments Dollar, D., & Kraay, A. (2004). Trade. Growth and Poverty. Economic Journal, 114, 22–49. Dollar, D., & Kraay, A. (2004). Trade. Growth and Poverty. Economic Journal, 114, 22–49. We thank two anonymous referees for their valuable comments on the initial draft of this paper. The usual disclaimer applies and views are our sole responsibility. Dufrenot, G., Mignon, V., & Tsangarides, C. (2010). The trade- growth nexus in the developing countries: A quantile regression approach. Review of World Economics, 146, 731–761. https://doi.org/10.1007/s10290-010-0067-5 https://doi.org/10.1007/s10290-010-0067-5 Author details Yaya Keho1 Fenira, M. (2015). Trade openness and growth in developing countries: An analysis of the relationship after comparing trade indicators. Asian Economic and Financial Review, 5, 468–482. E-mail: yayakeho@yahoo.fr 1 Department of Applied Economics, Ecole Nationale Supérieure de Statistique et d’Economie Appliquée (ENSEA), 08 BP 03 Abidjan 08, Abidjan, Côte d’Ivoire. 1 Department of Applied Economics, Ecole Nationale Supérieure de Statistique et d’Economie Appliquée (ENSEA), 08 BP 03 Abidjan 08, Abidjan, Côte d’Ivoire. 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https://openalex.org/W3184754250	https://www.dora.lib4ri.ch/wsl/islandora/object/wsl%3A27681/datastream/PDF/Gebremedhin-2021-Quest_for_new_space_for-%28published_version%29.pdf	English	null	Quest for New Space for Restricted Range Mammals: The Case of the Endangered Walia Ibex	Frontiers in ecology and evolution	2,021	cc-by	9,564	Quest for New Space for Restricted Range Mammals: The Case of the Endangered Walia Ibex Berihun Gebremedhin1†, Desalegn Chala2†, Øystein Flagstad3†, Afework Bekele4, Vegar Bakkestuen2,5, Bram van Moorter3, G. Francesco Ficetola6,7, Niklaus E. Zimmermann8, Christian Brochmann2 and Nils Chr. Stenseth1,4* 1 Department of Biosciences, Centre for Ecological and Evolutionary Synthesis (CEES), University of Oslo, Oslo, Norway, 2 Natural History Museum, University of Oslo, Oslo, Norway, 3 Norwegian Institute of Nature Research, Trondheim, Norway, 4 Department of Zoological Science, Addis Ababa University, Addis Ababa, Ethiopia, 5 Norwegian Institute for Nature Research, Oslo, Norway, 6 Department of Environmental Sciences and Policy, Università degli Studi di Milano, Milan, Italy, 7 Laboratoire d’Écologie Alpine (LECA), Univ. Grenoble Alpes, CNRS, Grenoble, France, 8 Swiss Federal Research Institute WSL, Birmensdorf, Switzerland Populations of large mammals have declined at alarming rates, especially in areas with intensiﬁed land use where species can only persist in small habitat fragments. To support conservation planning, we developed habitat suitability models for the Walia ibex (Capra walie), an endangered wild goat endemic to the Simen Mountains, Ethiopia. We calibrated several models that differ in statistical properties to estimate the spatial extent of suitable habitats of the Walia ibex in the Simen Mountains, as well as in other parts of the Ethiopian highlands to assess potentially suitable areas outside the current distribution range of the species. We further addressed the potential consequences of future climate change using a climate model with four emission scenarios. Model projections estimated the potential suitable habitat under current climate to 501– 672 km2 in Simen and 6,251–7,732 km2 in other Ethiopian mountains. Under projected climate change by 2,080, the suitable habitat became larger in Simen but smaller in other parts of Ethiopia. The projected expansion in Simen is contrary to the general expectation of shrinking suitable habitats for high-elevation species under climate warming and may partly be due to the ruggedness of these particular mountains. The Walia ibex has a wide altitudinal range and is able to exploit very steep slopes, allowing it to track the expected vegetation shift to higher altitudes. However, this potential positive impact may not last long under continued climate warming, as the species will not have much more new space left to colonize. Our study indicates that the current distribution range can be substantially increased by reintroducing and/or translocating the species to other areas with suitable habitat. Keywords: connectivity, corridors, endemic species, distribution range, habitat loss, habitat suitability modeling, reintroduction, translocation Quest for New Space for Restricted Range Mammals: The Case of the Endangered Walia Ibex Indeed, to increase the viability and prospects for survival of this ﬂagship species, we strongly recommend human-assisted reintroduction to other Ethiopian mountains. Emulating the successful reintroduction of the Alpine ibex that has spread from a single mountain in Italy to its historical ranges of the Alps in Europe might contribute to saving the Walia ibex from extinction. ORIGINAL RESEARCH published: 20 July 2021 doi: 10.3389/fevo.2021.611632 Edited by: Jon Fjeldså, University of Copenhagen, Denmark Reviewed by: Mirko Di Febbraro, University of Molise, Italy Francesco Carotenuto, University of Naples Federico II, Italy Correspondence: Nils Chr. Stenseth n.c.stenseth@mn.uio.no †These authors share ﬁrst authorship Edited by: Jon Fjeldså, University of Copenhagen, Denmark Reviewed by: Mirko Di Febbraro, University of Molise, Italy Francesco Carotenuto, University of Naples Federico II, Italy Correspondence: Nils Chr. Stenseth n.c.stenseth@mn.uio.no †These authors share ﬁrst authorship Specialty section: This article was submitted to Conservation and Restoration Ecology, Specialty section: This article was submitted to Conservation and Restoration Ecology, a section of the journal Frontiers in Ecology and Evolution a section of the journal Frontiers in Ecology and Evolution Received: 29 September 2020 Accepted: 24 June 2021 Published: 20 July 2021 Gebremedhin B, Chala D, Flagstad Ø, Bekele A, Bakkestuen V, van Moorter B, Ficetola GF, Zimmermann NE, Brochmann C and Stenseth NC (2021) Quest for New Space for Restricted Range Mammals: The Case of the Endangered Walia Ibex. Front. Ecol. Evol. 9:611632. doi: 10.3389/fevo.2021.611632 INTRODUCTION There is increasing evidence that anthropogenic land use change poses imminent threats to biodiversity globally (Leadley, 2010; Watson et al., 2014). Indeed, humans have greatly altered the distribution ranges of endangered species and directly impacted their survival (Thuiller et al., 2005). Degradation of ecosystem services goes hand in hand with species extinctions, declining species abundances, and widespread shifts in species distributions (Leadley, 2010). The current rate of global extinction in biodiversity due to human activities is orders of magnitude higher than natural extinction rates (Pimm et al., 1995). Natural rates are calculated as approximately 1.8 extinctions per year per million species, whereas anthropogenic rates during the last 500 years have reached 28–100 extinctions per year per million species (Barnosky et al., 2011). y While land use change was considered the main driver of terrestrial biodiversity loss during the twenteith century, climate change is increasingly becoming a threat (Leadley, 2010). Climate change has started to impose multiple impacts on species, such as demographic and phenological changes, species range shifts and range size changes (Pounds et al., 1999; Bellard et al., 2012). Many species are shifting their ranges toward higher elevations and latitudes (Lenoir et al., 2008; Lovari et al., 2020), but migration is not always possible, because suitable areas can be lacking or because habitat loss has disrupted connectivity (Early and Sax, 2011). Given current trends, climate change could surpass habitat destruction as the greatest global threat to biodiversity over the coming decades (Leadley, 2010). The populations of large mammals have declined at alarming rates, especially in areas of intense land use where species can only persist in small habitat fragments, and often inside protected areas (Morrison et al., 2007). However, even protected areas face strong pressures from increasing human populations (McNeely, 1994). Hence, ensuring the survival of large mammals in human-dominated landscapes is challenging (Morrison et al., 2007; Gordon, 2009). In this study, we analyzed and estimated the spatial extent of the suitable habitats of the Walia ibex in the Simen Mountains, and projected our models to other Ethiopian mountains to assess whether areas with potentially suitable bioclimatic conditions exist. Being a mountain specialist, we expect the ibex to be negatively aﬀected by climate warming in all mountains due to shrinkage of its habitat. To test this hypothesis and in order to understand the spatiotemporal dynamics of suitable areas, we addressed the potential consequences of future climate change. INTRODUCTION We implemented three diﬀerent strategies to improve the reliability of our species distribution predictions. Speciﬁcally, (i) when predicting models under present and projected future climate conditions, we excluded any territory where the models extrapolated; (ii) we accounted for imperfect detection of presences; and (iii) we measured habitat features that are indicative of an increase or decrease in total suitable areas under present and projected future conditions. Our results are discussed and evaluated in a conservation context. Modeling the suitability of habitats by means of species distribution models (SDMs) has become increasingly important to understand species ecology and develop conservation strategies (Morrison et al., 2007; Lobo et al., 2010), particularly to address the impact of climate change on species distributions (Araújo et al., 2004). SDMs assess the relationships between species occurrence data and environmental predictors to evaluate the suitability of a given area for a target species (Guisan and Zimmermann, 2000), and allow for identifying suitable areas for species conservation management (Zielinski et al., 2006; Thuiller, 2007). Multiple statistical algorithms are used for modeling species distributions. The combination of multiple models (ensemble forecasting) for suitability projections are a means to visualize uncertainty, thus providing more robust conclusions for conservation planning (Thuiller et al., 2006; Araújo and New, 2007; Marmion et al., 2009; Meller et al., 2014). Citation: Gebremedhin B, Chala D, Flagstad Ø, Bekele A, Bakkestuen V, van Moorter B, Ficetola GF, Zimmermann NE, Brochmann C and Stenseth NC (2021) Quest for New Space for Restricted Range Mammals: The Case of the Endangered Walia Ibex. Front. Ecol. Evol. 9:611632. doi: 10.3389/fevo.2021.611632 July 2021 \| Volume 9 \| Article 611632 1 Frontiers in Ecology and Evolution \| www.frontiersin.org New Space for Restricted Range Mammals Gebremedhin et al. a direct response to the high conservation signiﬁcance of the species. Main threats resulting in population decline of the Walia ibex originated from habitat loss due to agricultural expansion, overgrazing, constructions, and occasional poaching (Galvin and Haller, 2008). Low genetic variability, with an eﬀective population size (Ne) much smaller than the census population size (Gebremedhin et al., 2009), is an additional threat. Genetic data have documented a past population bottleneck, which combined with its single small distribution area has led to extremely low genetic diversity; in fact, one of the lowest ever recorded in any endangered mammal species (Gebremedhin et al., 2009). The Walia ibex has the smallest current distribution of all wild goat species (<100 km2), but it had a much larger range, including mountains beyond the park boundary, in the past (Figure 1). However, anthropogenic factors pushed the population to the verge of extinction, with an estimated total population of 150 individuals in the early 1990’s. Due to conservation measures, the population is recovering, and current estimates suggest a population size of >957 (Ejigu et al., 2017). Given this trend, the protected area may not have adequate suitable habitat to support a larger population in the future. Proposed conservation actions include the establishment of viable populations in other suitable areas, for instance by facilitating movements or through translocations to adjacent unoccupied habitats (Hirzel et al., 2002; IUCN, 2013). Such actions require the identiﬁcation of suitable habitat area outside the current park boundaries (Seddon et al., 2007). SDMs allow for identifying such suitable areas, thereby supporting a detailed planning of translocations or assisted colonization of new habitats. Given that climate change will further modify the distribution of potentially suitable habitats in the future, such scenarios should additionally be taken into account in conservation management planning. MATERIALS AND METHODS The Walia ibex (C. walie) is an endangered (EN) species restricted to the montane, sub-afro-alpine and afro-alpine habitats of the Simen Mountains in northern Ethiopia. It inhabits rugged and steep terrain in a single conservation area, the Simen Mountains National Park. The park was established as Frontiers in Ecology and Evolution \| www.frontiersin.org Study Area The Simen Mountains National Park (with area of 412 km2), situated in northern Ethiopia at 13◦9′57′′–13◦19′58′′ northern Frontiers in Ecology and Evolution \| www.frontiersin.org July 2021 \| Volume 9 \| Article 611632 2 New Space for Restricted Range Mammals Gebremedhin et al. latitude and 37◦54′48′′–38◦24′43′′ eastern longitude, is the only refuge for the Walia ibex (Figure 1). The park is topographically diverse with an elevation ranging from 1,900 to 4,543 m above sea level (a.s.l.). Agriculture is the dominant land use system. The natural vegetation is divided into three main zones: the montane forest belt, the transitional ericaceous belt, and the uppermost afro-alpine belt (Puﬀand Nemomissa, 2005). elevation model was resampled by using the nearest neighbor resampling algorithm by means in Google Earth Engine (GEE) to the same resolution as the WordClim variables before derivation of slope and aspect. Aspect was kept as a continuous variable in the analyses. We used GlobCover (Arino et al., 2012), resampled to 1,000 m using the majority nearest neighbor resampling algorithm in GEE, to represent land cover. Each land cover class was converted to 0 and 1 by specifying each category data as a dummy variable. Occurrence Data To limit multicollinearity eﬀects, we evaluated pairwise Pearson correlations among predictor variables, and selected only variables with r < \|0.7\|, selecting the ones with higher biological importance. We retained eight variables: slope angle, slope aspect, land cover, isothermality (BIO3), temperature annual range (BIO7), precipitation of the wettest month (BIO13), precipitation of the warmest month (BIO18) and precipitation of the coldest quarter (BIO19). The Walia ibex inhabits altitudes between 2,700 and 4,300 m a.s.l. (Ejigu et al., 2015), but historical data indicate that the species has inhabited altitudes as low as 2,300 m (Nievergelt, 1981). Here, we followed (Nievergelt, 1981) and considered all areas above 2,300 m in the Simen and in other Ethiopian mountains (Figure 1) as our study area. The extant range of the Walia ibex is well known, and is the subject of regular monitoring (Gebremedhin et al., 2009, 2010). We performed repeated surveys of the Walia ibex habitats through the whole range of the species from March to April 2011. We used binoculars and telescopes to spot the animal from a distance. Presence points were recorded at the spot an individual or a group of Walia ibex were seen or fecal samples were observed. Individuals or groups of Walia ibex that were seen within less than about 100 m distance were considered as the same group. In such a way, we documented 214 occurrence points of Walia ibex. All models from the three algorithms were projected to all Ethiopian highlands (areas >2,300 m; Figure 1), both for current and projected future climates, using the same criteria as described above to assess suitability. We projected the models to future climates representing the year 2,080 using data from the Community Climate System Model version 4 (CCSM4) relying on all the four emission scenarios (RPCs 2.6, 4.5, 6.0, and 8.5 Wm2 of radiative forcing). Spatial and Temporal Projections of Models The Gradient Boosting Model (GBM), a resampling (boosting) approach (Friedman et al., 2000; Elith et al., 2008). The Gradient Boosting Model (GBM), a resampling (boosting) approach (Friedman et al., 2000; Elith et al., 2008). The diﬀerent modeling approaches calls for diﬀerent way of creating absence points (Barbet-Massin et al., 2012). For MaxEnt and GAM we used the thickening procedure proposed by Vollering et al. (2019a) for collection of absences (background points and pseudo-absences). We generated 10,000 weighted points within discrete thickening distances by analyzing geostatistical properties of the predictors such as semi-variance. The weighting was performed by using the relative probability of absences to be equal to the number of presences within a radius length of each location determined of the spatial-autocorrelation range of the predictor variables (Vollering et al., 2019b). A minimum separation distance was set to 10 km. In the third algorithm (GBM), we randomly generated pseudo-absence points equivalent in number to the presence points that were collected during the ﬁeld work. We then re-run the algorithm 10 times as recommended by Barbet-Massin et al. (2012), as tree-based machine learning algorithms requires this amount of runs at least when the number of presence points is less than 1,000 (Barbet-Massin et al., 2012). All models from the three algorithms were projected to all Ethiopian highlands (areas > 2,300 m; Figure 1), both for current and projected future climates representing the year 2080 using data from the Community Climate System Model version 4 (CCSM4) by relying on all the four emission scenarios (RPCs 2.6, 4.5, 6.0, and 8.5 Wm2 of radiative forcing). The probabilistic prediction maps obtained from models runs as well as projections were split into binary presence- absence maps using three threshold criteria: (1) Minimum presence threshold, selecting the minimum probability value among occurrence points; (2) Maximum sum threshold (Maximum Sensitivity plus Speciﬁcity), maximizing the sum of sensitivity and speciﬁcity (Liu et al., 2005); and (3) Maximum Kappa, using the threshold that provides highest Cohen’s Kappa values. All nine binary ensemble maps (three algorithms × three probability threshold criteria) were combined to deﬁne three habitat suitability classes: Unsuitable habitat, where less than 30% (maximum two out of nine) binary maps predict presence; Uncertain suitability, where 30–60% (3–5) maps predict presence; and Suitable habitat, where >60% (more than ﬁve) maps predict presence following Chala et al. (2016). Predictor Variables Three statistical modeling approaches were applied, diﬀering in general performance and statistical properties: (1) The Maximum Entropy Model (MaxEnt), a parametric maximum likelihood approach (Phillips et al., 2006; Halvorsen, 2013; Halvorsen et al., 2015); (2) The Generalized Additive Model (GAM), a non- parametric maximum likelihood approach (Wood, 2011); and (3) Nineteen bioclimatic candidate variables at the 30 arc-seconds resolution (about 1 km2) were obtained from WorldClim (Hijmans et al., 2005). Slope and aspect were additionally derived from the 90 m resolution Shuttle Radar Topography Mission digital elevation model (SRTM DEM) (Jarvis et al., 2008). The FIGURE 1 \| Study area showing the Ethiopian highland masses above 2,300 m (A) and Simen Mountains (B), the only place where the Walia ibex is extant today. The park boundary and the occurrence points used in the models are indicated in (B). FIGURE 1 \| Study area showing the Ethiopian highland masses above 2,300 m (A) and Simen Mountains (B), the only place where the Walia ibex is extant today. The park boundary and the occurrence points used in the models are indicated in (B). July 2021 \| Volume 9 \| Article 611632 Frontiers in Ecology and Evolution \| www.frontiersin.org Frontiers in Ecology and Evolution \| www.frontiersin.org 3 New Space for Restricted Range Mammals Gebremedhin et al. Spatial and Temporal Projections of Models Twenty-one replicates of MaxEnt models were ﬁt with diﬀerent regularization multiplier values (0–10 with an interval of 0.5) to regulate model complexity, using the DISMO package (Hijmans et al., 2013). We used the MIAmaxent R package for variable transformation and model selection for species distribution models. We ﬁnally retained the model with regularization multiplier of 3.5 due to the best cross-validation performance. We used the caret package in R to tune the parameters in the GAM models for choosing the optimal model across our parameters. Accuracy was used to select the optimal model using the largest value as criteria. Future habitat suitability ensembles were produced by combining 36 maps (three algorithms, three threshold criteria and four emission scenarios) and produced the three habitat suitability classes following the same approach that was applied to the ensemble current habitat suitability map. We performed a MESS analyses (Elith et al., 2010) by using the R-package “dismo” (Hijmans et al., 2013). The MESS analyses show how far pixel values in the predictors are outside the range of presences in the training data. Negative values indicate that one or more predictors are outside this range and the MESS values decreases in accordance with the dissimilarity. Thus pixels with negative MESS values were ruled out and not included in the results. For the GBM approach, we built several models by setting the back fraction to the default value (0.75; Elith et al., 2008) and by using diﬀerent combinations of tree complexity levels (1–5) and learning rates (slightly varying values ranging from 0.0001 to 0.05) from the “gbm.step” function in the gbm package (Ridgeway, 2013). From models with more than 1,000 trees, the least number of trees by default; see Elith et al. (2008), we selected the combination of tree complexity levels and learning rates that provided the lowest cross-validation deviance. Correction for Lack of Species Occurrences in Steep Areas Ibex species are specialized to steep cliﬀs up to 60◦(Nievergelt, 1981), which are diﬃcult to access for predators and other organisms (Shackelton, 1997). The steepest areas (>45◦) are, however, not likely to be well covered by species occurrence data obtained through visual observation, as in our case. Thus, we assumed that our presence-based models mainly captured the climate requirement of Walia, without fully capturing the topographic requirements, due to under-sampling of the steepest slopes. In order to take into account this source of bias, we repeated the whole analyses such that occurrences of Walia ibex in gentle slopes far away from steep cliﬀs were removed from the presence data set. We also repeated the whole analyses and built models without slope as predictor. We compared the two model versions, and ﬁltered ﬂat areas from both. To ﬁlter ﬂat areas far away from steep cliﬀs, we calculated the average slope among occurrence points. Then we ﬂagged all occurrence For each of the performed algorithms, we ran a ﬁvefold cross-validation procedure and repeated it 10 times. To this end, we divided our data into ﬁve equal subsets, ran the cross-validation procedure, and repeated these steps 10 times. For each of the mentioned algorithms, we then calculated predictions from the weighted mean across all repeats and folds. All the three ﬁnal models were validated against the test data set by means of the AUC and TSS accuracy metrics (Swets, 1988). The reported AUC of each algorithm is the average AUC computed across the repeated ﬁvefold cross-validation. We also report TSS (true skill statistics) as an average across the repeated ﬁvefold cross- validation. TSS was calculated as “sensitivity” + “speciﬁcity” – 1 (Allouche et al., 2006). July 2021 \| Volume 9 \| Article 611632 Frontiers in Ecology and Evolution \| www.frontiersin.org 4 New Space for Restricted Range Mammals Gebremedhin et al. where the species is not currently known to exist. The model projections suggested that there is 501–672 km2 of suitable habitat in the Simen Mountains (Figure 2 and Table 1), considerably larger than the currently delineated protected area (Figure 1). points for which the slope was lower than the mean value and calculated the distance between these points and the nearest cliﬀwith slope above the mean. We computed the ﬁrst (lower) quartile, the third (upper) quartile, and the interquartile range (IQR). Landscape Connectivity We performed a landscape connectivity analyses in order to provide an estimation of the habitat that can be spontaneously colonized by the Walia ibex under present and projected future climate conditions. Thus, we calculated two diﬀerent connectivity indices in the program package FRAGSTATS (McGarigal, 2002): the proximity index and the connectance index. This analysis was limited to suitable habitats inside a “landscape” of 100 × 100 km with the current occurrence points of Walia ibex in the center, because available data on species distribution and movements do not support the idea of spontaneous dispersal over broader scales. Other typologies of connectivity analyses were hampered by limited data on species movements and lack of high-resolution information on ﬁne-scale landscape elements that could act as barriers. Overall, the modeling approaches indicate that there are fragmented patches with potentially high habitat suitability, mostly in the central northern highlands, west of the Rift Valley, and that most of these patches are isolated (Figure 3 and Supplementary Figure 2). Correction for Lack of Species Occurrences in Steep Areas In this way, we restricted the presence points for the Walia ibex to include only suitable habitats that overlap with the cliﬀs that are above mean values of the slope angle and any suitable habitat within the distance of the sum of the upper quartile value plus 1.5 times the IQR from these cliﬀs. Overall, for both current and future climate conditions we present two versions of habitat suitability maps for the Simen Mountains and other Ethiopian mountains, i.e., maps with and without steep area correction. Projecting the models to all Ethiopian highland areas suggested that suitable habitat is not only present in the Simen mountains, but also in the several other mountain fragments in northwestern Ethiopia (Supplementary Figure 2). The GAM model also predicted suitable habitats in the southern highlands of Ethiopia, in the Bale and Arsi Mountains, southeast of the Rift Valley (Supplementary Figure 2). In the other two models, this was only the case when the minimum presence threshold was used. The models without slope as predictor resulted in reduced suitable habitat area, namely a 21.4% reduction in the Simen Mountains and a 20.4% reduction across all Ethiopian highlands (Table 1). The impact of under-sampling of the steepest slopes was lower in the Simen Mountains (−13.6%) than in other Ethiopian mountains (−28.4%; Table 1). Predictions Under Projected Climate Change Model projections under future emission scenarios indicated a tendency of shifting suitable habitats toward higher altitudes, both in the Simen and in the other Ethiopian mountains (Supplementary Figure 1). Upward range shifts resulted in an expansion ranging from 501–672 to 1,164–1,243 km2 of suitable habitat in Simen, depending on model and scenario applied. The opposite pattern was detected in other Ethiopian mountains (a reduction ranging from 6,251–7,732 to 4,922–5,812 km2), with consistent results across models. Correction for under-sampling of the steepest slopes had less pronounced impact under climate change scenarios, especially in mountain areas outside Simen, reﬂecting that less ﬂat area is found to be available or suitable toward mountain summits (Table 1). The MESS analyses showed four mountain areas outside Simen where our predictions may be unreliable (Figure 4). One of these areas was in the northern part of Ethiopia close to Dessie and three other areas in the South. The prediction cells in these territories were ruled out and excluded from the reported results. In this analysis, suitable habitats are deﬁned as the habitats currently used by the Walia ibex. The proximity index is calculated between a given habitat spot (patch) and all other habitat spots of the same type. The sum of the area of the habitat spots (m2) is divided by the square of nearest edge-to-edge distance (m2). Therefore, the proximity index has higher values at increasing amounts of suitable areas and if suitable areas are nearby. The connectance index is the number of connections (c) between all habitat spots of the same type (i) (sum of cijk, where cijk = 0 if spot j and k are not within the speciﬁed distance from each other, and cijk = 1 if spot j and k are within the landscape, divided by the total number of possible connections between all spots of the same type, multiplied by 100 to give a percentage value (values between 0 and 100). Habitat Connectivity Predictions Under Current Climate The three statistical models showed similarly high performance, with average AUC and TSS values from all model runs per algorithm: AUC = 0.902 and TSS = 0.846 for GAM, AUC = 0.905 and TSS = 0.853 for GBM, and AUC = 0.906 and TSS = 0.855 for MaxEnt. The predicted suitability patterns were also similar for the models (Supplementary Figures 1, 2). Our results consistently showed that suitable habitat for the Walia ibex is available in the Simen Mountains, also in areas The proximity analysis identiﬁed that several suitable areas are well connected to currently occupied patches (Figure 5). Under scenarios of climate change, some increase of connectivity levels are expected, with an increase up to 5.86%, particularly in the central Simen area. This is likely caused by the prediction of more future suitable habitats in this area. Slight increases of the proximity index under climate change may suggest that larger suitable patches in areas with high elevation would become better Frontiers in Ecology and Evolution \| www.frontiersin.org July 2021 \| Volume 9 \| Article 611632 New Space for Restricted Range Mammals Gebremedhin et al. FIGURE 2 \| Comparison of habitat suitability under current and future climate in the Simen Mountains. Current suitability maps are produced by averaging nine maps (3 algorithms × 3 threshold values). Future habitat suitability is produced by averaging 36 (3 algorithms × 3 threshold values × 1 climate model × 4 emission scenarios). The numbers on the legend bar refer to the degree of agreement among these maps in predicting presence: 0–0.3 unsuitable with high certainty, 0.3–0.6 uncertain suitability, >0.6 suitable with high certainty. Slope is excluded as a predictor variable and correction is made for under-sampling of steep slopes in the maps on the right panels. FIGURE 2 \| Comparison of habitat suitability under current and future climate in the Simen Mountains. Current suitability maps are produced by averaging nine maps (3 algorithms × 3 threshold values). Future habitat suitability is produced by averaging 36 (3 algorithms × 3 threshold values × 1 climate model × 4 emission scenarios). The numbers on the legend bar refer to the degree of agreement among these maps in predicting presence: 0–0.3 unsuitable with high certainty, 0.3–0.6 uncertain suitability, >0.6 suitable with high certainty. Habitat Connectivity Slope is excluded as a predictor variable and correction is made for under-sampling of steep slopes in the maps on the right panels TABLE 1 \| The total area (km2) of suitable habitat predicted with high certainty (>60% agreement for presence among models) in the Simen Mountains and among all Ethiopian highland masses (above 2,300 m), and correction for under-sampling of the steepest slopes. TABLE 1 \| The total area (km2) of suitable habitat predicted with high certainty (>60% agreement for presence among models) in the Simen Mountains and among all Ethiopian highland masses (above 2,300 m), and correction for under-sampling of the steepest slopes. Simen mountains Whole Ethiopia With slope Without slope With slope Without slope Current Future Current Future Current Future Current Future Suitable habitat 738 1,418 580 1,345 10,970 6,626 8,731 7,100 Suitable habitat* 672 1,243 501 1,164 7,732 4,922 6,251 5,812 *Area of suitable habitat corrected for under sampling of the steepest slopes. DISCUSSION connected in the future. In the opposite direction, connectance tended to decrease (Table 2), suggesting increasing distances between the total of patches, where the ibex at lower elevation would have to traverse larger areas of less suitable habitat. This study is the ﬁrst to examine both the current and future potential suitable habitats of the charismatic Walia ibex for Frontiers in Ecology and Evolution \| www.frontiersin.org July 2021 \| Volume 9 \| Article 611632 6 New Space for Restricted Range Mammals Gebremedhin et al. all Ethiopian highlands, assessing in particular the expected impacts of climate change on habitat suitability. Our ﬁndings have important bearings on conservation management under changing climate and land use in Ethiopia. Indeed, our results demonstrate that there is suitable habitat available in the Simen Mountains and also in other Ethiopian mountains that is not yet realized, both under current and projected future climates. The particularly high suitability within the Simen Mountains conﬁrms the signiﬁcance of the area as a refuge for this endangered mountain mammal. All projected areas of high habitat suitability are located west of the Ethiopian Rift Valley, while suitability is generally lower on the eastern side (Figure 3). The projected area of suitable habitat varies depending on the predictors used in the model calibration, as models without slope tended to predict smaller areas of suitable habitat (Table 1). of suitable habitat within the Simen Mountains, the only area where the species occurs today. In contrast, we found the opposite trend for all other parts of the Ethiopian highlands (Table 1). The projected expansion in the Simen Mountains is contrary to the general expectation of a shrinking of suitable areas for high-elevation species in response to climate warming and may be attributed to the ruggedness of the mountains, as has also been suggested in other areas of the world (Elsen and Tingley, 2015). The wide altitudinal range of the Walia ibex, and its ability to exploit the steepest slopes, might allow the species to track the expected vegetation shift to higher altitudes. The Simen is a complex mountain range with rugged topography presenting steep slopes compared to other Ethiopian mountains. As a consequence, the ibex could ﬁnd additional, currently unexploited habitats under climate change (see Supplementary Figure 1). DISCUSSION Frontiers in Ecology and Evolution \| www.frontiersin.org Frontiers in Ecology and Evolution \| www.frontiersin.org July 2021 \| Volume 9 \| Article 611632 7 New Space for Restricted Range Mammals Gebremedhin et al. in a loss of up to 32% in range size (Dunbar, 1998). Such a shift is primarily driven by the dietary behavior of this primate, which mainly feeds on grasses (Dunbar and Bose, 1991). The niche of the gelada baboon partially overlaps with the Walia ibex (Nievergelt, 1981) and it is documented that the Walia ibex previously could have inhabited lower altitudes, as low as 2,300 m a.s.l. (Nievergelt, 1981). In our surveys, however, tracks of the species were never observed below 2,700 m, which could imply that a shift toward higher elevations has already started. It is diﬃcult to determine whether such a shift was caused by climate change or by direct anthropogenic impact. However, in the last few decades, agricultural expansion at low altitudes has been intensive, suggesting that habitat loss and/or competition with domestic goats (Gebremedhin et al., 2016) could have forced the ibex to move upwards. In our study, we did not include projections of land-use change, given the lack of high-resolution projections for the study area. However, regional analyses suggest that agricultural increase could cause further habitat shrinkage in the future (Williams et al., 2021), thus the prevention of habitat loss from agriculture is a conservation priority for the next years. FIGURE 4 \| MESS analysis. Negative MESS values showed by red colors have one or more predictors outside the range of present in the training data. Deeper red colors show larger differences and predictions here should be treated with strong caution. FIGURE 4 \| MESS analysis. Negative MESS values showed by red colors have one or more predictors outside the range of present in the training data. Deeper red colors show larger differences and predictions here should be treated with strong caution. IUCN’s guidelines for planning and managing protected areas recommend that protected areas (PAs) should be large and continuous with limited edge eﬀects and with high connectivity to other areas of suitable habitat (Hamilton and McMillan, 2004). The Simen Mountains National Park satisﬁes none of these criteria, as it has been aﬀected by considerable human intervention (Hurni et al., 2010). In addition to direct 2080, as the species will not have much more new space left to colonize. DISCUSSION However, the potentially positive impact in Simen may not last very long under continued climate warming after Notably, our projections under climate change scenarios for the year 2080 suggest the possibility of an increase in the area FIGURE 3 \| Comparison of habitat suitability under current (upper panels) and future (lower panels) climates in Ethiopian highland masses (>2,300 m). Current suitability maps are produced by average nine maps (3 algorithms × 3 threshold values). Future habitat suitability is produced by averaging 72 maps (3 algorithms × 3 threshold values × 1 climate model × 4 emission scenarios). The numbers in the legend bar refer to the degree of agreement among maps in predicting presence: 0–0.3 unsuitable with high certainty, 0.3–0.6 uncertain suitability, >0.6 suitable with high certainty. Slope is excluded as a predictor variable and correction is made for under-sampling of steep slopes in the maps on the right panels. FIGURE 3 \| Comparison of habitat suitability under current (upper panels) and future (lower panels) climates in Ethiopian highland masses (>2,300 m). Current suitability maps are produced by average nine maps (3 algorithms × 3 threshold values). Future habitat suitability is produced by averaging 72 maps (3 algorithms × 3 threshold values × 1 climate model × 4 emission scenarios). The numbers in the legend bar refer to the degree of agreement among maps in predicting presence: 0–0.3 unsuitable with high certainty, 0.3–0.6 uncertain suitability, >0.6 suitable with high certainty. Slope is excluded as a predictor variable and correction is made for under-sampling of steep slopes in the maps on the right panels. FIGURE 3 \| Comparison of habitat suitability under current (upper panels) and future (lower panels) climates in Ethiopian highland masses (>2,300 m). Current suitability maps are produced by average nine maps (3 algorithms × 3 threshold values). Future habitat suitability is produced by averaging 72 maps (3 algorithms × 3 threshold values × 1 climate model × 4 emission scenarios). The numbers in the legend bar refer to the degree of agreement among maps in predicting presence: 0–0.3 unsuitable with high certainty, 0.3–0.6 uncertain suitability, >0.6 suitable with high certainty. Slope is excluded as a predictor variable and correction is made for under-sampling of steep slopes in the maps on the right panels. DISCUSSION A study on the impact of climate change on the gelada baboon (Theropithecus gelada) suggests that baboons could shift their lower altitudinal limit (>2,000 m) upward by about 500 m for every 2◦C of increase in mean annual temperature, resulting FIGURE 5 \| Landscape proximity between present and suitable habitats for Walia ibex. The raster colors indicate the present-day proximity values. The dots represent the centroid of each suitable habitat patch. The color of the dots show how much the proximity value in each suitable patch would change (in percent) in the Simen mountain according to the prediction model Simen future corrected for slope showed in Figure 2 lower right. FIGURE 5 \| Landscape proximity between present and suitable habitats for Walia ibex. The raster colors indicate the present-day proximity values. The dots represent the centroid of each suitable habitat patch. The color of the dots show how much the proximity value in each suitable patch would change (in percent) in the Simen mountain according to the prediction model Simen future corrected for slope showed in Figure 2 lower right. Frontiers in Ecology and Evolution \| www.frontiersin.org Frontiers in Ecology and Evolution \| www.frontiersin.org July 2021 \| Volume 9 \| Article 611632 8 New Space for Restricted Range Mammals Gebremedhin et al. TABLE 2 \| Average values for Proximity index and Connectance index for suitable habitat patches in the Simen Mountains, respectively, under current climate and future projection. Proximity Connectance Simen current 22.0695 7.8886 Simen future projection 23.2189 7.2981 TABLE 2 \| Average values for Proximity index and Connectance index for suitable habitat patches in the Simen Mountains, respectively, under current climate and future projection. to multiple sites within the historical range of the Alpine ibex in 1911 allowed populations to grow and to colonize many areas across all of the Alps (Hirzel et al., 2002). Such eﬀorts are needed in order to conserve the Walia ibex in the highlands of the Ethiopian mountains. Reintroductions are complex management actions that can have impacts also on the species that currently are resident in the target areas. Hence, it is important to consider the possible interactions between translocated species and the resident ones. Interspeciﬁc competition is one of the major potential issues. In a study conducted to assess the spatial overlap between reintroduced Bison (Bison bison) and resident ungulates, Jung et al. DISCUSSION (2015) conclude that the overall potential for competition between reintroduced bison and resident ungulates is low, still they highlight that competition could occur across multiple niche dimension axes. A study on seven species of herbivores in the Simen Mountains suggested limited levels of competition (Dunbar, 1978). This could be because several of the species were in the process of re−establishing themselves following periods of absence or reduced density, so that competition was more likely to be incipient rather than actual. anthropogenic factors, we show that climate warming will force the species to move further upwards. Under these circumstances, the pressure on small-sized protected areas such as the Simen Mountains National Park will become extremely high. Given the recent increase in population size of the Walia ibex, animals may already soon have to expand beyond the protected areas (Mason et al., 2014). The combination of anthropogenic factors, competition with livestock, and climate change may lead to further changes in the species’ range dynamics that may render the small-sized currently protected area of insuﬃcient quality in future conservation strategies and prompt the need for expansion of such areas. Indeed, future consideration of areas for reintroduction or translocation should focus on those that are well connected with other afro-alpine, sub-afro-alpine and montane ecosystems. p The potential wild competitors for the Walia ibex in the Simen Mountains include the gelada baboon and the klipspringer (Oreotragus oreotragus) (Nievergelt, 1981). However, the density of klipspringer in the park is very low and the competition that may arise should not signiﬁcantly aﬀect the Walia ibex population (Dunbar, 1978). On the other hand, the density of domestic livestock in the study area has increased to a higher level over the last four decades (Gebremedhin et al., 2016). Studies performed on related species in India (Bagchi et al., 2004) observed very strong interactions between the Himalayan ibex (C. sibirica) and livestock species, suggesting that domestic livestock deplete the density and diversity of wild herbivores in the cold deserts of the Trans−Himalaya by imposing resource limitations. Similarly, using metabarcoding methodology Gebremedhin et al. (2016) documented potential competition with domestic goats in the Simen mountains. Even though reintroductions have a great potential to allow the Walia ibex regaining its historical range, accurate analyses of competition with both native and domestic species will be pivotal for the success of conservation actions. DISCUSSION In response to the increasing human pressure on the habitats of the Walia ibex and the overall afro-alpine biodiversity, the total area of the Simen Mountains National Park was recently expanded from ∼225 to 412 km2. With the enlargement of the protected area, the boundaries were redesigned to connect the currently occupied areas to the historical species distribution. So far, no animal movement to the reconnected areas has been observed. Our connectivity analysis suggests that proximity values between the Simen and some nearby suitable areas are not low, thus the expansion to nearby areas is in principle possible (Figure 5). However, our connectivity analyses focused on broad-scale suitability values, and did not take into account ﬁne-scale landscape elements, such as barriers, that could actually hamper the movements of ibexes. More accurate assessments of potential colonization pathways would require high-resolution data on the occurrence of barriers, and better information on the actual movements of ibexes, for example through radiotracking. Such data would allow to identify areas where additional corridors and protected areas could be deﬁned in order to increasing the chance for spontaneous re-colonization of historical sites. Frontiers in Ecology and Evolution \| www.frontiersin.org FUNDING This work was funded by CEES and NHM at the University of Oslo and the Ruﬀord Small Grants for Nature Conservation (RSG 8960-1). This work was funded by CEES and NHM at the University of Oslo and the Ruﬀord Small Grants for Nature Conservation (RSG 8960-1). REFERENCES range and genetic diversity in Lobelia rhynchopetalum. Ecol. Evol. 6, 8931–8941. doi: 10.1002/ece3.2603 range and genetic diversity in Lobelia rhynchopetalum. Ecol. Evol. 6, 8931–8941. doi: 10.1002/ece3.2603 Allouche, O., Tsoar, A., and Kadmon, R. (2006). Assessing the accuracy of species distribution models: prevalence, kappa and the True Skill Statistic (TSS). J. Appl. Ecol. 43, 1223–1232. doi: 10.1111/j.1365-2664.2006.01214.x Clark, J. D., Huber, D., and Servheen, C. 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Selecting pseudo-absences for species distribution models: how, where and how many? Methods Ecol. Evol. 3, 327–338. doi: 10.1111/j.2041-210x.2011.00172.x Ejigu, D., Bekele, A., Powell, L., and Lernould, J. M. (2015). Habitat preference of the endangered Ethiopian walia ibex (Capra walie) in the simien mountains national park, Ethiopia. Anim. Biodivers. Conserv. 38, 1–10. doi: 10.32800/abc. 2015.38.0001 Elith, J., Kearney, M., and Phillips, S. (2010). The art of modelling range-shifting species. Methods Ecol. Evol. SUPPLEMENTARY MATERIAL Ethical review and approval was not required for the animal study because the study didn’t require animal handling. The ﬁeld survey and data collection were conducted without disturbing the animals. The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fevo.2021. 611632/full#supplementary-material AUTHOR CONTRIBUTIONS translocations and the building of suitable migration corridors (Stüwe and Nievergelt, 1991; Clark et al., 2002; Hirzel et al., 2002), the species could expand its population further, especially in the Simen Mountains, but potentially also in other areas west of the Ethiopian Rift Valley. A future presence of the Walia ibex in several interconnected mountain fragments that ideally form a functional metapopulation would certainly increase the viability and future prospects of this charismatic ﬂagship species. The historically successful strategy of reintroduction of the once-nearly extinct species of the Alpine ibex that has roamed to the whole Alps mountains should be taken as a lesson to enable the Walia ibex to occupy suitable habitat in the Simen and other mountains west of the Rift Valley. BG collected the data in the ﬁeld. DC conducted the data analyses with input from VB, NZ, and GF. BG and ØF drafted the manuscript and was improved and revised by all co- authors. All authors contributed to the intellectual conception and design of the study. DATA AVAILABILITY STATEMENT We thank Ethiopian Wildlife Conservation Authority (EWCA) and Simen Mountains National Park (SMNP) for the permission to carry out the ﬁeldwork. We thank to the two reviewers for their constructive comments and suggestions. The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s. CONCLUSION Our study has identiﬁed potential key sites that can support re-established populations of the Walia ibex outside its current distribution range and has pointed to areas in which the species will also ﬁnd suitable habitats under projected future climates. Planning and implementing conservation actions like reintroducing and translocating animals to other mountain areas have been successful for several close relatives of the Walia ibex, such as e.g., the Alpine ibex, C. ibex (Maudet et al., 2002). The Alpine ibex, endemic to the Alps in Europe, was driven very close to extinction in the early nineteenth century, when less than 100 individuals were estimated to be left in a single protected area in Italy (Stüwe and Nievergelt, 1991). The reintroduction The population size of the Walia ibex recently increased from 150 individuals in the early 1990s to a minimum of 957 individuals at present. Although the population increase is good news for the conservation of this iconic species, new tasks are ahead. The area currently protected by the National Park is very small, potentially leading to ﬁerce competition for resources within the population as well as with the increasing number of domestic animals (Gebremedhin et al., 2016). 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https://openalex.org/W4318067656	http://ijmscr.org/index.php/ijmscrs/article/download/555/470	English	null	The Relationship between Noise Level and Work Stress	International journal of medical science and clinical research studies	2,023	cc-by	3,699	INTRODUCTION It is stress that causes harmful effects for individuals who experience it such as: unpleasant or excessive demands that drain the energy of the individual. Work stress is a source or work stressor that causes individual reactions in the form of physiological, psychological, and behavioral reactions. In simpler terms, stress due to work occurs due to the inability of workers to face a job demand (Tarwaka et al., 2004). KEYWORDS: noise, work stress KEYWORDS: noise, work stress KEYWORDS: noise, work stress d. Hypostress It is stress that arises due to lack of stimulation. For example, stress due to boredom or due to regular work. One of the physical stressors at work is caused by noise. High intensity noise is noise that exceeds the threshold value or is more than 85 dB while low intensity noise is noise below the threshold value or less than 85 dB (Harrianto, 2010). In a survey conducted by researchers at the Gotri Gentong Factory in Semarang, 7 out of 10 employees complained of headaches, difficulty concentrating, impatience, and irritability when in noisy places. Some of these complaints are symptoms of work stress. Berney and Selye (Asih et al., 2018) reveal there are four types of stress: Corresponding Author: Depri Ardiyansyah c. Hyperstress That is stress that has a tremendous impact on those who experience it. Although it can be positive or negative, this stress still makes individuals limited their adaptability. An example is the stress caused by terrorist attacks. Stress that is often experienced by employees due to the environment around the workplace will affect their work performance, so the organization or company needs to improve or assess the organizational quality for employees. The decrease in stress experienced by employees will definitely improve health or quality in the organization. Work stress can have detrimental consequences for employees or companies (Aldi & Susanti, 2019). The Relationship between Noise Level and Work Stress Depri Ardiyansyah1, Fitriani Kahar2, Suratman3, Dwi Sarwani Sri Rejeki4, Siwi Pramatama5, Muh. Yusuf 6 1,3,4,5Jenderal Soedirman University Megister Health Study Program 2Poltekkes Kemenkes Semarang 6SMPN 15 Makassar City 2https://orcid org/my-orcid?orcid=0000-0001-8787-4015 Depri Ardiyansyah1, Fitriani Kahar2, Suratman3, Dwi Sarwani Sri Rejeki4, Siwi Pramatama5, Muh. Yusuf 6 1,3,4,5Jenderal Soedirman University Megister Health Study Program 2Poltekkes Kemenkes Semarang 6SMPN 15 Makassar City 2https://orcid org/my orcid?orcid=0000 0001 8787 4015 2https://orcid.org/my-orcid?orcid=0000-0001-8787-4015 Published On: 25 January 2023 International Journal of Medical Science and Clinical Research Studie ISSN(print): 2767-8326, ISSN(online): 2767-8342 Volume 03 Issue 01 January 2023 Page No: 100-104 Volume 03 Issue 01 January 2023 Page No: 100-104 a. Eustres (good stress) It is stress that causes stimulus and excitement, so it has a beneficial effect on the individual who experiences it. For example: the challenges that arise from increased responsibility. According to WHO work stress occurs a lot in almost every job around the world and has become a "global epidemic". Based on research in several companies in Indonesia, around 15% to 30% of workers have experienced mental health b. Distress 100 Volume 03 Issue 01 Janaury 2023 ABSTRACT This study aims to determine the relationship between noise levels and work stress levels in employees of the Gotri Gentong Factory in Semarang. The research location in the working area of the Gotri Gentong Factory in Semarang in the administration and production department. The research sample was 36 samples in employees in the production department and 36 samples of employees in the administrative department. Analysis of the relationship between noise levels and work stress levels in employees of the Gotri Gentong Factory Semarang using the chi-square test. The results showed that there was a meaningful relationship between noise intensity and stress levels with a prevalence value of 0.000 (< 0.05). For further research, it is necessary to use different types of treatment with different time periods. Available on: https://ijmscr.org/ b. Distress 100 Volume 03 Issue 01 Janaury 2023 The Relationship between Noise Level and Work Stress problems ranging from mild complaints to those that cause disease (Ikrimadhani, 2015). In a survey conducted in Europe, around 40 million people in Europe experienced work stress. According to the European Risk Observatory Report Around 50% to 60% of workers experience work stress (ILO, 2016). In the previous study conducted by Pradana regarding the Relationship between Noise and Work Stress, it was found that 32% of the workforce experienced light work stress, 44% of the workforce experienced moderate work stress, and 22% of the workforce experienced severe work stress (Pradana, 2013). According to the European Foundation for The Improvement of Living and Working Conditions, work stress is the second largest case in Europe related to work. Health problems caused by stress include heart disease, back pain, hypertension, stroke, and musculoskeletal disorders. Physiological disorders caused by this stress also contribute to the rate of pain, disability, and death. About 75% to 90% of visits to corporate doctors are due to stress (Yunus, 2011). In addition, other negative impacts of stress are in the form of psychological symptoms such as dissatisfaction, boredom, and tension. If these symptoms continue, it can reduce work efficiency and productivity and has the potential to cause work accidents (Tarwaka et al., 2004). Physiologically, when the body is exposed to a situation that is considered threatening or the presence of a stressor, there will be a response to face it. The stress response is closely related to two systems in the body, namely the sympathetic adrenomedullary (SAM) system and the hypothalamic pituitary adrenocortical (HPA) axis which can cause physiological changes in the body. The earliest response is an increase in sympathetic adrenomedullary (SAM) system activity. This increase in sympathetic activity will stimulate the medulla of the adrenal glands so that the release of catecholamines such as epinephrine and norepinephrine occurs. Exposure to stressors not only increases the sympathetic adrenomedullary (SAM) system but also the hypothalamic pituitary adrenocortical (HPA) axis. The hypothalamus will secrete corticotropin releasing factor (CRF). CRF will stimulate the pituitary gland to secrete adrenocorticotropic hormone (ACTH). employees such as irritability, headaches, sleep disturbances, psychomotor reaction disorders, loss of concentration, communication disorders, and decreased work performance (Tarwaka et al., 2004). This study aims to determine the relationship between noise levels and work stress levels in employees of the Gotri Gentong Factory in Semarang. Inclusion criteria: Inclusion criteria: 1. Age 20-40 years. 2. In good health. 3. Have no previous history of hearing disease. 4. Service life ≥ 2 years. 5. Work shifts 8 hours per day. The removal of ACTH will trigger the cortex of the adrenal glands to secrete glucocorticoids, especially cortisol. The function of the hormone cortisol is to help the body maintain itself during stress (Sherwood, 2011). Noise is one of the stressors that can cause stress. The effect of noise on stress depends on the intensity of the noise. High-intensity noise or noise that exceeds the threshold value causes physical disorders such as increased blood pressure, increased pulse, indigestion, disturbances in work, and increased fatigue. The influence of low intensity noise or below the threshold value can cause psychological disorders in 1. Have a previous history of hearing disease. 2. Have a previous history of stress. 3. Respondents are sick e.g. high heat. 4. Respondents did not come during the study The data were analyzed univariately and bivariately. Analysis of the relationship between noise levels and work stress levels in employees of the Gotri Gentong Factory in Semarang using the chi-square test (Dahlan, 2014). 101 Volume 03 Issue 01 Janaury 2023 METHOD The type of research used in research is observational analytics with a case control approach. The research site at the Gotri Gentong Factory in Semarang is divided into two locations, namely in the administrative department and in the production department. The sample of this study was 36 samples in employees in the production department and 36 samples of employees in the administrative department. Approximate size of the research sample based on calculations by using formulas : 𝑛1 = 𝑛2 = (𝑍𝛼 √2𝑃𝑄+ 𝑍𝛽√𝑃1𝑄1 + 𝑃2𝑄2)2 (𝑃1𝑄1)2 = (1,96√2x0,575x0,425 + 0,842√0,5x0,5 + 0,65x0,35) 2 (0,5x0,5)2 = 36,08 𝑛1 = 𝑛2 = (𝑍𝛼 √2𝑃𝑄+ 𝑍𝛽√𝑃1𝑄1 + 𝑃2𝑄2)2 (𝑃1𝑄1)2 = (1,96√2x0,575x0,425 + 0,842√0,5x0,5 + 0,65x0,35) 2 (0,5x0,5)2 = 36,08 So 36 samples were needed in the production department and 36 samples in the administrative department. Information: P1 = standard effect proportions (from the library) P2 = proportion of effects studied (clinical judgement) Zα = degree of meaningfulness (set) Zβ = power (set) P = ½ (P1+P2) Q1 = 1 – P1 Q2 =1 – P2 P1 = standard effect proportions (from the library) 2 = proportion of effects studied (clinical judgement) P2 = proportion of effects studied (clinical judgement) Zα = degree of meaningfulness (set) Zβ = power (set) P = ½ (P1+P2) Q1 = 1 – P1 Q2 =1 – P2 Characteristics of Respondents employees exposed to low intensity noise (Administration section). The following is a description of the characteristics This study involved 36 samples in employees exposed to high intensity noise (Production section) and 36 samples of The Relationship between Noise Level and Work Stress of respondents, namely the distribution of the frequency of research samples according to age Table 1. Distribution of Research Sample Frequency by Age Age (years) Production Section Administration Section Age group; a. 20 – 25 years old b. 26 – 30 years old c. 31 – 35 years old d. 36 – 40 years old 4 (11,11%) 10 (27,78%) 10 (27,78%) 12 (33,33%) 1 (2,78%) 13 (36,11%) 11 (30,56%) 11 (30,56%) Total 36 (100%) 36 (100%) Table 1. Distribution of Research Sample Frequency by Age The table above provides information regarding the characteristics of research samples by age. Based on age The distribution of research samples based on the age group of 22- 38 years, at least in the age group of 20 - 25 years as many as 4 samples (11.11%) in the production department and there was only one sample (2.78%) in the administrative department. Meanwhile, the distribution of research samples in the production section in the age group of 36-40 years was 12 samples (33.33%), and in the administrative section as many as 13 samples (36.11%) in the age group of 26-30 years. The following is a description of the characteristics of respondents, namely the distribution of the frequency of research samples according to gender Table 2. Frequency Distribution of Research Samples By Gender Gender Production Section Administration Section a. Man b. Woman 24 (66,7%) 12 (33,3%) 14 (38,89%) 22 (61,11%) Total 36 (100%) 36 (100%) Table 2. Frequency Distribution of Research Samples By Gender administration department, there were 14 samples of male sex (38.89%), and samples of female sex as many as 22 samples (61.11%). administration department, there were 14 samples of male sex (38.89%), and samples of female sex as many as 22 samples (61.11%). The distribution of research samples according to gender based on the table above, namely, in the production section of male sex samples there were 24 people (66.7%), and samples with female sex there were 12 people (33.3%). In the Corresponding Author: Depri Ardiyansyah The Relationship between Noise Level and Work Stress The Relationship between Noise Level and Work Stress Corresponding Author: Depri Ardiyansyah Univariate Analysis The following is a table of the distribution of research sample frequencies according to noise levels in this study: ble 3. Frequency Distribution of Research Samples According to Noise Level Noise Level Intensity (db) Production Section Administration Section a. Point 1 b. Point 2 c. Point 3 96 92 93 58 63 61 e table above describes the measurements at 3 noise level nts. In the production section measured at three asurement points of 96db each; 92db; 93dB, all three points are at high noise levels (> 85dB). The administrative part was measured at three measurement points of 58db each; 63db; 61dB, all three points are at low noise levels (< 85DB). Corresponding Author: Depri Ardiyansyah A. Univariate Analysis A. Univariate Analysis Table 3. Frequency Distribution of Research Samples According to Noise Level Table 3. Frequency Distribution of Research Samples According to Noise Level stribution of Research Samples According to Noise Level Noise Level Intensity (db) Production Section Administration Section a. Point 1 b. Point 2 c. Point 3 96 92 93 58 63 61 Noise Level Intensity (db) Production Section Administration Section Point 1 Point 2 Point 3 96 92 93 58 63 61 points are at high noise levels (> 85dB). The administrative part was measured at three measurement points of 58db each; 63db; 61dB, all three points are at low noise levels (< 85DB). points are at high noise levels (> 85dB). The administrative part was measured at three measurement points of 58db each; 63db; 61dB, all three points are at low noise levels (< 85DB). The table above describes the measurements at 3 noise level points. In the production section measured at three measurement points of 96db each; 92db; 93dB, all three Corresponding Author: Depri Ardiyansyah 102 Volume 03 Issue 01 Janaury 2023 Corresponding Author: Depri Ardiyansyah The Relationship between Noise Level and Work Stress Table 4. Distribution of Research Sample Frequency According to Stress Level Stress Levels Description a. Normal b. Light c. Medium d. Heavy e. Very Heavy 18 (25%) 33 (45,8%) 20 (27,8%) 1 (1,4%) - Total 72 (100%) The Relationship between Noise Level and Work Stress samples that experienced moderate stress levels (27.8%). And there was only one sample that experienced severe stress levels (1.4%). And in this study there were no samples that had very heavy stress levels. samples that experienced moderate stress levels (27.8%). And there was only one sample that experienced severe stress levels (1.4%). And in this study there were no samples that had very heavy stress levels. The distribution of research samples according to stress levels based on the table above, namely, 18 samples (25%) experienced normal stress levels. There were 33 samples that experienced mild stress levels (45.8%). There were 20 Bivariate Analysis Based on the results of bivariate analysis with chi square test, a relationship can be obtained between noise intensity and stress level, as follows: Table 5. Relationship between Noise Intensity and Stress Level Table 5. Relationship between Noise Intensity and Stress Level Bivariate Analysis Stress Levels Total P value Normal Light Medium Heavy Very Heavy Noise intensity n % n % n % N % n % n % High (Production) 5 13,9 12 33,3 18 50 1 2,8% - 0 36 100 0,000 Low (Administration) 13 36,1 21 58,3 2 5,6 - 0 - 0 36 100 Total 18 25 33 45,8 20 27,8 1 1,4 - 0 72 100 DISCUSSION The table above shows the distribution of research samples based on the relationship between noise intensity and stress levels. In high noise intensity, there were 5 samples that experienced normal stress levels (13.9%), there were 12 samples that experienced mild stress levels (33.3%), there were 18 samples that experienced moderate stress levels (50%), there was one sample that experienced severe stress levels (2.8%). At high noise intensity the majority of the study sample experienced moderate stress levels. Based on the research that has been done, the following results were obtained 1. Noise Level The results showed that at the measurement of the noise of each field was carried out at three points. In accordance with the Decree of the Minister of Manpower No. 13/MEN/2011 on the Threshold Value of Noise in the workplace is 85 db. In the production section, from the three points measured regarding the noise level, successive results were obtained, namely 96db; 92db; 93db. As per its stipulation that all three points exceed the noise level threshold, so they can be categorized at high noise levels. This is natural, because in the field or production room there are various factory machines which of course are one of the producers of the source of noise. From tobacco sorting machines, the provision of cigarettes to filters in making cigarettes, using engine power which of course is the main source of noise. In low noise intensity, there were 13 samples that experienced normal stress levels (36.1%), there were 21 samples that experienced mild stress levels (58.3%), and there were 2 samples that experienced moderate stress levels (5.6%). At low noise intensity the majority of the study sample experienced mild stress levels. Meanwhile, statistically, the pattern of relationships between variables results in the form of a significance value of 0.000. The significance value is smaller than the specified significance limit of 0.05 or (0.000<0.05). With these results, it can be interpreted that there is a meaningful relationship between noise intensity and stress levels. Things are different in measuring noise levels in the room or administration. From the three measurement points, a result of 58db was obtained; 63db; 61db. All three points are still below the prescribed standard of 85db. In other 103 Volume 03 Issue 01 Janaury 2023 CONCLUSION words, the runag or administration section is in the category of low noise level. In the administrative part, the work run is indeed separate and has a partition with the production room. Based on the results of research that has been carried out, the conclusion of this study is that there is a meaningful relationship between noise intensity and stress levels with a prevalence value of 0.000 (< 0.05). Based on the results of research that has been carried out, the conclusion of this study is that there is a meaningful relationship between noise intensity and stress levels with a prevalence value of 0.000 (< 0.05). Employees who work in this administrative department do need quiet space conditions. So that the partition between spaces is designed to minimize noise coming from the production runag or the surrounding environment. The Relationship between Noise Level and Work Stress words, the runag or administration section is in the category of low noise level. In the administrative part, the work run is indeed separate and has a partition with the production room. Employees who work in this administrative department do need quiet space conditions. So that the partition between spaces is designed to minimize noise coming from the production runag or the surrounding environment. REFERENCES I. Aldi, Y., & Susanti, F. (2019). The Effect of Work Stress and Work Motivation on Employee Performance at Pt. Frisian Flag Indonesia Padang Region 2. Work Stress Level In measuring work stress using a questionnaire measuring instrument. The results showed that of the 72 respondents who were sampled in the study, most of them were included in the category of light work stress. Broadly speaking, indicators of work stress include depression, anxiety and stress. The scale of depression contains statements about no positive feelings, can not develop, no hope, sad, moody, depressed, no interest, feel worthless, feel life useless and meaningful, do not get pleasure, are not enthusiastic, it is difficult to take the initiative. The anxiety scale consists of statements about dry mouth, shortness of breath, frequent trembling, anxiety, dizziness, causeless sweating, fear, difficulty swallowing, panic, helplessness. The stress scale consists of statements about being irritated at a small thing, overreactions, difficulty relaxing, wasted energy, impatience, tension, and anxiety (Kholifah, 2015). II. Asih, G. Y., Widhiastuti, H., & Dewi, R. (2018). Work stress. Semarang University Press. II. Asih, G. Y., Widhiastuti, H., & Dewi, R. (2018). Work stress. Semarang University Press. III. Braun, C., & Ising, H. (2000). Acute And Chronic Endocrine Effects Of Noise : Review Of The Research Conducted At The Institute For Water, Soil And Air Hygiene. Noise Health, 2(7), 7–24. IV. Dahlan, M. S. (2014). Statistics For Medicine And Health (6th ed.). Salemba Medika. V. Guyton, A. C., & Hall, J. E. (2014). Textbook of Medical Physiology. 9th Edition . EGC. VI. Harrianto, R. (2010). Textbook of Occupational Health. EGC Doctor Book Publishers. VII. Ikrimadhani, T. (2015). Differences in Work Stress Levels Between Morning, Afternoon and Night Shifts in Inpatient Nurses at Banyudono Boyolali Regional General Hospital. 1–10. Corresponding Author: Depri Ardiyansyah B. Bivariate Analysis VIII. ILO. (2016). Workplace Stress a Collective Challenge, World Day for Safety and Health at Work. In Workplace Stress: A collective challenge World. International Labour Organization. The results showed that there was a meaningful relationship between noise intensity and stress levels with a prevalence value of 0.000 (< 0.05). The effect of noise on stress depends on the intensity of the noise. High-intensity noise or noise that exceeds the threshold value causes physical disorders such as increased blood pressure, increased pulse rate, indigestion, disturbances in work, and increased fatigue. The influence of low intensity noise or below the threshold value can cause psychological disorders in employees such as irritability, headaches, sleep disturbances, psychomotor reaction disorders, loss of concentration, communication disorders, and decreased work performance (Tarwaka et al., 2004). IX. Kholifah, A. (2015). Description of Stress Levels in School-Age Children Facing First Menstruation (Menarche) at Gegerkalong Girang Public Elementary School 2. Jurnal Pendidikan Keperawatan Indonesia, 1(2), 125–130. X. Pradana, A. (2013). The Relationship Between Noise and Work Stress in Gravity Workers at PT. Two Rabbits. Unnes Journal of Public Health, 2(3), 1–9. The higher the intensity of the noise, the more the cortisol hormone will increase. Cortisol suppresses the immune system because it can reduce T lymphocytes so that in high noise exposure cortisol levels will increase and decrease the immune system. In addition, the effect of continuous stress due to an increase in cortisol levels will cause sympathetic nerve stimulation for vasoconstriction so that at high noise intensity exposure one of the symptoms is an increase in blood pressure (Braun & Ising, 2000) (Guyton & Hall, 2014). In addition to noise exposure, work stress is also influenced by several factors such as age, work shifts, workload, health conditions, and work mass (Sarwendah, 2013). XI. Sarwendah, E. (2013). The Relationship between Workload and Work Stress Levels for Social Workers as Caregivers at the Tresna Werdha Budi Mulia Social Institution DKI Jakarta. 1–56. XII. Sherwood, L. (2011). Human Physiology (6th ed.). EGC. XIII. Tarwaka, Bakri, S. H., & Sudiajeng, L. (2004). Ergonomics for Occupational Safety, Health and Productivity (1st ed.). UNIBA PRESS. XIV. Yunus, M. (2011). Factors Associated with the Occurrence of Work Stress in Work Unit Employees in Laundry Work Unit Employees at Pasar Rebo Hospital. 104 Volume 03 Issue 01 Janaury 2023 Corresponding Author: Depri Ardiyansyah
https://openalex.org/W4361942442	https://figshare.com/articles/journal_contribution/Supplementary_Table_S2_from_Reversine_a_novel_Aurora_kinases_inhibitor_inhibits_colony_formation_of_human_acute_myeloid_leukemia_cells/22485806/1/files/39937370.pdf	Turkish	null	Supplementary Table S1 from Reversine, a novel Aurora kinases inhibitor, inhibits colony formation of human acute myeloid leukemia cells	null	2,023	cc-by	78	Supplemental Table 2 Supplemental Table 2 Supplemental Table 2 Pt Age/Sex FAB % Blast Reversine IC50 VX-680 IC50 P1 38/F M0 97 26±4 215±20 P2 30/F M1 77 153±4 184±3 P3 53/M M1 N.A. 64±13 25±8 P4 81/M M1 N.A. 131±4 113±3 P5 77/F M0 47 91±5 100±3 P6 74/M M1 N.A. 125±3 99±7 P7 47/M M2 98 548±5 770±2 P8 73/F M2 N.A. 133±11 35±15 P9 64/M M1 90 128±9 102±5 P10 70/F M0 N.A. 120±5 72±7
W4320352492.txt	https://zenodo.org/record/7632588/files/Lerch-Neg.pdf	en		Lerch's $\Phi$ and the Polylogarithm at the Negative Integers	Zenodo (CERN European Organization for Nuclear Research)	2,021	cc-by	3,617	Lerch’s Φ and the Polylogarithm at the Negative Integers Jose Risomar Sousa September 17, 2021 Abstract At the negative integers, there is a simple relation between the Lerch Φ function and the polylogarithm. Starting from that relation and a formula for the polylogarithm at the negative integers known from the literature, we can deduce a simple closed formula for the Lerch Φ function at the negative integers, where the Stirling numbers of the second kind are not needed. Leveraging that finding, we also produce alternative formulae for the k-th derivatives of the cotangent and cosecant (ditto, tangent and secant), as simple functions of the negative polylogarithm and Lerch Φ, respectively, which is evidence of the importance of these functions (they are less exotic than they seem). Lastly, we extend formulae for the Hurwitz zeta function only valid at the positive integers to the complex half-plane using this novelty. 1 Introduction As seen in reference [4], it’s possible to derive a formula for the Hurwitz zeta function at the positive integers with results from two previous papers that introduced new formulae for generalized harmonic numbers and progressions, [2] and [3], respectively. To greatly summarize the reasoning presented in [4], if k is a positive integer greater than one, then: Z 0 1 H(n) k! u (1 − cos 2π n u) cot πu du ∼ − − π π k b(k−1)/2c X j=1 (−1)j (2π)−2j ζ(2j + 1) , (k − 2j)! which implies the following approximation, Z 0 1 k! (u −u)(1−cos 2π n u) cot πu du ∼ − π k b(k−1)/2c X j=1 (−1)j (2π)−2j ζ(2j + 1) = (k − 2j)! Z 1 (uk −u) cot πu du, 0 with the equality on the right only valid for integer k. And this approximation in turn justifies the formula1 shown next. 1 The formulae that can be derived with this method are not unique and the one shown may be the simplest. 1 For every integer k greater than one and every non-integer complex b: ! k −2πi b X Li−k+1 e−2πi b δ + Li (e ) 1j −j+1 + e−2πi b (k − 1)! (j − 1)!(k − j)! j=1 Z k i(2πi)k 1 X δ1j + Li−j+1 (e−2πi b ) uk−j e−2πi b u − e−2πi b − cot πu du 2 (j − 1)!(k − j)! 0 j=1 1 (2πi)k ζ(k, b) = k + 2b 4 The discovery of a new, possibly first, closed formula for the Lerch transcendent function at the negative integers was made possible through the analysis of the above formula. The big breakthrough is really in the following straightforward identity, which only works for the analytic continuation of the Lerch Φ and the polylogarithm functions at the negative integers, and makes it possible to obtain the former as a sum of the latter: k X Li−j eb uk−j eb (1) = Φ(eb , −k, u + 1) j!(k − j)! k! j=0 In fact, this identity also applies to the sum of Lerches: k X Φ eb , −j, v uk−j 1 = Φ(eb , −k, u + v) j!(k − j)! k! j=0 (2) Since formulae (1) and (2) break down when eb = 1, from two known facts from the literature, namely, a recurrence for the Bernoulli polynomials and a relation between them and the Hurwitz zeta function: Bk (u + v) = k! k X Bj (v) uk−j j=0 j!(k − j)! and Bj (v) = −j ζ(1 − j, v), one can conclude that, k X uk+1 ζ(−j, v) uk−j ζ(−k, u + v) = − + k! , k+1 j!(k − j)! j=0 and from this recurrence, one can obtain a natural expression relating the Hurwitz and the Riemann zeta functions, which completes the picture: ζ(−k, u + 1) = − k X uk+1 ζ(−j) uk−j + k! k+1 j!(k − j)! j=0 (3) Relation (1) may be the counterpart to the functional equation that relates the Lerch Φ to the Hurwitz zeta at the positive integers, as demonstrated in a previous paper 5 . This new relation therefore presupposes the existence of another relation between the polylogarithm and the zeta function at the negative integers. 2 2 Hurwitz zeta analytic continuation Going back to the Hurwitz zeta formula, from relation (1) one obtains, k X Li−j+1 e−2πi b uk−j e−2πi b = Φ(e−2πi b , −k + 1, u + 1), (j − 1)!(k − j)! (k − 1)! j=1 which leads to the below analytic continuation for the Hurwitz zeta function, ζ(k, b) = 1 (2πi)k e−2πi b + e−4πi b Φ(e−2πi b , −k + 1, 2) + Li−k+1 e−2πi b + k 2b 4(k − 1)! Z 1 i(2πi)k − uk−1 e−2πi b u − e−2πi b 2(k − 1)! 0 −2πi b(u+1) −2πi b −4πi b −2πi b +e Φ(e , −k + 1, u + 1) − e Φ(e , −k + 1, 2) cot πu du, (4) which then holds for <(k) > 1. Since this formula depends on the Lerch’s Φ analytic continuation at the negative integers, if we can figure out the Lerch’s Φ at the negative integers, we can rewrite (4). 3 Stirling numbers of the second kind The existing formula for the polylogarithm, Li−j+1 (z), available in the literature, makes use of the Stirling numbers of the second kind2 : j X q j z Li−j+1 (z) = (q − 1)! q 1−z q=1 (5) Let’s see how to obtain the Lerch Φ from it. Making z = e−2πi b , one has: 1 + i cot π b z =− 1−z 2 Therefore, back to equation (1): q j k k X Li−j+1 e−2πi b u−j X X j 1 + i cot π b = (q − 1)! − ⇒ (j − 1)!(k − j)! q 2 j=1 j=1 q=1 j k X X j=1 q=1 q (q − 1)!u−j j 1 + i cot π b − ⇒ (j − 1)!(k − j)! q 2 q k k X 1 + i cot π b X u−j j (q − 1)! − 2 (j − 1)!(k − j)! q q=1 j=q 2 The S(j, q) in the curly brackets. 3 (6) Now we need to inspect the second sum from equation (6) and see if it’s possible to rewrite it. An expression for a similar sum exists in the literature, k X j=q 1 j 1 k+1 = , j!(k − j)! q k! q + 1 but this new one is much more complicated. 4 Binomial formula for Stirling numbers Let’s try and rewrite the below finite sum, k X j=q u−j j (j − 1)!(k − j)! q (7) First off, the literature provides us with the below relation: k j X j x j=q q j! = (ex − 1)q , q! therefore, ∞ X k j X j x k=q j=q y k−j ey (ex − 1)q = , q! q j! (k − j)! and differentiating with respect to x, k ∞ X X j xj−1 ey ex (ex − 1)q−1 y k−j = q (j − 1)! (k − j)! (q − 1)! k=q j=q Now, making x = u z and y = z, one has: ∞ X z k−1 j=q k=q q−1 k X j e(u+1)z (eu z − 1)q−1 X (−1)q−1−j e(u+1+j u)z uj−1 = = , (q − 1)! j!(q − 1 − j)! q (j − 1)!(k − j)! j=0 and hence differentiating k − 1 times with respect to z, we conclude that, k X j=q q X uj j u (−1)q−j (j u + 1)k−1 = (j − 1)!(k − j)! q (k − 1)! j=1 (j − 1)!(q − j)! To obtain a more appropriate version of this formula, we integrate as below, Z 0 u k X j=q Z uX q xj−1 j 1 (−1)q−j (j x + 1)k−1 dx = dx, (j − 1)!(k − j)! q (k − 1)! 0 j=1 (j − 1)!(q − j)! 4 (8) which gives the neater expression below, k X j=q q uj j 1 X (−1)q−j (j u + 1)k = , j!(k − j)! q k! j=0 j!(q − j)! (9) which holds for every non-negative integer q and every u. Though it’s not going to be used here, another pattern similar to the binomial theorem emerges in the factorial power of the sum of two numbers, x and y: (k) (x + y) = k! k X x(j) y (k−j) j=0 5 j!(k − j)! , where x(j) = x! (x − j)! Lerch’s Φ at the negative integers Now, if equations (1), (6) and (8) are combined, one obtains: −2πi b Φ(e 2πi b , −k + 1, u + 1) = e k X (q − 1)! q=1 1 + i cot π b 2 q X q j=1 (−1)j (j + u)k−1 (j − 1)!(q − j)! (10) For integer b, the formula is not defined as the cotangent is infinity, so we can’t extract the Hurwitz zeta at the negative integers from it. But from the relation, Φ(e−2πi b , −k + 1, 1) = e2πi b Li−k+1 e−2πi b , we can derive the polylogarithm, which however is just a rewrite of equation (5) with an expression for S(k, q) known from the literature, which nonetheless confirms equation (10): Li−k+1 e −2πi b = k X (q − 1)! q=1 1 + i cot π b 2 q X q j=1 (−1)j j k−1 (j − 1)!(q − j)! (11) Looking at formulae (10) and (11) now, it might look simple to go from the latter straight to the former without having to figure (8), but that’s misleading. Finally, we can turn (10) into a neater form, which holds for every non-negative integer k: k 1 X Φ(z, −k, u) = − q! z − 1 q=0 z z−1 q X q j=0 (−1)j (j + u)k j!(q − j)! (12) It’s interesting to note how much simpler the analytic continuation of the Lerch Φ at the negative integers is than the proper function at the positive integers. And also how strikingly similar it is to the power series for the Lerch Φ at the positive integers available in the literature: q X q ∞ 1 X z (−1)j (j + u)−k Φ(z, k, u) = − q! z − 1 q=0 z − 1 j=0 j!(q − j)! 5 6 Derivatives of trigonometric functions In his paper On the Hurwitz function for rational arguments 1 , Victor Adamchik provides the first ever formula for the intricate patterns of the k-th derivatives of the cotangent. It looks like this: q k X dk (cot a x) k −1 + i cot a x k = (2 i a) (−i + cot a x) q! d xk 2 q q=1 It’s possible to express this formula as a simple function of the polylogarithm. First, we rewrite it as: q q k X dk (cot a x) 1 − i cot a x X (−1)j j k−1 k (13) = (2 i a) (−i + cot a x) q! d xk 2 (j − 1)!(q − j)! q=1 j=1 Secondly, we note how similar it looks to the polylogarithm from (11): q q k X 1 − i cot a x X (−1)j j k−1 2iax Li−k+1 e = (q − 1)! 2 (j − 1)!(q − j)! q=1 j=1 If the above polylog is differentiated once with respect to x and transformed, an alternative expression is obtained for the polylogarithm of order k, q q k X 1 − i cot a x X (−1)j j k−1 1 2iax q! , (14) Li−k e = 1 − e2 i a x q=1 2 (j − 1)!(q − j)! j=1 which, however, is not exactly equal to form (11). That stems from a property of polylogs, that when differentiated they yield the next order polylog. Finally, comparing the two expressions, (13) and (14), we conclude that: dk (cot a x) k 2iax = −i δ − 2 i(2 i a) Li e , where δ0 k = 1 iff k = 0 0 k −k d xk (15) To obtain the cosecant, we can resort to a simple logic: ei a x 1 cos a x + i sin a x = = i + cot a x ⇒ = e−i a x (i + cot a x), sin a x sin a x sin a x and then the Leibniz rule for the derivative of a product of two functions leads to: k X dk 1 k! (−i a)k−q −i a x q 2iax = −2 i e (2 i a) Li e −q d xk sin a x q!(k − q)! q=0 Lastly, formula (1) allows us to rewrite the above expression as: dk 1 1 2iax k iax , −k, = −2 i(2 i a) e Φ e , d xk sin a x 2 which holds for every non-negative integer k. 6 (16) 6.1 Tangent and secant To be able to obtain the tangent and secant, first we need to produce a formula for the cotangent and cosecant of a translated arc. Adamchik’s formula 1 becomes: q q k X dk (cot (a x + b)) 1 − i cot (a x + b) X (−1)j j k−1 k = (2 i a) (−i + cot (a x + b)) q! d xk 2 (j − 1)!(q − j)! q=1 j=1 The polylog formula then changes to: 2 i(a x+b) Li−k e = k X 1 1 − e2 i(a x+b) q=1 q! 1 − i cot (a x + b) 2 q X q j=1 (−1)j j k−1 , (j − 1)!(q − j)! and then the final formula is not too different from the simple case: dk (cot (a x + b)) = −i δ0 k − 2 i(2 i a)k Li−k e2 i(a x+b) , where δ0 k = 1 iff k = 0 k dx Similarly, the cosecant of a translated arc is: dk 1 1 k i(a x+b) 2 i(a x+b) = −2 i(2 i a) e Φ e , −k, d xk sin (a x + b) 2 Finally, to obtain the tangent and secant, we just need to set b to π/2. And since the formulae for the translated arc are not very different from b = 0, for the tangent one has: dk (tan (a x + b)) k 2 i(a x+b) = i δ + 2 i(2 i a) Li −e , where δ0 k = 1 iff k = 0, 0 k −k d xk (17) and for the secant: dk d xk 1 cos (a x + b) k i(a x+b) = 2 (2 i a) e 1 2 i(a x+b) Φ −e , −k, 2 (18) It’s surprising that these derivatives can be expressed by means of negative Lerch and polylogs. For example, the negative polylog is known to yield the derivatives of a simple exponential function at a point, but not the derivative itself: k−1 x dk b = −k δ + Li e a 1 k −k+1 d xk ea x+b − 1 x=0 7 7 Hurwitz zeta at the positive integers Now that fomula (10) is known, the Hurwitz zeta formula at the positive integers greater than one from (4) can be rewritten with only references to elementary functions:   q k (2πi)k  −2πi b X 1 1 + i cot π b q X (−1)j e−2πi b (j + 1)k−1 + j k−1  ζ(k, b) = k + e + (q − 1)! 2 (j − 1)!(q − j)! 2b 4(k − 1)! q=1 j=1 Z 1 i(2πi)k uk−1 e−2πi b u − e−2πi b + 2(k − 1)! 0 ! q k X 1 + i cot π b q X (−1)j e−2πi b u (j + u)k−1 − e−2πi b (j + 1)k−1 (q − 1)! cot πu du 2 (j − 1)!(q − j)! − q=1 j=1 The pattern of the above formula becomes clearer and easier to grasp if we include the outside terms into the summation symbol, assuming the convention that (−1)!/(−1)! = 1: k 1 (2πi)k X ζ(k, b) = k + (q − 1)! 4(k − 1)! 2b q=0 i(2πi)k − 2(k − 1)! Z k 1X (q−1)! 0 q=0 1 + i cot π b 2 1 + i cot π b 2 q X q j=0 q X q j=0 (−1)j e−2πi b (j + 1)k−1 + j k−1 (j − 1)!(q − j)! (−1)j e−2πi b u (j + u)k−1 − e−2πi b (j + 1)k−1 cot πu du (j − 1)!(q − j)! One of the advantages of this new formula is the fact it allows one to get rid of its non-real parts more easily, though the resulting formula is inevitably more complicated. 7.1 When the parameter b is a half-integer The below result stems from e−2πi b = −1 and cot π b = 0 when b is a half-integer:   q X q k X (−1)j (j + 1)k−1 − j k−1 1 (2πi)k  1  ζ(k, b) = k − 1+ (q − 1)! 4(k − 1)! 2 (j − 1)!(q − j)! 2b q=1 j=1   q X Z 1 q k j −2πi b u k−1 k−1 k X (−1) e (j + u) + (j + 1) i(2πi) 1 uk−1 e−2πi b u + 1 +  cot πu du − (q − 1)! 2(k − 1)! 0 2 (j − 1)!(q − j)! q=1 8 j=1 A new formula for the Hurwitz zeta In [4], we had created a generating function for the Hurwitz zeta function, f (x). When b is not a half-integer, the expression is: ∞ X x2 1 πx sin πx f (x) = x ζ(k, b) = − − 2b(x − b) 2 sin πb sin π(x − b) k=2 Z 1 sin 2πu(x − b) sin 2πbu − πx − cot πu du (19) sin 2π(x − b) sin 2πb 0 k 8 The k-th derivative of f (x) yields the Hurwitz zeta function of order k: ζ(k, b) = f (k) (0) k! And now that we know how to differentiate the cosecant successively, it’s possible to produce an explicit formula from f (x), again through the Leibniz rule. However, to make this process simpler, we resort to two artifices. First, to get rid of the extra x factor in the integral, we divide f (x) by x and take the (k − 1)-th derivative instead of the k-th. Second, to avoid the complications of differentiating the sine, we replace it with an equivalent sum of exponential functions. The first and second parts of (19) are straightforward, they coincide with the terms outside of the integral from (4), that is: 1 dk k! d xk 1 πx sin πx − 2 sin πb sin π(x − b) = x=0 (2πi)k −2πi b e + e−4πi b Φ(e−2πi b , −k + 1, 2) + Li−k+1 e−2πi b 4(k − 1)! The same isn’t true for the third part of (19), since f (x) was created using a different process than (4) (see [4] for details). The integrals evaluate to the same number, but the integrands are not the same. After all is put together, the final formula holds for any positive integer k greater than one, and any b that’s not an integer or a half-integer: (2πi)k −2πi b 1 −2πi b −4πi b −2πi b + e + e + e Φ(e , −k + 1, 2) + Li −k+1 4(k − 1)! 2bk Z k i(2πi)k e−2πi b 1 X 2j uk−j e−2πi b u − (−1)k−j e2πi b u 1 −4πi b Φ e , −j + 1, cot πu du (20) − 4 (j − 1)!(k − j)! 2 0 ζ(k, b) = j=1 Finally, using the relation (2), an analytic continuation can be produced: 1 (2πi)k −2πi b −2πi b −4πi b −2πi b e + e Φ(e , −k + 1, 2) + Li e + + −k+1 4(k − 1)! 2bk Z i(4πi)k e−2πi b 1 −2πi b u u+1 −u + 1 −4πi b 2πi b u −4πi b − e Φ e , −k + 1, −e Φ e , −k + 1, cot πu du 4(k − 1)! 2 2 0 ζ(k, b) = 9 References [1] Adamchik, Victor S. On the Hurwitz function for rational arguments, Applied Mathematics and Computation, Volume 187, Issue 1, 2007, Pages 3–12. [2] Risomar Sousa, Jose Generalized Harmonic Numbers, eprint arXiv:1810.07877, 2018. [3] Risomar Sousa, Jose Generalized Harmonic Progression Part II, eprint arXiv:1902.01008, 2019. [4] Risomar Sousa, Jose The Hurwitz Zeta Function at the Positive Integers, eprint arXiv:1902.06885, 2019. [5] Risomar Sousa, Jose Lerch’s Φ and the Polylogarithm at the Positive Integers, eprint arXiv:2006.08406, 2020. 10
https://openalex.org/W4307362178	https://ccforum.biomedcentral.com/counter/pdf/10.1186/s13054-022-04199-3	English	null	Extracorporeal versus conventional cardiopulmonary resuscitation for refractory out-of-hospital cardiac arrest: a secondary analysis of the Prague OHCA trial	Critical care	2,022	cc-by	6,876	Abstract Background: Survival rates in refractory out-of-hospital cardiac arrest (OHCA) remain low with conventional advanced cardiac life support (ACLS). Extracorporeal life support (ECLS) implantation during ongoing resuscitation, a method called extracorporeal cardiopulmonary resuscitation (ECPR), may increase survival. This study examined whether ECPR is associated with improved outcomes. Methods: Prague OHCA trial enrolled adults with a witnessed refractory OHCA of presumed cardiac origin. In this secondary analysis, the effect of ECPR on 180-day survival using Kaplan–Meier estimates and Cox proportional hazard model was examined. Results: Among 256 patients (median age 58 years, 83% male) with median duration of resuscitation 52.5 min (36.5–68), 83 (32%) patients achieved prehospital ROSC during ongoing conventional ACLS prehospitally, 81 (32%) patients did not achieve prehospital ROSC with prolonged conventional ACLS, and 92 (36%) patients did not achieve prehospital ROSC and received ECPR. The overall 180-day survival was 51/83 (61.5%) in patients with prehospital ROSC, 1/81 (1.2%) in patients without prehospital ROSC treated with conventional ACLS and 22/92 (23.9%) in patients without prehospital ROSC treated with ECPR (log-rank p < 0.001). After adjustment for covariates (age, sex, initial rhythm, prehospital ROSC status, time of emergency medical service arrival, resuscitation time, place of cardiac arrest, percutaneous coronary intervention status), ECPR was associated with a lower risk of 180-day death (HR 0.21, 95% CI 0.14–0.31; P < 0.001). Conclusions: In this secondary analysis of the randomized refractory OHCA trial, ECPR was associated with improved 180-day survival in patients without prehospital ROSC. Trial registration: ClinicalTrials.gov Identifier: NCT01511666, Registered 19 January 2012. Keywords: Out-of-hospital cardiac arrest, Extracorporeal life support, Extracorporeal membrane oxygenation, Extracorporeal cardiopulmonary resuscitation, Return of spontaneous circulation Rob et al. Critical Care (2022) 26:330 https://doi.org/10.1186/s13054-022-04199-3 Rob et al. Critical Care (2022) 26:330 https://doi.org/10.1186/s13054-022-04199-3 Extracorporeal versus conventional cardiopulmonary resuscitation for refractory out‑of‑hospital cardiac arrest: a secondary analysis of the Prague OHCA trial Daniel Rob1, Jana Smalcova1, Ondrej Smid1, Ales Kral1, Tomas Kovarnik1, David Zemanek1, Petra Kavalkova1, Michal Huptych2, Arnost Komarek3, Ondrej Franek4, Stepan Havranek1, Ales Linhart1 and Jan Belohlavek1* Background © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://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. Background Out-of-hospital cardiac arrest (OHCA) is one of the leading causes of death in Western countries [1]. Patients without prehospital return of spontane- ous circulation (ROSC) bear a grave prognosis with Correspondence: jan.belohlavek@vfn.cz 1 2nd Department of Medicine – Department of Cardiovascular Medicine, First Faculty of Medicine, Charles University and General University Hospital in Prague, U Nemocnice 2, 128 00 Prague, Czech Republic Full list of author information is available at the end of the article Correspondence: jan.belohlavek@vfn.cz g Out-of-hospital cardiac arrest (OHCA) is one of the leading causes of death in Western countries [1]. Patients without prehospital return of spontane- ous circulation (ROSC) bear a grave prognosis with 1 2nd Department of Medicine – Department of Cardiovascular Medicine, First Faculty of Medicine, Charles University and General University Hospital in Prague, U Nemocnice 2, 128 00 Prague, Czech Republic Full list of author information is available at the end of the article Population and study designh This study is a secondary analysis of the Prague OHCA study, a randomized clinical trial which was conducted at a single center in Prague, Czech Republic, from March 1, 2013, to October 25, 2020. Adult patients resuscitated for witnessed OHCA of presumed cardiac etiology after at least 5 min of ACLS were eligible for enrollment in the trial. A web-based secured randomi- zation system was used to assign patient number and intervention group prehospitally during ongoing CPR in the field. The methodology and results of the inten- tion to treat analysis were published in detail elsewhere [8, 9]. In the present analysis, all 256 enrolled patients were included and pooled into three groups (regardless of their original randomization assignment) according to their prehospital ROSC and ECPR status (Fig. 1). The first group (prehospital ROSC) is formed by all patients who achieved prehospital ROSC and thus were not can- didates for ECPR. The second group is formed by all patients without prehospital ROSC despite prolonged ACLS who did not receive ECPR. This group includes patients who died during prolonged ACLS in the field as well as the group of patients admitted to the hospital who died during ACLS or achieved ROSC in the hos- pital. Finally, the third group is formed by all patients who received ECPR after arrival to the hospital. To date, two prospective randomized trials on ECPR in refractory OHCA were published, both testing “load and go” strategy with in-hospital ECLS cannulation. The first is the ARREST trial [7] which randomized 30 patients with refractory ventricular fibrillation only and was prematurely stopped due to superior- ity of ECPR and showed that ECPR is a feasible res- cue option after prolonged unsuccessful ACLS, where standard approach has negligible chance for success [7]. The second prospective trial is the recently pub- lished Prague OHCA study [8] which enrolled 256 patients during on-scene ongoing ACLS to invasive arm (including intra-arrest transport for in-hospital ECPR and immediate invasive assessment) or stand- ard ACLS. The invasive treatment did not significantly improve survival with good neurologically outcome at 180 days compared to standard ACLS in the inten- tion to treat analysis but showed a beneficial effect of the invasive approach in 30-day neurological out- come and a subgroup of patients with prolonged CPR over 45 min [8]. Methods survival rates as low as 4% [2–4]. An increasing num- ber of cardiac arrest centers worldwide have estab- lished a collaboration with emergency medical services using early transport from the field and extracorporeal life support (ECLS) implantation during ongoing car- diopulmonary resuscitation (CPR) when ROSC is not achieved conventionally, a method called extracor- poreal cardiopulmonary resuscitation (ECPR). How- ever, the current 2020 American Heart Association as well as the recent European Resuscitation Coun- cil guidelines provide a weak recommendation for ECPR which may be considered as a rescue method in selected patients when conventional CPR is failing, with low certainty of evidence [5, 6]. In addition, both guidelines highlighted the need for further research to define patients who would benefit from this interven- tion most [5, 6]. © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://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. Rob et al. Critical Care (2022) 26:330 Page 2 of 9 Page 2 of 9 Population and study designh Importantly, the anticipated statistical scenario of expected benefit provided by invasive approach was not reached due to higher-than- expected survival in the standard group [8]. Further, crossovers were allowed and 4 out of 10 (40%) patients crossed from standard to invasive ECPR treatment survived 180 days [8]. In addition, part of the prehos- pitally randomized patients in both arms experienced ROSC before reaching the hospital and thus were not candidates for ECPR [8]. All these factors might have influenced the effect of the ECPR treatment in the intention to treat analysis. Therefore, we performed this secondary analysis of the Prague OHCA study to evaluate whether successful ECPR might have been associated with improved outcomes. The original study as well as secondary analyses was approved by the Institutional Review Board of the Gen- eral University Hospital and First Faculty of Medicine, Charles University in Prague (192/11 S-IV). Each par- ticipant’s legal representative was informed of the par- ticipant’s study enrollment and was asked for written informed consent as soon as possible. All patients who regained normal neurological function were asked to provide their written consent regarding the use of their data. Consent requirements were waived for patients who died at the scene and never reached the hospital and for participants without known legal representa- tives. The research was carried out in accordance with requirements stated above (192/11 S-IV) and the Hel- sinki Declaration of 1964, revised in 2008. Outcomes Th The primary outcome of the current analysis was all- cause 180-day survival. The secondary outcome was good neurological outcome at 180 days. A CPC of 1–2 was considered a good neurological outcome, and a CPC of 3–5 was considered a poor neurological outcome. Rob et al. Critical Care (2022) 26:330 Page 3 of 9 4345 Patients with out-of-hospital cardiac arrest assessed for eligibility 3987 Excluded 1601 Declared dead at scene before randomization 1263 Return of spontaneous circulation before enrollment considered 677 Unwitnessed cardiac arrest 363 Non cardiac cause 49 Age below 18 years 34 Outcome data not available 264 Randomized 94 Excluded 36 Age known > 65-years old 29 Physician decision not to enroll 19 Referred to other institutions 4 ECLS or ICU bed capacity not available 4 Reason not known 1 Mechanical CPR device not functional 1 Polymorbid patient 358 Patients without return of spontaneous circulation assessed for inclusion 8 Excluded 7 Consent not obtained 1 Randomized after DSMB stopped the study 83 Prehospital ROSC 81 No ROSC and ACLS 92 No ROSC and ECPR Fig. 1 Modified consort flow diagram of the Prague OHCA study. ACLS advanced cardiac life support, CPR cardiopulmonary resuscitation, DSMB data safety monitoring board, ICU intensive care unit, ECLS extracorporeal life support, ECPR extracorporeal membrane resuscitation, ROSC return of spontaneous circulation 4345 Patients with out-of-hospital cardiac arrest assessed for eligibility 8 Excluded 7 Consent not obtained 1 Randomized after DSMB stopped the study Fig. 1 Modified consort flow diagram of the Prague OHCA study. ACLS advanced cardiac life support, CPR cardiopulmonary resuscitation, DSMB data safety monitoring board, ICU intensive care unit, ECLS extracorporeal life support, ECPR extracorporeal membrane resuscitation, ROSC return of spontaneous circulation Statistical analysish https://www.medcalc.org; 2021), and the Cox pro- portional hazards model [10] analysis was performed using the R (R Core Team, 2021) software, version 4.1.0 (2021–05-18). The continuous data were tested to normal distribution by Shapiro–Wilk test. Categorical values are expressed as count and percentage, and the continuous variables are expressed as median and intra-quartile range. The Kruskal–Wallis test was used to compare the continu- ous values over all groups. Categorical values were tested using the chi-square test over all groups. Sur- vival rates were compared using Kaplan–Meier analysis and Cox proportional hazards regression. Multivariate Cox proportional hazard model included all enrolled patients, and variables were age, sex, initial rhythm, prehospital ROSC status, time from collapse to EMS arrival, resuscitation time, place of cardiac arrest, per- cutaneous coronary intervention (PCI) status and ECPR status. A 2-sided p < 0.05 was considered sta- tistically significant. Overall statistical analyses were performed with MedCalc® Statistical Software ver- sion 20.014 (MedCalc Software Ltd, Ostend, Belgium; Baseline characteristics Baseline characteristics of patients according to the ROSC and ECPR status are presented in Table 1. There were no differences in age, sex, medical history or loca- tion of cardiac arrest between the three analyzed groups. Importantly, patients without prehospital ROSC with ACLS only compared to the ECPR group and prehos- pital ROSC group had significantly less common initial shockable rhythm (44.4% vs. 62% vs. 75.9%, p < 0.001) and consequently received more adrenalin doses (median 6 vs. 4 vs. 3, p < 0.001) and less defibrillations prehospi- tally (median 1 vs. 3 vs. 3, p = 0.02) (Table 1). Further, Rob et al. Baseline characteristics Critical Care (2022) 26:330 Page 4 of 9 Table 1 Baseline and resuscitation characteristics Table 1 Baseline and resuscitation characteristics Highlighted in bold are the values which are statistically significant (less than 0.05) For patients with initial VF f Parameter Prehospital ROSC (n = 83) No ROSC and ACLS (n = 81) No ROSC and ECPR (n = 92) P value Age (years) 55 (49–63.8) 58 (48–66) 58.5 (45–65) 0.69 Sex Woman 14 (16.9%) 14 (17.3%) 16 (17.4%) 1.0 Man 69 (83.1%) 67 (82.7%) 76 (82.6%) Medical history Hypertension 38 (48.1%) 13 (43.3%) 38 (46.3%) 0.9 Coronary artery disease 14 (17.9%) 5 (17.2%) 15 (18.8%) 0.98 Chronic heart failure 4 (5.1%) 3 (11.5%) 9 (11.1%) 0.35 Diabetes 14 (17.9%) 9 (31%) 13 (16.3%) 0.21 Chronic kidney disease 3 (3.8%) 0 (0%) 2 (2.5%) 0.58 COPD 7 (9.0%) 1 (3.8%) 2 (2.5%) 0.19 ICD implanted 0 (0.0%) 1 (2.7%) 2 (2.2%) 0.37 Location of cardiac arrest Home 26 (31.3%) 24 (29.6%) 26 (28.3%) 0.15 Public place 38 (45.8%) 29 (35.8%) 31 (33.7%) EMS 4 (4.8%) 12 (14.8%) 20 (21.7%) Health facility 0 (0%) 1 (1.2%) 1 (1.1%) Car 2 (3.6%) 5 (6.2%) 7 (7.6%) Hotel 4 (4.8%) 5 (6.2%) 1 (1.1%) Workplace 8 (9.6%) 5 (6.2%) 6 (6.5%) Initial rhythm VF 63 (75.9%) 36 (44.4%) 57 (62%) < 0.001 Asystole 14 (16.9%) 23 (28.4%) 18 (19.6%) PEA 6 (7.2%) 22 (27.2%) 17 (18.5%) Time of CPR (time to death/ROSC or ECPR) (min) 31 (24–39.8) 66 (46–82.3) 60 (51–70) < 0.001 Bystander CPR 81 (97.6%) 80 (98.8%) 91 (98.9%) 0.75 Time from collapse to EMS arrival (min) 9 (7–11) 9 (6–12) 8 (6–10) 0.6 Time from collapse to ACLS (physician arrival) (min) 11 (8.3–14) 11 (8–14.3) 10 (6–13) 0.06 Time from collapse to randomization (min) 24 (19–29.8) 26 (21–34.3) 24.5 (19.5–30) 0.27 Time to ECLS (min) NA NA 61 (55–70) NA Time of implantation (door to ECLS) (min) NA NA 12 (11–14) NA Number of epinephrine doses prehospitally (mg) 3 (2–4.8) 6 (5–9) 4 (2–6) < 0.001 Dose of amiodarone prehospitally (mg) 300 (0–300) 225 (0–300) 300 (0–300) 0.6 Number of defibrillations prehospitally 3 (2–5) 1 (0–4) 3 (0–6) 0.02 For patients with initial VF ACLS advanced cardiac life support, CPR cardiopulmonary resuscitation, COPD chronic obstructive pulmonary disease, ECPR extracorporeal cardiopulmonary resuscitation, ECLS extracorporeal life support, EMS emergency medical service, ICD implantable cardioverter defibrillator, PEA pulseless electrical activity, ROSC return of spontaneous circulation, VF ventricular fibrillation without prehospital ROSC treated with ACLS only died during initial CPR or the first hours after hospi- tal admission (Table 2), they received less target thera- peutic hypothermia (TTM) (28.6% vs. Baseline characteristics 97.8% vs. 94%, p < 0.001) and coronary angiography (CAG) (40% vs. 97.8% vs. 94%, p < 0.001) than patients treated with ECPR and patients with prehospital ROSC. Accord- ingly, patients without ROSC treated with ACLS patients without ROSC treated with ACLS only as well as the ECPR group had much longer CPR times compared to patients with prehospital ROSC (median 66 and 60 vs. 31 min, p < 0.001) (Table 1). Hospitalization characteristics, procedures, and cause of death Admission characteristics and in-hospital interven- tions are described in Table 2. As most of the patients Rob et al. Hospitalization characteristics, procedures, and cause of death Critical Care (2022) 26:330 Page 5 of 9 Table 2 Hospitalization characteristics, procedures and cause of death Table 2 Hospitalization characteristics, procedures and cause of death Highlighted in bold are the values which are statistically significant (less than 0.05) ECLS therapy indicated during hospitalization for arrhythmic storm with cardiogenic shock ECLS therapy indicated for refractory OHCA (ECPR) *Bleeding complications were assessed based on Thrombolysis in Myocardial Infarction classification under “major” category, defined as any intracranial hemorrhage (excluding microhemorrhages < 10 mm), fatal bleeding directly resulting in death within 7 days or overt bleeding associated with a decrease in hemoglobin concentration of 5 g/dL or a 15% absolute decrease in hematocrit ACLS advanced cardiac life support, ECLS extracorporeal life support, ECPR extracorporeal cardiopulmonary resuscitation, ICU intensive care unit, MODS multiple organ dysfunction syndrome, NA not applicable, PCI percutaneous coronary intervention, ROSC return of spontaneous circulation, TTM target therapeutic management, UNK unknown, WLST withdrawal of life-sustaining therapy Parameter Prehospital ROSC (n = 83) No ROSC and ACLS (n = 81) No ROSC and ECPR (n = 92) P value Admitted to the hospital 83 (100%) 35 (43.2%) 92 (100%) < 0.001 Achieved ROSC 83 (100%) 9 (11.1%) NA 0.002 Laboratory on admission pH 7.13 (7–7.19) 6.85 (6.75–6.91) 6.86 (6.75–6.98) < 0.001 Lactate (mmol/L) 8.2 (6.2–11.5) 13.6 (11.1–17.5) 13.7 (10.95–17.0) < 0.001 ECLS therapy 1 (1.2%) 0 92 (100%) < 0.001 TTM used 78 (94%) 10 (28.6%) 90 (97.8%) < 0.001 Coronary angiography 78 (94%) 14 (40%) 89 (97.8%) < 0.001 PCI 37 (47.4%) 4 (28.6%) 51 (57.3%) 0.1 Successful 31 (83.8%) 2 (50%) 47 (92.2%) 0.04 Unsuccessful 6 (16.2%) 2 (50%) 4 (7.8%) Cause of death Refractory arrest 1 (2.9%) 72 (90%) 7 (9.9%) < 0.001 Brain death 9 (26.5%) 2 (2.5%) 19 (26.8%) MODS 17 (50%) 4 (5%) 31 (43.7%) Cardiogenic shock 3 (8.8%) 1 (1.3%) 10 (14.1%) UNK 4 (11.8%) 0 (0%) 1 (1.4%) Bleeding 0 (0%) 1 (1.3%) 3 (4.2%) WLST 13 (15.7%) 2 (2.5%) 20 (21.7%) < 0.001 Complications Bleeding—any* 5 (6.1%) 1 (8.3%) 40 (44%) < 0.001 Fatal 0 (0%) 1 (100%) 3 (7.5%) 0.03 Intracranial 1 (20%) 0 (0%) 9 (22%) Overt 4 (80%) 0 (0%) 28 (70%) Organ lacerations 2 (2.7%) 2 (3.3%) 3 (3.6%) 0.95 Technical 0 (0%) 0 (0%) 3 (3.3%) 0.07 Length of ICU stay (days) Survivors 11 (8–15) 5 (5–5) 16 (11–29) 0.007 Deceased 6 (2–9.5) 1 (1–1) 3 (2–8) < 0.001 Highlighted in bold are the values which are statistically significant (less than 0.05) ACLS advanced cardiac life support, ECLS extracorporeal life support, ECPR extracorporeal cardiopulmonary resuscitation, ICU intensive care unit, MODS multiple organ dysfunction syndrome, NA not applicable, PCI percutaneous coronary intervention, ROSC return of spontaneous circulation, TTM target therapeutic management, UNK unknown, WLST withdrawal of life-sustaining therapy Survival at 180 daysh only died mostly due to refractory cardiac arrest and patients treated with ECPR died primarily due to multiorgan dysfunction syndrome and brain death (Table 2). Only one patient in the prehospital ROSC group received ECLS for an arrhythmic storm with cardiogenic shock during hospitalization (Table 2). Further, patients treated with ECPR had a significantly higher rate of bleeding complications and a longer stay in the intensive care unit compared to others (Table 2). only died mostly due to refractory cardiac arrest and patients treated with ECPR died primarily due to multiorgan dysfunction syndrome and brain death (Table 2). Only one patient in the prehospital ROSC group received ECLS for an arrhythmic storm with cardiogenic shock during hospitalization (Table 2). Further, patients treated with ECPR had a significantly higher rate of bleeding complications and a longer stay in the intensive care unit compared to others (Table 2). The overall 180-day survival was 1/81 (1.2%) in patients without prehospital ROSC treated with ACLS only com- pared to 22/92 (23.9%) in patients without prehospital ROSC treated with ECPR and 51/83 (61.5%) in patients with prehospital ROSC (log-rank p < 0.001) (Fig. 2). Cox proportional hazards model of 180‑day survival After adjusting for the most important covariates in the Cox proportional hazards model for all 256 enrolled Rob et al. Critical Care (2022) 26:330 Page 6 of 9 Fig. 2 Kaplan–Meier survival curve in the study according to ROSC and ECPR status of 180-day survival in the study (HR 0.10, CI 0.06–0.16, p < 0.001). In addition, shockable initial rhythm, younger age and shorter time of resuscitation were all signifi- cantly associated with better 180-day survival (Table 3). ACLS advanced cardiac life support, CPC cerebral performance category, ECPR extracorporeal cardiopulmonary resuscitation, PEA pulseless electrical activity, ROSC return of spontaneous circulation, VF ventricular fibrillation Neurological outcome at 180 days Favorable neurological outcome of CPC 1 or 2 at 180 days was achieved in 1/81 (1.2%) in patients without pre- hospital ROSC treated with ACLS only, 20/92 (21.7%) in patients treated with ECPR and 47/83 (56.6%) in patients with prehospital ROSC (p < 0.001) (Table 4). Patients with an initial shockable rhythm had a better neurological out- come compared to patients with non-shockable rhythms in the prehospital ROSC (69.8% vs 15%) and ECPR group (33.3% vs 2.9%) (Table 4). Only 2/22 (9.1%) survivors in the ECPR group and 4/51 survivors (7.8%) in the prehos- pital ROSC group had poor neurological outcome (CPC 3 or 4) at 180 days (Table 4). Fig. 2 Kaplan–Meier survival curve in the study according to ROSC and ECPR status Table 3 The Cox proportional hazards model for 180-day mortality Table 3 The Cox proportional hazards model for 180-day mortality Highlighted in bold are the values which are statistically significant (less than 0.05) CPR cardiopulmonary resuscitation, CI confidence interval, ECPR extracorporeal cardiopulmonary resuscitation, EMS emergency medical service, ROSC return of spontaneous circulation, PCI percutaneous coronary intervention, PEA pulseless electrical activity Factor Hazard ratio 95% CI P value Sex (female) 0.89 0.6–1.3 0.55 Age (per year) 1.02 1.01–1.03 0.008 Initial rhythm (PEA/Asystole) 2.19 1.59–3.0 < 0.001 Prehospital ROSC (yes) 0.10 0.06–0.16 < 0.001 Collapse to EMS arrival (per minute) 1.02 0.99–1.05 0.22 CPR time (per minute) 1.01 1.01–1.02 < 0.001 Place of cardiac arrest (public) 1.01 0.72–1.42 0.95 Successful PCI (yes) 0.77 0.52–1.12 0.18 ECPR (yes) 0.21 0.14–0.31 < 0.001 Factor Hazard ratio 95% CI P value Highlighted in bold are the values which are statistically significant (less than 0.05) S advanced cardiac life support, CPC cerebral performance category, ECPR extracorporeal cardiopulmonary resuscitation, PEA pulseless el urn of spontaneous circulation, VF ventricular fibrillation Discussion The most rel- evant finding regarding the intra-arrest transport timing is derived from an observational study suggesting that ECPR should be considered between 8 and 24 min of professional on-scene resuscitation, with 16 min balanc- ing the risks and benefits of early and later transport [19]. Prague OHCA was the first r-OHCA trial randomizing patients prehospitally during ongoing CPR in the field [8]. Patients were randomized on average after 25 min of ongoing OHCA including 15 min of ACLS, reflect- ing a truly refractory cardiac arrest [8]. Despite that, almost one-third of enrolled patients in the invasive arm still achieved sustained ROSC prehospitally, en route or immediately after admission, most of them having initial shockable rhythm. This highlights the need of continu- ous high-quality ACLS during transport to cardiac arrest center and also the urgency of further research in this area as the key question (whether conventional CPR non- responders and candidates for ECPR can be identified early during CPR) remains unanswered. Moreover, our results confirmed that patients with- out prehospital ROSC have very low chances to survive even with prolonged (median time 66 min) conventional ACLS without ECPR which is in line with previous find- ings [3, 4, 7, 13]. Based on the current evidence from observational study [15] as well as the randomized trials [7, 8], it is obvious that the subgroup of patients with an initial shockable rhythm and prolonged CPR over 45 min benefit most from the ECPR approach [8]. However, it is important to underline that ECPR must be consid- ered early and provided in a well-established system with close cooperation between EMS and ECPR cardiac arrest center to achieve good outcomes [7, 8] as survival rates lower than 4% were reported in patients transported without field ROSC from observational studies [2, 3]. i Further, almost all randomized patients in both pro- spective ECPR studies [7, 8] had witnessed arrest with high rate of bystander CPR which is another important prerequisite for good outcomes. Moreover, only 6% of all OHCA patients were enrolled in the Prague OHCA trial which is in line with previous reports [21, 22]. This confirms that ECPR is not a substitute for conventional ACLS but rather complementary method for properly selected refractory OHCA patients provided in the well- organized system [7, 8]. Discussion Continuous efforts to achieve maximum rates of bystander CPR are extremely impor- tant as these are associated with favorable long-term outcomes and may also increase the pool of patients con- sidered for ECPR [7, 8, 23]. In addition, the current analysis confirms high rates of bleeding complications associated with invasive approach and ECPR [2]. Bleeding is an important limita- tion of ECLS therapy in all indications, especially among ECPR patients who underwent prolonged resuscitation attempts. Despite the substantial rates of bleeding, these complications were the leading cause of death in a small proportion of ECPR patients in our study (4.2%). The overall 180-day survival rate for patients treated with ECPR was 23.9% in this study, which is compa- rable to prior observational studies reporting survival rates from 12 to 33% [11–15]. It is lower compared to the results of the ARREST trial where 6 out of 14 patients survived (43%) as this study included patients with an initial shockable rhythm only [7]. Nonetheless, survival rate of patients with an initial shockable rhythm in the invasive arm of the Prague OHCA study was actually 48.6% [8] corresponding to the ARREST trial. The Prague OHCA trial also provided randomized data confirming a vast difference in r-OHCA outcomes between patients with initial shockable and non-shockable rhythms [8]. Results of this secondary analysis further confirm poor outcomes of non-shockable rhythms despite ECPR treatment. These findings are supporting current clini- cal practice in many systems which limit ECPR ser- vice to patients with an initial shockable rhythm [7, 15]. Neurological outcome results at 180 days in this study revealed that majority of survivors had good neurological outcome (mainly CPC 1), and only few patients treated with ECPR survived 180 days with a poor neurological outcome, similarly to patients treated conventionally. However, brain death was the third most common cause of death in the study, and irreversible brain damage is a major barrier to achieve better outcomes in refractory OHCA [24]. Further, data regarding long-term outcomes and quality of life in ECPR survivors are scarce [25] and more information is needed. Discussion In this secondary analysis of the randomized refractory OHCA trial, ECPR increased both 180-day survival and favorable neurological outcome in patients without pre- hospital ROSC compared to patients treated with pro- longed conventional ACLS only. In a multivariate Cox regression analysis, the use of ECPR was significantly associated with 180-day survival. This result is further supporting ECPR as an increasingly used method for r-OHCA and is consistent with previously published observational studies as well as the one randomized trial [7, 11–15]. Although proper selection of patients who will ben- efit from ECPR is essential, to date there is no consensus about the criteria for starting intra-arrest transport and implementing ECPR [5, 6]. In addition, significant dif- ferences in ECPR protocols between cardiac arrest cent- ers exist [5, 7, 8] and currently published data regarding patients (Table 3), ECPR was associated with a lower risk of 180-day death (HR 0.21, CI 0.14–0.31, p < 0.001). Fur- ther, prehospital ROSC status was the strongest factor Table 4 Neurological outcome at 180 days according to the groups and initial rhythms Highlighted in bold are the values which are statistically significant (less than 0.05) Parameter Prehospital ROSC (n = 83) No ROSC and ACLS (n = 81) No ROSC and ECPR (n = 92) P value Good neurological Outcome CPC 1 + 2 47 (56.6%) 1 (1.2%) 20 (21.7%) < 0.001 Initial VF 44/63 (69.8%) 0/36 (0%) 19/57 (33.3%) < 0.001 Initial PEA/Asystole 3/20 (15%) 1/45 (2%) 1/35 (2.9%) 0.07 CPC of 180-day survivors CPC 1 44 (86.3%) 1 (100%) 18 (81.8%) 0.91 CPC 2 3 (5.9%) 0 2 (9.1%) CPC 3 2 (3.9%) 0 0 CPC 4 2 (3.9%) 0 2 (9.1%) Table 4 Neurological outcome at 180 days according to the groups and initial rhythms Rob et al. Critical Care (2022) 26:330 Rob et al. Critical Care (2022) 26:330 Page 7 of 9 Page 7 of 9 predictors of survival in r-OHCA were based on evidence from observational studies only [17, 18]. The results of multivariate analysis in this study indicate that pre- hospital ROSC, shockable initial rhythm, shorter time of resuscitation as well as younger age are all positively associated with 180-day survival in r-OHCA confirm- ing findings from observational studies and systematic reviews [15–19]. Discussion However, further research is needed to achieve consensus regarding optimal ECPR strategy as excluding certain subgroup of patients without suffi- cient data may inappropriately limit patient care [20]. The same is true for ECPR timing as too early transport may decrease chances of achieving prehospital ROSC [4, 19], a major determinant of survival, but later transport and longer low flow time are associated with decreased sur- vival despite ECLS implantation [15, 19]. The most rel- evant finding regarding the intra-arrest transport timing is derived from an observational study suggesting that ECPR should be considered between 8 and 24 min of professional on-scene resuscitation, with 16 min balanc- ing the risks and benefits of early and later transport [19]. Prague OHCA was the first r-OHCA trial randomizing patients prehospitally during ongoing CPR in the field [8]. Patients were randomized on average after 25 min of ongoing OHCA including 15 min of ACLS, reflect- ing a truly refractory cardiac arrest [8]. Despite that, almost one-third of enrolled patients in the invasive arm still achieved sustained ROSC prehospitally, en route or immediately after admission, most of them having initial shockable rhythm. This highlights the need of continu- ous high-quality ACLS during transport to cardiac arrest center and also the urgency of further research in this area as the key question (whether conventional CPR non- responders and candidates for ECPR can be identified early during CPR) remains unanswered. predictors of survival in r-OHCA were based on evidence from observational studies only [17, 18]. The results of multivariate analysis in this study indicate that pre- hospital ROSC, shockable initial rhythm, shorter time of resuscitation as well as younger age are all positively associated with 180-day survival in r-OHCA confirm- ing findings from observational studies and systematic reviews [15–19]. However, further research is needed to achieve consensus regarding optimal ECPR strategy as excluding certain subgroup of patients without suffi- cient data may inappropriately limit patient care [20]. The same is true for ECPR timing as too early transport may decrease chances of achieving prehospital ROSC [4, 19], a major determinant of survival, but later transport and longer low flow time are associated with decreased sur- vival despite ECLS implantation [15, 19]. Funding Th d This study was supported by MH CZ-DRO-VFN64165, VFN: General University Hospital in Prague and the Charles University Research program “Cooperatio – Intensive Care Medicine.” 9. Belohlavek J, Kucera K, Jarkovsky J, et al. Hyperinvasive approach to out- of hospital cardiac arrest using mechanical chest compression device, prehospital intraarrest cooling, extracorporeal life support and early invasive assessment compared to standard of care. A randomized parallel groups comparative study proposal.“ Prague OHCA study.” J Transl Med. 2012;10:1–13. Limitationsh The present study has several limitations. First, this was a secondary analysis of the randomized trial and despite Rob et al. Critical Care (2022) 26:330 Page 8 of 9 Page 8 of 9 Page 8 of 9 Ethics approval and consent to participate pp p p The original study as well as secondary analyses was approved by the Insti- tutional Review Board of the General University Hospital and First Faculty of Medicine, Charles University in Prague (192/11 S-IV). 13. Haneya A, Philipp A, Diez C, et al. A 5-year experience with cardiopulmo- nary resuscitation using extracorporeal life support in non-postcardiot- omy patients with cardiac arrest. Resuscitation. 2012;83:1331–7. 14. Maekawa K, Tanno K, Hase M, Mori K, Asai Y. Extracorporeal cardiopul- monary resuscitation for patients with out-of-hospital cardiac arrest of cardiac origin: a propensity-matched study and predictor analysis. Crit Care Med. 2013;41:1186–96. Abbreviations C S d d 4. Grunau B, Kime N, Leroux B, et al. Association of intra-arrest trans- port vs continued on-scene resuscitation with survival to hospital discharge among patients with out-of-hospital cardiac arrest. JAMA. 2020;324:1058–67. 4. Grunau B, Kime N, Leroux B, et al. Association of intra-arrest trans- port vs continued on-scene resuscitation with survival to hospital discharge among patients with out-of-hospital cardiac arrest. JAMA. 2020;324:1058–67. ACLS: Advanced cardiac life support; CAG: Coronary angiography; CPC: Cerebral performance category; CPR: Cardiopulmonary resuscitation; ECPR: Extracorporeal cardiopulmonary resuscitation; ECLS: Extracorporeal life sup- port; EMS: Emergency medical service; OHCA: Out-of-hospital cardiac arrest; PCI: Percutaneous coronary intervention; r-OHCA: Refractory out-of-hospital cardiac arrest; ROSC: Return of spontaneous circulation; TTM: Targeted tem- perature management. 5. Panchal AR, Bartos JA, Cabañas JG, et al. Part 3: adult basic and advanced life support: 2020 American Heart Association guidelines for cardiopul- monary resuscitation and emergency cardiovascular care. Circulation. 2020;142(16_Suppl_2):S366–468. 5. Panchal AR, Bartos JA, Cabañas JG, et al. Part 3: adult basic and advanced life support: 2020 American Heart Association guidelines for cardiopul- monary resuscitation and emergency cardiovascular care. Circulation. 2020;142(16_Suppl_2):S366–468. 6. Lott C, Truhlář A, Alfonzo A, ERC Special Circumstances Writing Group Collaborators, et al. European Resuscitation Council Guidelines 2021: Cardiac arrest in special circumstances. Resuscitation. 2021;161:152–219. 6. Lott C, Truhlář A, Alfonzo A, ERC Special Circumstances Writing Group Collaborators, et al. European Resuscitation Council Guidelines 2021: Cardiac arrest in special circumstances. Resuscitation. 2021;161:152–219. Availability of data and materials The datasets used and analyzed during the current study are available from the corresponding author on reasonable request. 10. Cox DR. Regression models and life-tables. J Roy Stat Soc: Ser B (Meth- odol). 1972;34:187–202. 11. Nagao K, Kikushima K, Watanabe K, et al. Early induction of hypothermia during cardiac arrest improves neurological outcomes in patients with out-of-hospital cardiac arrest who undergo emergency cardiopulmonary bypass and percutaneous coronary intervention. Circ J. 2010;74:77–85. Conclusions In this secondary analysis of the randomized r-OHCA trial, ECPR was associated with improved 180-day sur- vival in patients without prehospital ROSC. Initial shockable rhythm, younger age and shorter time of resus- citation were all associated with better 180-day survival in r-OHCA. Majority of r-OHCA survivors treated by ECPR had good neurological outcome at 180 days. Acknowledgements The authors express their gratitude to the Prague Emergency Medical Service teams and the coronary care unit, and the catheterization laboratory teams of the 2nd Department of Internal Medicine, Cardiovascular Medicine, General University Hospital in Prague, for their efforts in providing quality care. 7. Yannopoulos D, Bartos J, Raveendran G, et al. Advanced reperfusion strategies for patients with out-of-hospital cardiac arrest and refractory ventricular fibrillation (ARREST): a phase 2, single centre, open-label, randomised controlled trial. The Lancet. 2020;396:1807–16. References 1. Gräsner JT, Herlitz J, Tjelmeland IB, et al. European Resuscitation Council Guidelines 2021: epidemiology of cardiac arrest in Europe. Resuscitation. 2021;161:61–79. 1. Gräsner JT, Herlitz J, Tjelmeland IB, et al. European Resuscitation Council Guidelines 2021: epidemiology of cardiac arrest in Europe. Resuscitation. 2021;161:61–79. 2. Wampler DA, Collett L, Manifold CA, et al. Cardiac arrest survival is rare without prehospital return of spontaneous circulation. Prehosp Emerg Care. 2012;16:451–5. 3. Drennan IR, Lin S, Sidalak DE, et al. Survival rates in out-of-hospital cardiac arrest patients transported without prehospital return of spontaneous cir- culation: an observational cohort study. Resuscitation. 2014;85:1488–93. 3. Drennan IR, Lin S, Sidalak DE, et al. Survival rates in out-of-hospital cardiac arrest patients transported without prehospital return of spontaneous cir- culation: an observational cohort study. Resuscitation. 2014;85:1488–93. Author contributions 8. Belohlavek J, Smalcova J, Rob D, et al. Effect of Intra-arrest transport, extracorporeal cardiopulmonary resuscitation, and immediate invasive assessment and treatment on functional neurologic outcome in refrac- tory out-of-hospital cardiac arrest: a randomized clinical trial. JAMA. 2022;327:737–47. All authors of this manuscript fulfill the authorship criteria. All authors read and approved the final manuscript. Competing interests h d h The corresponding author (JB) has received lecture honoraria from Maquet Company, Czech Republic. Remaining authors report no conflict of interests. 12. Sakamoto T, Morimura N, Nagao K, SAVE-J Study Group, et al. Extracorpor- eal cardiopulmonary resuscitation versus conventional cardiopulmonary resuscitation in adults with out-of-hospital cardiac arrest: a prospective observational study. Resuscitation. 2014;85:762–8. 10. Cox DR. Regression models and life-tables. J Roy Stat Soc: Ser B (Meth- odol). 1972;34:187–202. Author details 1 adjusting for covariates in multivariate analysis, there might have been other uncontrolled confounding vari- ables influencing the results. Second, this is a single‐ center study with limited enrollment. Third, these are the results of tertiary cardiac arrest center with considerable ECPR experience located in the urban area and the study included selected refractory OHCA population which limits the generalizability of our results. 1 2nd Department of Medicine – Department of Cardiovascular Medicine, First Faculty of Medicine, Charles University and General University Hospital in Prague, U Nemocnice 2, 128 00 Prague, Czech Republic. 2 Czech Institute of Informatics, Robotics and Cybernetics (CIIRC), Czech Technical University in Prague, Prague, Czech Republic. 3 Department of Probability and Mathemat- ical Statistics, Faculty of Mathematics and Physics, Charles University in Prague, Prague, Czech Republic. 4 Emergency Medical Service Prague, Prague, Czech Republic. Received: 13 September 2022 Accepted: 7 October 2022 Rob et al. Critical Care (2022) 26:330 Rob et al. Critical Care (2022) 26:330 Rob et al. Critical Care (2022) 26:330 15. Bartos JA, Grunau B, Carlson C, et al. Improved survival with extracor- poreal cardiopulmonary resuscitation despite progressive metabolic derangement associated with prolonged resuscitation. Circulation. 2020;141:877–86. 16. Wang J, Ma Q, Zhang H, Liu S, Zheng Y. Predictors of survival and neuro- logic outcome for adults with extracorporeal cardiopulmonary resuscita- tion: a systemic review and meta-analysis. Medicine. 2018;97:48. 17. D’Arrigo S, Cacciola S, Dennis M, et al. Predictors of favourable outcome after in-hospital cardiac arrest treated with extracorporeal cardiopulmo- nary resuscitation: a systematic review and meta-analysis. Resuscitation. 2017;121:62–70. 18. Goto T, Morita S, Kitamura T, et al. Impact of extracorporeal cardiopulmo- nary resuscitation on outcomes of elderly patients who had out-of- hospital cardiac arrests: a single-centre retrospective analysis. BMJ Open. 2018;8:e019811. 19. Grunau B, Reynolds J, Scheuermeyer F, et al. Relationship between time- to-ROSC and survival in out-of-hospital cardiac arrest ECPR candidates: when is the best time to consider transport to hospital? Prehosp Emerg Care. 2016;20:615–22. 20. Mørk SR, Stengaard C, Linde L, et al. Mechanical circulatory support for refractory out-of-hospital cardiac arrest: a Danish nationwide multicenter study. Crit Care. 2021;25:1–13. 21. Poppe M, Weiser C, Holzer M, et al. The incidence of “load&go” out-of-hos- pital cardiac arrest candidates for emergency department utilization of emergency extracorporeal life support: a one-year review. Resuscitation. 2015;91:131–6. 22. Alm-Kruse K, Sørensen G, Osbakk SA, et al. Outcome in refractory out- of-hospital cardiac arrest before and after implementation of an ECPR protocol. Resuscitation. 2021;162:35–42. 23. Kragholm K, Wissenberg M, Mortensen RN, Hansen SM, Malta Hansen C, Thorsteinsson K, Rasmussen BS. Bystander efforts and 1-year outcomes in out-of-hospital cardiac arrest. N Engl J Med. 2017;376(18):1737–47. 24. Migdady I, Rice C, Deshpande A, et al. Brain injury and neurologic outcome in patients undergoing extracorporeal cardiopulmonary resuscitation: a systematic review and meta-analysis. Crit Care Med. 2020;48:e611–9. 25. Spangenberg T, Schewel J, Dreher A, et al. Health related quality of life after extracorporeal cardiopulmonary resuscitation in refractory cardiac arrest. Resuscitation. 2018;127:73–8. Consent for publication All patients who regained normal neurological function were asked to provide their written consent regarding the use of their data. Consent requirements were waived for patients who died at the scene and never reached the hospi- tal and for participants without known legal representatives. Page 9 of 9 Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. 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https://openalex.org/W2759305342	http://jssidoi.org/jesi/article/download/133	English	null	The impact of financial management on innovation	Entrepreneurship and sustainability issues	2,017	cc-by	8,948	Additional disciplines ecology and environment; finance Additional disciplines ecology and environment; finance Marcus Illmeyer1, Dietmar Grosch2, Maria Kittler3, Pamela Priess 4 1,2,3,4 Рan-European University, Faculty of Economics and Business, Tematínska 10, 851 05, Bratislava, Slovakia -mails: 1marcus.illmeyer@gmx.at; 2 dietmar.grosch@gmx.net; 3mariakittler@yahoo.de; 4office@p Received 15 March 2017; accepted 14 August 2017 Abstract. In the current competitive market, innovation has become a crucial element for organizations, willing to grow. Financial management in this regard is playing a significant role in improving firms’ innovation capacity. This research paper evaluates the impact of financial management components on innovativeness of Austrian SMEs. Using data of 118 employees from 41 SMEs operating in Austria, the research finds a significant impact of the financial management model on firms’ innovation. The three components: liquidity, controlling, and financial literacy are statistically significant is explaining innovativeness at 1% level. The study suggests focus on the three financial management constructs in order to improve their innovation capability and capacity. Keywords: Financial Management, Financial Controlling, Liquidity, Financial Literacy, Innovativeness, Innovation Capacity Reference to this paper should be made as follows: Illmeyer, M.; Grosch, D.; Kittler, M.; Pamela Priess. P. 2017. The impact of financial management on innovation, Entrepreneurship and Sustainability Issues 5(1): 58-71. http://doi.org/10.9770/jesi.2017.5.1(5) Reference to this paper should be made as follows: Illmeyer, M.; Grosch, D.; Kittler, M.; Pamela Priess. P. 2017. The impact of financial management on innovation, Entrepreneurship and Sustainability Issues 5(1): 58-71. http://doi.org/10.9770/jesi.2017.5.1(5) JEL Classifications: M11, M160, G200, O10 The International Journal ENTREPRENEURSHIP AND SUSTAINABILITY ISSUES ISSN 2345-0282 (online) http://jssidoi.org/jesi/ 2017 Volume 5 Number 1 (September) http://doi.org/10.9770/jesi.2017.5.1(5) Publisher THE IMPACT OF FINANCIAL MANAGEMENT ON INNOVATION Marcus Illmeyer1, Dietmar Grosch2, Maria Kittler3, Pamela Priess 4 1,2,3,4 Рan-European University, Faculty of Economics and Business, Tematínska 10, 851 05, Bratislava, Slovakia E-mails: 1marcus.illmeyer@gmx.at; 2 dietmar.grosch@gmx.net; 3mariakittler@yahoo.de; 4office@priessreal.at R i d 15 M h 2017 t d 14 A t 2017 The International Journal ENTREPRENEURSHIP AND SUSTAINABILITY ISSUES ISSN 2345-0282 (online) http://jssidoi.org/jesi/ 2017 Volume 5 Number 1 (September) http://doi.org/10.9770/jesi.2017.5.1(5) Publisher THE IMPACT OF FINANCIAL MANAGEMENT ON INNOVATION Marcus Illmeyer1, Dietmar Grosch2, Maria Kittler3, Pamela Priess 4 1,2,3,4 Рan-European University, Faculty of Economics and Business, Tematínska 10, 851 05, Bratislava, Slovakia E-mails: 1marcus.illmeyer@gmx.at; 2 dietmar.grosch@gmx.net; 3mariakittler@yahoo.de; 4office@priessreal.at R i d 15 M h 2017 t d 14 A t 2017 The International Journal ENTREPRENEURSHIP AND SUSTAINABILITY ISSUES ISSN 2345-0282 (online) http://jssidoi.org/jesi/ 2017 Volume 5 Number 1 (September) http://doi.org/10.9770/jesi.2017.5.1(5) Publisher The International Journal ENTREPRENEURSHIP AND SUSTAINABILITY ISSUES ISSN 2345-0282 (online) http://jssidoi.org/jesi/ 2017 Volume 5 Number 1 (September) http://doi.org/10.9770/jesi.2017.5.1(5) Publisher 1. Introduction Organisations are anticipated to act promptly and appropriately towards the high market pressures to meet the customers' changing demands due to the increasing competition in the dynamically growing market. In this regard, the basic but the most critical aspect is of adequate financial controlling, in terms of planning the finances and managing the liquidity aspects as well (Kozubikova, Homolka, & Kristalas, 2017; Belas & Sopková, 2016; Ključnikov, Kozubíková, & Sopková, 2017). Meanwhile, it has also been contended that the organisations these days are more concerned towards turnover and profit margins, rather than keeping the financial aims and liquidity as the prime objectives (Upadhaya, Munir, & Blount, 2014; Frame, & White, 2014). Besides, there is another notable aspect of the increasingly competitive market that demands the organisations to be considerate enough, in order to gain the maximum level of competitive advantage within the respective industry (Hristov, & Reynolds, 2015). Innovation management is the most crucial element that has acquired potential magnitude in an integrated manner with the management of finances if the firm intends to be a leading entity even in the incessantly increasing competitive marketplace (Hausman, & Johnston, 2014; Schrage, 2013; Laužikas et al., 2017). 58 The International Journal ENTREPRENEURSHIP AND SUSTAINABILITY ISSUES ISSN 2345-0282 (online) http://jssidoi.org/jesi/ 2017 Volume 5 Number 1 (September) http://doi.org/10.9770/jesi.2017.5.1(5) The International Journal ENTREPRENEURSHIP AND SUSTAINABILITY ISSUES ISSN 2345-0282 (online) http://jssidoi.org/jesi/ 2017 Volume 5 Number 1 (September) http://doi.org/10.9770/jesi.2017.5.1(5) With respect to the element of managing the innovation within an organisational framework, it has been stated that these innovations are employed to mitigate the likely adversities within the technical areas. If managed appropriately, the resulting outcomes are ultimately beneficial both in terms of economic and ecological achievements. Considering the increasing trend of technological integrations across the business domain, the implications of innovation management are undeniable. However, financing of these innovations has been a challenging situation for the business entities, regardless of being a potential driver of firms' competitive advantage in the long run (Botric, & Bozic, 2017). Accordingly, this particular element of managing the finances of the organisations with respect to the innovations' deployment has been a debatable issue of the current literature. Primarily, the reason of financing constraints towards integration innovations has been the uncertainty prevailing across the domain towards the asymmetric information regarding the innovation activities (Hall & Lerner, 2010; Botric, & Bozic, 2017). 1. Introduction In order to sustain competitiveness in the market, the firms must be continuous in innovating their business processes, which requires the firms to manage sustained investment in both the tangible and intangible aspects of business innovations (Fang, Tian, & Tice, 2014). Lee, Sameen and Cowling (2015) have documented that mainly Small and Medium-sized Enterprises (SMEs) or Entrepreneurial start-ups having potential innovation opportunities face financial constraints that eventually limits the adoption of technological innovations. Sufficient financial resources are essential for the successful exploitation of innovatively diverging growth patterns, particularly in terms of advancing the Research & Development (henceforth; R & D) areas. In this regard, it has been established that the option of external capital (preferably stock market) is significantly favourable for such firms since it tends to reduce the asymmetry in the information regarding the innovation activities (Agénor, Canuto, & Jelenic, 2014; Agénor, & Canuto, 2017). However, multiple other perspectives regarding managing the financials and liquidity of the organization in relation to the deployment of innovations have also been presented in the literature; yet, the studies are observed to be deficient in facilitating credible outcomes. Problems have been there due to the inefficient identification of valued patterns in the data, along with the use of inappropriate controls, and sampling inadequacies as the main reasons. Consequently, this particular study intends to fill the typical gap in the literature, by means of being vigilant in adopting the research method and all other associated elements of the research design. Besides, it has been recognized at first that all the competitive outcomes of innovation element are second to the assurance of the most proficient financial controls within the organizational activities. It leads to the assertion that innovation is dependent on the finances; thus, financial controlling and management turns out to be significantly critical for the competitiveness of the organisations. 2. Literature Review In the current competitive market, innovation has become a crucial element for organizations, willing to grow. In fact, in some industries like technology, an entity cannot survive without innovation. This is the reason why global giants like Volkswagen, Samsung, Intel, and Microsoft spent a material ratio of their earnings (5.2%, 6.4%, 20.1% & 13.4% respectively) in research and development. Hence the need to bring up new ideas for supporting industry growth through fulfilling the expectation of the consumer is always a concerned factor. It is important to know that no matter how important the innovation element is, it always comes after the primary objectives of business, which are profitability and survival. Therefore, it is crucial to lead the research only if appropriate financial controls are present. Thus, the innovation is in a sense dependent on finance. 2.1. Relationship between Finance and Innovation Thus the financing risk in innovative projects exists, therefore the estimated Net Present Value of any such project may be lower than its actual worth, due to the high-risk element of financing. This risk can be eliminated if the investor commits, to provide more investment until the end of the project, however, in any case, the risk of uncertainty will remain to impact the financial risk in equilibrium. As a result, the investor keeps a close track on his investments, and require regular updates about the potential of the project, possibility of the achievement and time required for completion, in order to preserve his investments and exclude the option of terminating the research (see Bergemann & Hege, 2005). Thus, low financing risk leads to more interest of investors into funding experiments (Nanda & Rhodes- Kropf, 2016). As much important an innovation is for the growth of any business for the purpose of competitive advantage and strategic gain, finding and maintaining finance stream is still a difficult and considerable part. One of the useful external sources of funding is the stock market. Sometimes a firm does not have the ability to conduct a quality research, and other times the capable firm holds itself back due to the unavailability of funds required for experiments. Listed firms can easily access Stock market, to raise funds for investing in their research activities, unlike private entities which do not have any access to the stock exchange, unless under the capacity of a trader. Public listed companies that are externally financed dependent (EFD) are known for being more efficient in their research programs than other private or non-listed companies. However, the internal finance dependent (IFD) types of both public and private companies have no material difference while supporting their research and development divisions. The extent that listed companies can reach to for the development of their operations or their brand name. It is considered as the best option for public companies to go for EFD, and due to unavailability of this option to private investors, the suggestion is to go for other sources, like private investors or more likely for internal development of fund, so the innovation, experiment or research programs continue for the sake of company’s future. 2.1. Relationship between Finance and Innovation p The nature of finance is often dependent on the purpose of funding and nature of the project. Research and development is a risky business and does not always comes with a favourable outcome, there is no certainty of the 59 The International Journal ENTREPRENEURSHIP AND SUSTAINABILITY ISSUES ISSN 2345-0282 (online) http://jssidoi.org/jesi/ 2017 Volume 5 Number 1 (September) http://doi.org/10.9770/jesi.2017.5.1(5) outcome, the collective nature requires finance from a different source, and the cumulativeness indicates that innovation may lead to a merger of different research results (Lazonick and Mazzucato, 2013). All these characters direct us to the facts that Innovation demands finance from various sources, of patient nature and with acceptability of high-risk. Thus, the nature of investment is dependent on the type of finance. From the perspective of investors (specifically private investors) funding research and development is not the best investment (Turner, 2015), due to its’ long-term nature. Instead, they believe that investing in short-term projects, for example, share trading is considered more profitable. This is caused by the modern corporate governance structure that prefers earning short-term returns (Kay, 2012) and reinvesting in companies existing activities or support expansion, plus the uncertainty of achievement is a scary concept for investors. On the contrary, many technological revolutions are resulted through high-risk investment, even though those experiments are more task- based in nature. These sorts of investments are made with the aim of the betterment of some target industrial landscape, for achieving some heights that have never been met before (Mowery, 2010; Foray et al., 2012). Such investments are put in motion to accommodate two kinds of purposes, for meeting the expectations and demand in the market or for developing a new product that will be desired by the market (Climate Policy Initiative, 2013; Mazzucato & Semieniuk, 2017). As mentioned above, most of the research projects are cumulative and require multiple investments throughout the process. Even in the mission-oriented innovation projects, where the objective and the deadline is set, the risk of financing, and so the innovation is high. This is because the investor has to rely on the hope that the funding will keep flowing in as long as the project requires it, but if for any reason, funding is not available at any time, the whole previous investment will probably turn to a waste, with no return. 2.2. Financial Dependence of Innovation Firstly, in comparing public and private companies, it is observed that public firms with dependency on external finance are more sensitive to quality, quantity, and novelty of their patents (for research projects), but ones which rely on internal financial sources are not that concerned about these factors. In fact, when it comes to internal financial sources private firms are better in preserving the researchers than public companies. Hence it can be considered that including outside finance (through public listing), is a good for the innovation factor. This element can further be proved through observing the benefit of listing on the stock exchange, and the ultimate effect of this listing on company’s research and development division. Another important factor is the pressure of investors for early returns. With the excess of the low cost of capital, the public listing also puts the firm under pressure of investors who require the entity to lower the risk approaches and generate returns in short intervals, which is against the nature of innovation (which requires long-term trust and patience) (Stein,1989). Another observed difference is the ability of the firm for carrying its R&D, and affordability of allocating time and money on something other than its core business activity. But the better presentation of patent profile could merely the cause of acquisition of patents outside firm’s own R&D division, these are mostly one of the benefits of mergers, and acquisitions of firms (Bena & Li, 2014; Seru, 2013). A more accurate outcome can be observed through measurement of spending on innovations including the acquisition of patents in comparison to the returns of the entity after the spending. The factors of concern here are the number of patents acquired after a successful innovation, and these must be further divided into categories and planned future usage, the franchising of the patent (to evaluate the actual worth and demand of the developed research in the market), the intensity of these citations vary industry to industry, therefore, it is better to compare each patent with average number of patents yearly issued in relevant industry. Hence a mere count of developments or patents for innovations in a year is not a sufficient measure (Hall et al, 2001; Hall et al, 2005; Acharya & Xu, 2017). 2.1. Relationship between Finance and Innovation There are many ways to evaluate the results, but the most common one is the comparison between cost and benefit of the research that is in the case of achievement of required results. In the case of absence of a proper cost-benefit analysis, a private company may not be able to survive the aftermath of research, 60 The International Journal ENTREPRENEURSHIP AND SUSTAINABILITY ISSUES ISSN 2345-0282 (online) http://jssidoi.org/jesi/ 2017 Volume 5 Number 1 (September) http://doi.org/10.9770/jesi.2017.5.1(5) The International Journal ENTREPRENEURSHIP AND SUSTAINABILITY ISSUES ISSN 2345-0282 (online) http://jssidoi.org/jesi/ 2017 Volume 5 Number 1 (September) http://doi.org/10.9770/jesi.2017.5.1(5) if the expected results are not achieved, a public or large company, on the other hand, can bear the after results and go on to the proceeding operations. if the expected results are not achieved, a public or large company, on the other hand, can bear the after results and go on to the proceeding operations. 2.3. The Role of Financial Management in Innovation g A relevant example of the effect of financial management on innovation industry could be China, which has successfully turned the living style of its locals, increased GDP and has maintained a reputable and leading rank in world's top most business countries and all this was done within a span of few decades. There is more than one reason behind the sudden growth of China, and the incline in financial literacy of local investors is one of the major reasons. China realized that one does not need a 4-year degree to read the profit figures, and in order to operate successfully the person must at least have sufficient knowledge or literacy of the finance, to understand the ups and downs of business and the causes of the volatility. And so the Chinese government provided the tool to its inhabitants leading to the growth in business sectors in various industries. Coming to our main topic, the innovation industry in China before the growth in the financial sector was too low, but with the increasing trend in financing sector and availability of more investors, the innovation industry also followed the rise-up. A detailed analysis, including figure based assessments, are presented in a recent article “Financial Literacy in China as an Innovation Opportunity” (Brejcha, Wang & Zhang, 2016 July). Another example of the rapid growth of innovation industry is of Germany in 1930s and 40s when German government gave its scientist a free hand and unlimited access to finance for conducting researches and innovation products. Thus, it has been proved by many scholars, theorists and researchers that for firms engaged in innovation activities and dependent on external finance to maintain the flow of their operations, effective financial management policies and potential willing financiers are vital elements. Overview of the relationship between Financial Management and Innovation. Author Description Lazonick and Mazzucato Research and Development are risky business and have three critical elements. The uncertainty of outcome, collective and cumulative nature and requirement of the long-term commitment, through patience. Turner From the perspective of investors (specifically private investors) funding research and development is not the best investment, due to its’ long-term nature Kay Modern corporate governance structure, prefer earning short-term returns to keep the up with the market competition. 2.2. Financial Dependence of Innovation Firms in the innovation business are more exposed to the financial implications as compared to the companies which carry R&D in addition to their primary business activities. Therefore, the R&D firms are more at risk and are challenged in seeking external finance to keep fueling the innovation process. The main reason being the majority assets of being intangible natures (Patents), which are too volatile in trading markets and lose value as soon as a more relevant and updates innovation arrives in the market. Possessing intangible assets does not relieve a R&D firm from regular expenses for instance, wages and salaries for the staff, patent application fees, research expenses (if outsources), and maintenance of equipment to keep them on up-to-date with current technologies, and out of all these wages of scientist are the most crucial and therefore the most expensive part. Studies from an early age are intact with the idea that financial management has no adverse effect on innovation activities of the firm, but as the researchers developed more theories, this concept began to revert. In the most current studies, researchers have agreed to the concept of the relationship between financial management and the innovation, and have evident that financial frictions have its adverse effects on innovation (Hall & Lerner, 2010). As Discussed earlier, innovation is not a very attractive sector for short time investors and this hazard further grows due to the fact that the investor may not be able to understand the real value of the income. In other words, only the firm engaged in innovation knows for real, whether the innovation truly serves the purpose or not, hence the idea of being in the dark is not appreciated by the investors. However, this hesitance can be eliminated by building up extra monitoring measures, for timely checks of investments, innovations and the returns in monitory terms, encouraging the investors to further involve in the investing activities. The relationship between financiers and innovation is modeled by many theorist, and researchers, giving their own view for measuring the correlation between the two. 2.2. Financial Dependence of Innovation One of the theories is given in Morales 2003, in which 61 The International Journal ENTREPRENEURSHIP AND SUSTAINABILITY ISSUES ISSN 2345-0282 (online) http://jssidoi.org/jesi/ 2017 Volume 5 Number 1 (September) http://doi.org/10.9770/jesi.2017.5.1(5) the theorist mentioned the monitoring technologies which enable lenders to keep a close eye on n his investment, through regular updates and expected returns, this technique, in addition, allows the financier to force the firm to maintain a fast running cycle, creating outcomes within lesser intervals than they would in absence of his/her investment. (Morales, 2003; Agénor & Canuto, 2017). 3. Methodology The successful execution of a research process demands the collected data to be credible and authentic. It brings in the undeniable importance of selecting the most appropriate method of collecting and analysing the data (Feilzer 2010; Flick 2011). Accordingly, Bryman (2015) has affirmed that needs of being selective towards the research method, as the accomplishment of the study objectives are greatly dependent on the adopted method. In this regard, research approach is identified at first due to its feasibilities of categorising the data collection and analysis methods to be easily selected (Fowler Jr, 2013). Taylor Bogdan & DeVault (2015) have documented three types of research approaches as being Quantitative, Qualitative, and the combination of both as a Mixed research approach. Reviewing both the qualitative and quantitative approaches within the study context, the researcher has selected Quantitative approach as the most preferred one. It has been preferred over the Qualitative approach based on the fact that the study objectives of assessing the relation in between the management of financial aspects of the organisation and the investments in the innovation deployment for being competitive. Recognising the notable gap in the literature, qualitative and thus, mixed research approach has been disregarded. The significance of Quantitative research approach is validated from the study of Creswell (2013), which asserts that this particular approach facilitates the researcher by means of its unique aspect of "cause and effect thinking”. Rovai, Baker, & Ponton, (2014) have affirmed this unique thinking pattern leads to the accurate and logical interpretation of the problem focus. Consequently, the selected approach has been remarkable in helping the attainment of cohesive results pertaining to the relationship in between the identified variables. Subsequently, there comes the selection of research purpose that depicts the strict abidance of the devised aim of the study. The study context could be either a new idea or analysis of the already identified variables within the extensive research scope. Therefore, the purpose of the study needs to be clearly articulated prior to the initiation of the study. Davies & Hughes (2014) have presented three main purposes of the research, as Exploratory, Descriptive, and Explanatory, depending on the study context. This particular study is explanatory in nature since the objectives are related to the assessment of relationship in between the identified variables of financial controlling aspects and the innovation management at the organisational level. 2.3. The Role of Financial Management in Innovation Mowery; Foray et al Investments are made with the aim of the betterment of some target industrial landscape, for achieving some heights that have never been met before. Climate Policy Initiative Investments are put in motion to accommodate two kinds of purposes, for meeting the expectations and demand in the market or for developing a new product that will be desired by the market. Bergemann & Hege The investor keeps a close track on his investments, and require regular updates about the potential of the project, possibility of the achievement and time required for completion, in order to preserve his investments and exclude the option of terminating the research. Nanda & Rhodes-Kropf Low financing risk leads to more interest of investors into funding experiments. Stein With the excess of the low cost of capital, the public listing also puts the firm under pressure of investors who require the entity to lower the risk approaches and generate returns in short intervals. From the perspective of investors (specifically private investors) funding research and development is not the best investment, due to its’ long-term nature Modern corporate governance structure, prefer earning short-term returns to keep the up with the market competition. With the excess of the low cost of capital, the public listing also puts the firm under pressure of investors who require the entity to lower the risk approaches and generate returns in short intervals. 62 The International Journal ENTREPRENEURSHIP AND SUSTAINABILITY ISSUES ISSN 2345-0282 (online) http://jssidoi.org/jesi/ 2017 Volume 5 Number 1 (September) http://doi.org/10.9770/jesi.2017.5.1(5) Bena & Li; Seru A better presentation of patent profile could merely the cause of acquisition of patents outside firm’s own R&D division, these are mostly one of the benefits of mergers and acquisitions of firms. Hall et al; Acharya & Xu The factors of concern are the number of patents acquired after a successful innovation, and these must be further divided into categories and planned future usage, the franchising of the patent. Hall & Lerner In the most current studies, researchers have agreed to the concept of relationship between financial management and the innovation, and have evident that financial frictions have its adverse effects on innovation Morales; Agénor & Canuto The monitoring technologies which enable lenders to keep a close eye on their investment, through regular updates and expected returns. 2.3. The Role of Financial Management in Innovation Brejcha, Wang & Zhang The innovation industry in China before the growth in the financial sector was too low, but with the increasing trend in financing sector and availability of more investors, the innovation industry also followed the rise-up. The innovation industry in China before the growth in the financial sector was too low, but with the increasing trend in financing sector and availability of more investors, the innovation industry also followed the rise-up. 3. Methodology For exploring innovativeness at country level, the study relies on secondary data sources, including World Banks Database and Global Competitiveness Report 2016-17. The distribution of the survey questionnaire incorporates the researchers' credibility of targeting the most appropriate population in this regard. It has been established from the study outcomes of Weiss & Weiss (2012) that the population of the study is also an essential aspect of the entire research plan, which needs to be carried out vigilantly. In the case of targeting incompetent or irrelevant population, the integrity and credibility of the study outcomes are at risk. Therefore, the researcher has been considerate in this regard, by means of targeting the SMEs at Austria on the basis of the fact that the challenges of financial controls in relation to the innovation management are mostly prevalent across the SMEs. Therefore, the employees from accounting and finance departments of 41 SMEs at Austria have been targeted, based on random sampling strategy. Random sampling has facilitated the collection of evidences across different sectors of businesses, along with entailing the prospects of flexibility and accessibility for the researcher as well. Accordingly, the research proceeded with the sample size of 148 employees, from finance and acconting departments, across the targeted SMEs. However, only 125 questionnaires were completely filled, with only 118 to be regarded as valuable and usable with respect to the research objectives. Aarons et al., (2001) defined questionnaire as a documented form of a number of questions related to the study context. The design of the questionnaire could be open-ended or close-ended, based on the nature of the study. In this particular study, the designed survey questionnaire comprised of close-ended questions, demanding the responders to select the respective answers based on five-point Likert scale. It has been selected due to the anticipated collection of the most relevant evidence that is not instilled within the open-ended questionnaires since open-ended questionnaire incorporates the concerns of irrelevant answers that would eventually be challenging towards the expected success of the study. Here it brings an important consideration of validating the quality of the collected data, and the abidance of ethical aspects of valuing the human participation. The participants were provided with a consent form, having clearly articulated objectives of the study to abide by their right-to-information. 3. Methodology In addition to this, another important element is the consideration of research design, for its potential contribution to addressing the research problem (Bryman & Bell 2015). Research design has its multiple forms, including review-based design, meta-analytic design, experimental and semi-experimental design, descriptive design, correlational design and others (Davies & Hughes, 2014; Lampard & Pole, 2015). Depending on the nature of the study objectives, the researcher adopts the most appropriate design of these all on the basis of their unique aspects. The current study has employed 63 The International Journal ENTREPRENEURSHIP AND SUSTAINABILITY ISSUES ISSN 2345-0282 (online) http://jssidoi.org/jesi/ 2017 Volume 5 Number 1 (September) http://doi.org/10.9770/jesi.2017.5.1(5) descriptive and correlational designs for their respective contribution towards the accomplishment of study objectives. The descriptive design has facilitated the analysis of the identified variables in terms of percentages, frequencies, and others. On the other side, the correlational design has assisted in recognising the relationship in between the identified variables under consideration. Even though, the researcher is expected to be considerate towards the selection of the most appropriate approach, purpose and the design of the study, the importance of data sources remains undeniable. It has been asserted based on the fact that the overall authenticity and reliability of the study is reliant over the feasibility of accessing the required data (Matthews & Ross, 2014). The sources of data are of importance since these sources direct the collection of evidence, in order to answer the formulated research questions that eventually lead towards the accomplishment of the study aim and objectives. Two types of data sources are prevalent across the research domain, entailing a distinct level of significance towards a study; primary sources of data and secondary sources of data (Zikmund et al, 2013; Mertens, 2014). In this particular study, the primary source of data has been used to collect the evidence regarding the objectives of analysing the relationship in between the identified variables. Primarily, this data source has its unique aspect of providing first-hand or direct information accessibility that eventually validates its reason of being selected. The study context already observes a notable research gap that reflects the preference of direct sources of data for mitigating this particular gap; thus, cohesive and coherent concluding remarks have been drawn. It is noteworthy to mention here that the primary or the direct data has been collected by means of Five-point Likert Survey Questionnaire. 4.1. Research Objectives The current research paper aims to determine the relationship between financial management and firm innovation. For this purpose, it focuses on small and medium enterprises (SMEs) operating in various business sectors in Austria (see Figure 1). Financial management, in this study, is represented by financial liquidity, financial controlling, and financial literacy. Following are the objectives of the study: 1. To examine the relationship between financial management components and innovativeness of Austrian SMEs. 1. To examine the relationship between financial management components and innovativeness of Austrian SMEs. develop a financial management-innovation model based on the data from Austrian SMEs 2. The develop a financial management-innovation model based on the data from A 2. The develop a financial management-innovation model based on the data from Austrian SMEs 4.3. Research Hypotheses 4.3. Research Hypotheses The statistical relationships between financial management constructs and innovativeness of the Austrian SMEs is based on the following hypotheses: yp The statistical relationships between financial management constructs and innovativeness of the Austrian SMEs is based on the following hypotheses: yp The statistical relationships between financial management constructs and innovativenes based on the following hypotheses: g yp H1: The impact of financial liquidity is significant on innovativeness of the Austrian SMEs. H Th i f fi i l lli i i ifi i i f h A i SME g yp 1: The impact of financial liquidity is significant on innovativeness of the Austrian SMEs. p q y g 2: The impact of financial controlling is significant on innovativeness of the Austrian SME H3: The impact of financial literacy is significant on innovativeness of the Austrian SME 4. Research Objectives and Hypotheses 4. Research Objectives and Hypotheses 4.2. Research Questions The research question of the current research paper is: What is the impact of financial management components on innovativeness of Austrian SMEs? Based on the above question following sub questions are proposed: The research question of the current research paper is: What is the impact of financial management components on innovativeness of Austrian SMEs? q p p What is the impact of financial management components on innovativeness of Austrian SMEs? Based on the above question, following sub-questions are proposed: 1. What is impact of financial liquidity on innovativeness? 2. What is impact of financial literacy on innovativeness? 3. What is impact of financial controlling on innovativeness? 3. Methodology Besides, the participants were also ensured in terms of their right-to-privacy as well, since the consent form included the assurance of confidentiality of their private information. Additionally, the participants were also given the right-to-withdraw, in the case on any concern or problem. After the collection of information through a questionnaire, statistical analysis techniques were employed for generating the results. Among the credible techniques of statistical analysis, the study has employed descriptive After the collection of information through a questionnaire, statistical analysis techniques were employed for generating the results. Among the credible techniques of statistical analysis, the study has employed descriptive statistics, factor analysis, reliability test, and regression analysis. Descriptive analysis techniques highlight the 64 The International Journal ENTREPRENEURSHIP AND SUSTAINABILITY ISSUES ISSN 2345-0282 (online) http://jssidoi.org/jesi/ 2017 Volume 5 Number 1 (September) http://doi.org/10.9770/jesi.2017.5.1(5) The International Journal ENTREPRENEURSHIP AND SUSTAINABILITY ISSUES ISSN 2345-0282 (online) http://jssidoi.org/jesi/ 2017 Volume 5 Number 1 (September) http://doi.org/10.9770/jesi.2017.5.1(5) basic trends or patterns that might foster the forecasting regarding the potential impacts on the study context (Davies & Hughes, 2014). Once the patterns within the collected data have been comprehended, factor analysis and reliability tests have facilitated the researcher in determining the hidden unpredictability within the identified and correlated variables (Kline, 2014). Consequently, regression analysis led to the identification of the relationship in between the dependent and independent variables; thus, fostering the success of the study (Punch & Oancea, 2014). As a result, the overall research process has been conducted in the most vigilant manner, since the credibility, objectivity, and integrity of the study could not be compromised at any cost. 5.1. Innovation in Austria For overall competitiveness, the factor of innovation plays a significant role not only at organizational level but also at the country level. According to Global Competitiveness Report 2016, Austria ranks 11th in the overall innovation and sophistication factors; 8th in business sophistication; and 14th in innovation in the world (Schwab, 2016). Moreover, Austria ranks high in the capacity for innovation (7th), PCT patent applications (11th) and company spending on research and development (19th) (Schwab, 2016). It suggests that the country and its businesses considers the significance of innovation, research and development. 65 The International Journal ENTREPRENEURSHIP AND SUSTAINABILITY ISSUES ISSN 2345-0282 (online) http://jssidoi.org/jesi/ 2017 Volume 5 Number 1 (September) http://doi.org/10.9770/jesi.2017.5.1(5) The International Journal ENTREPRENEURSHIP AND SUSTAINABILITY ISSUES ISSN 2345-0282 (online) http://jssidoi.org/jesi/ 2017 Volume 5 Number 1 (September) http://doi.org/10.9770/jesi.2017.5.1(5) Figure 1. R&D Expenditure Source: World Bank (2017) According to the Figure 1, in Austria, the expenditures for R&D on creative work (for increasing knowledge) as a percentage of GDP is constantly increasing. The R&D expenditure in Austria is also higher than average R&D expenditure in the European Union as % of the GDP. 5.3. Validity and Reliability y y For inferential testing, the researcher ensured validity and examined reliability of the test instrument i.e. the 5- point Likert Scale questionnaire. The validity of the questionnaire is tested through an expert review. Moreover, the validity of the innovativeness construct is ensured by adopting items from previous literature. The internal consistency reliability of the test items is examined via Cronbach’s alpha statistic, which reveal relatively high degree of internal consistency (greater than 0.7) of each construct (liquidity, controlling, financial literacy, and innovativeness). 5.2. Demographic analysis 5.2. Demographic analysis Table 1. Respondents’ business sector Business Sector Frequency Percent Food and Drink 20 16.9 Chemical and Automotive 10 8.5 Mechanical and Steel Engineering 16 13.6 Wood, Pulp and Paper 9 7.6 Financial Services 18 15.3 Electrics and Electronics 9 7.6 Textiles, clothing 15 12.7 Tourism 14 11.9 Others 7 5.9 Total 118 100.0 Source: Authors’ own estimation In the context of the current research study, which examines the impact of financial management on innovation in Austrian SMEs, majority of research participants (16.9%) are working in the Food and Drink sector, followed by Financial services (15.3%), and Mechanical and Steel Engineering sector (13.6%) (Table 1, Fig. 2). 5.2. Demographic analysis Table 1. Respondents’ business sector Business Sector Frequency Percent Food and Drink 20 16.9 Chemical and Automotive 10 8.5 Mechanical and Steel Engineering 16 13.6 Wood, Pulp and Paper 9 7.6 Financial Services 18 15.3 Electrics and Electronics 9 7.6 Textiles, clothing 15 12.7 Tourism 14 11.9 Others 7 5.9 Total 118 100.0 Source: Authors’ own estimation Table 1. Respondents’ business sector Business Sector In the context of the current research study, which examines the impact of financial management on innovation in Austrian SMEs, majority of research participants (16.9%) are working in the Food and Drink sector, followed by Financial services (15.3%), and Mechanical and Steel Engineering sector (13.6%) (Table 1, Fig. 2). In the context of the current research study, which examines the impact of financial management on innovation in Austrian SMEs, majority of research participants (16.9%) are working in the Food and Drink sector, followed by Financial services (15.3%), and Mechanical and Steel Engineering sector (13.6%) (Table 1, Fig. 2). 66 The International Journal ENTREPRENEURSHIP AND SUSTAINABILITY ISSUES ISSN 2345-0282 (online) http://jssidoi.org/jesi/ 2017 Volume 5 Number 1 (September) http://doi.org/10.9770/jesi.2017.5.1(5) The International Journal ENTREPRENEURSHIP AND SUSTAINABILITY ISSUES ISSN 2345-0282 (online) http://jssidoi.org/jesi/ 2017 Volume 5 Number 1 (September) http://doi.org/10.9770/jesi.2017.5.1(5) Figure 2. Respondents’ business sector Source: Authors’ own estimation 5 3 V lidit d R li bilit Figure 2. Respondents’ business sector Source: Authors’ own estimation Figure 2. Respondents’ business sector Source: Authors’ own estimation 5.3. Validity and Reliability 5.4. Factor Analysis y In order to summarise the questionnaire items, principal component analysis (PCA) technique is used, which applies varimax rotation method. The KMO value and Bartlett’s test suggests that the sample is adequate and appropriate for running factor analysis Table 2. Rotated Matrix Rotated Component Matrix Component 1 2 3 4 Your firm consider establishing and executing internal controls over financial and accounting procedures .958 It is important for your organisation to conduct appropriate financial planning and reporting .919 Your organisation performs in-depth financial analysis .898 Your organisation has sufficient free cash flows available .943 Your organisation has the ability to pay its short- term debts .903 Your organisation has a reasonable cash conversion cycle .841 Your organisation invests in research and development .835 Technical innovation is supported and readily .730 67 The International Journal ENTREPRENEURSHIP AND SUSTAINABILITY ISSUES ISSN 2345-0282 (online) http://jssidoi.org/jesi/ 2017 Volume 5 Number 1 (September) http://doi.org/10.9770/jesi.2017.5.1(5) accepted Management actively seeks innovative ideas .683 You are appreciated for innovative and new ideas. .764 You are aware of current financial needs of you company .758 There is appropriate expenditure and income management. .877 Your organisation focuses on long term financial goals .805 Extraction Method: PCA Rotation Method: Varimax Source: Authors’ own estimation The International Journal ENTREPRENEURSHIP AND SUSTAINABILITY ISSUES ISSN 2345-0282 (online) http://jssidoi.org/jesi/ 2017 Volume 5 Number 1 (September) http://doi.org/10.9770/jesi.2017.5.1(5) The Table 2 extracts four components (factors) from the total of 13 items (liquidity = 3; controlling = 3; literacy = 3; and innovativeness = 4). These factor loadings combined correlated items into unobservable (latent) constructs, which are used in regression analysis. ( ) ( ) ( ) ( ) The constant term is removed from the model due to its insignificant relationship with th Discussion and Recommendations Discussion and Recommendations The research paper aims to evaluate the impact of financial management components on firms’ innovation. These components include financial literacy, controlling, and liquidity. Based on the quantitative analysis (regression analysis), the study finds a significant impact of the overall financial management model on innovation capacity of the Austrian firms. The analysis of each component within the financial management model is based on their respective research hypotheses: H1: The impact of financial liquidity is significant on innovativeness of the Austrian SMEs – H1 is confirmed, which means that higher liquidity in terms of free cash flows, ability to pay short-term debts, and cash conversion cycle enhance innovation capacity of the firms. H2: The impact of financial controlling is significant on innovativeness of the Austrian SMEs. – H2 is accepted, which suggests that internal controls over financial and accounting procedures, financial planning and reporting, and in-depth financial analysis increases innovation capacity of the SMEs in Austria. H3: The impact of financial literacy is significant on innovativeness of the Austrian SMEs – the third hypothesis is also confirmed, showing higher awareness, appropriate expenditure and income management, and focus on long- term financial goals increases firms’ innovativeness. Based on the above findings, the Austrian SMEs are suggested to increase their focus on the three financial management constructs in order to improve their innovation capability and capacity. Future studies on the problem may consider a qualitative analysis (based on in-depth interviews), or a quantitative analysis based on secondary data. Moreover, future primary studies should increase the sample size (firms and respondents) for more representative and reliable results. Conclusions Financial management and controlling plays a significant role in the overall performance of businesses. In this paper, the significance financial management is assessed with respect to innovation. Based on the primary data of 118 respondents from 41 SMEs operating in Austria, the research finds a significant impact of financial management components on firms’ innovation. Individually, financial liquidity, literacy, and controlling are statistically significant in explaining firms’ innovation capacity. It suggests that Austrian firms are required to focus on these financial constructs for enhancing innovative capabilities and capacities. References References Aarons, G. A., Brown, S. A., Stice, E., & Coe, M. T. (2001). Psychometric evaluation of the marijuana and stimulant effect expectancy questionnaires for adolescents. Addictive behaviors, 26(2), 219-236 http://doi.org/10.1016/S0306-4603(00)00103-9 Acharya, V., & Xu, Z. (2017). Financial dependence and innovation: The case of public versus private firms. Journal of Financial Economics, 124(2), 223-243. http://pages.stern.nyu.edu/~sternfin/vacharya/public_html/pdfs/jfe14653_revision.pdf Agénor, P. R., & Canuto, O. (2017). Access to finance, product innovation and middle-income traps. Research in Economics. https://doi.org/10.1016/j.rie.2017.03.004 Agénor, P. R., Canuto, O., & Jelenic, M. (2014). Access to Finance, Product Innovation, and Middle-Income Growth Traps. 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Journal of Financial Economics, 124(2), 223-243. http://pages.stern.nyu.edu/~sternfin/vacharya/public_html/pdfs/jfe14653_revision.pdf Agénor, P. R., & Canuto, O. (2017). Access to finance, product innovation and middle-income traps. Research in Economics. https://doi.org/10.1016/j.rie.2017.03.004 Agénor, P. R., Canuto, O., & Jelenic, M. (2014). Access to Finance, Product Innovation, and Middle-Income Growth Traps. World Bank- Economic Premise, (137), 1-7. http://siteresources.worldbank.org/EXTPREMNET/Resources/EP137.pdf Belas, J. & Sopková, G. (2016). Significant determinants of the competitive environment for SMEs in the context of financial and credit risks. Journal of International Studies. 9(2), 139 – 149. http://dx.doi.org/10.14254/2071-8330.2016/9-2/10 Bena, J., & Li, K. (2014). Corporate innovations and mergers and acquisitions. The Journal of Finance, 69(5), 1923-1960. https://doi.org/110.1111/jofi.12059 Aarons, G. A., Brown, S. A., Stice, E., & Coe, M. T. (2001). Psychometric evaluation of the marijuana and stimulant effect expectancy questionnaires for adolescents. Addictive behaviors, 26(2), 219-236 http://doi.org/10.1016/S0306-4603(00)00103-9 Acharya, V., & Xu, Z. (2017). Financial dependence and innovation: The case of public versus private firms. Journal of Financial Economics, 124(2), 223-243. http://pages.stern.nyu.edu/~sternfin/vacharya/public_html/pdfs/jfe14653_revision.pdf Acharya, V., & Xu, Z. (2017). Financial dependence and innovation: The case of public versus private firms. Journal of Financial Economics, 124(2), 223-243. http://pages.stern.nyu.edu/~sternfin/vacharya/public_html/pdfs/jfe14653_revision.pdf Agénor, P. R., & Canuto, O. (2017). Access to finance, product innovation and middle-income traps. Research in Economics. https://doi.org/10.1016/j.rie.2017.03.004 Agénor, P. R., & Canuto, O. Bena, J., & Li, K. (2014). Corporate innovations and mergers and acquisitions. The Journal of Finance, 69(5), 1923-1960. https://doi.org/110.1111/jofi.12059 Belas, J. & Sopková, G. (2016). Significant determinants of the competitive environment for SMEs in the context of financial and credit risks. Journal of International Studies. 9(2), 139 – 149. http://dx.doi.org/10.14254/2071-8330.2016/9-2/10 5.5. Regression Analysis The statistical relationship between the components of financial management and the construct of innovation is tested using multiple regression analysis. The association between the variables can be mathematically represented as: I = α + β1L + β2C + β3FL+ e .......... (1) I = α + β1L + β2C + β3FL+ e .......... (1) Where I, L, C, and FL represent innovativeness, liquidity, controlling, and financial literacy. In this section, the research hypotheses of the study are tested. Table 3. Multiple Regression Analysis Table 3. Multiple Regression Analysis Table 3. Multiple Regression Analysis Dependent Variable: Innovativeness B t Sig. (Constant) .035 .097 .923 Liquidity .332 4.459 .000 Financial Literacy .222 4.034 .000 Controlling .435 9.953 .000 F Statistic 64.78 P Value 0.000 R Square 0.630 Adjusted R Square 0.621 Source: Authors’ own estimation Source: Authors’ own estimation The Table 3 reveals that the overall multiple regression model is statistically significant at 0.01 level, which suggests that financial management significantly affects innovativeness or innovation capacity of the Austrian SMEs. The financial management components of this study, that are financial liquidity, financial literacy, and financial controlling, are all statistically significant at 1%, with positive beta values. It means that liquidity, literacy, and controlling with respect to finance improve innovativeness or innovation capacity of the Austrian SMEs operating in diverse business sectors. Following model is established on the basis of the above results: I = 0.332(L) + 0.435(C) + 0.222(FL)+ e .......... (2) 68 The International Journal ENTREPRENEURSHIP AND SUSTAINABILITY ISSUES ISSN 2345-0282 (online) http://jssidoi.org/jesi/ 2017 Volume 5 Number 1 (September) http://doi.org/10.9770/jesi.2017.5.1(5) The International Journal ENTREPRENEURSHIP AND SUSTAINABILITY ISSUES ISSN 2345-0282 (online) http://jssidoi.org/jesi/ 2017 Volume 5 Number 1 (September) http://doi.org/10.9770/jesi.2017.5.1(5) The International Journal ENTREPRENEURSHIP AND SUSTAINABILITY ISSUES ISSN 2345-0282 (online) http://jssidoi.org/jesi/ 2017 Volume 5 Number 1 (September) http://doi.org/10.9770/jesi.2017.5.1(5) The International Journal ENTREPRENEURSHIP AND SUSTAINABILITY ISSUES ISSN 2345-0282 (online) http://jssidoi.org/jesi/ 2017 Volume 5 Number 1 (September) http://doi.org/10.9770/jesi.2017.5.1(5) The International Journal ENTREPRENEURSHIP AND SUSTAINABILITY ISSUES ISSN 2345-0282 (online) http://jssidoi.org/jesi/ 2017 Volume 5 Number 1 (September) http://doi.org/10.9770/jesi.2017.5.1(5) Bergemann, D., & Hege, U. (2005). The financing of innovation: Learning and stopping. RAND Journal of Economics, 719-752. http://citeseerx.ist.psu.edu/viewdoc/download?doi=10.1.1.414.9480&rep=rep1&type=pdf Bergemann, D., & Hege, U. (2005). The financing of innovation: Learning and stopping. RAND Journal of Economics, 719-752. http://citeseerx.ist.psu.edu/viewdoc/download?doi=10.1.1.414.9480&rep=rep1&type=pdf Botric, V., & Bozic, L. (2017). Access To Finance: Innovative Firms' perceptions In Post-Transition EU MEMBERS. E+ M Ekonomie a Management, 20(1), 129. https://doi.org/10.15240/tul/001/2017-1-009 Botric, V., & Bozic, L. (2017). Access To Finance: Innovative Firms' perceptions In Post-Transition EU MEMBERS. E+ M Ekonomie a Management, 20(1), 129. https://doi.org/10.15240/tul/001/2017-1-009 Brejcha, J., Wang, C., Wang, X., Wang, Z., Wang, L., Xu, Q., ... & Zhang, S. (2016, July). Financial Literacy in China as an Innovation Opportunity. 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https://openalex.org/W4392637198	https://aclanthology.org/2023.ijcnlp-short.4.pdf	English	null	The Impact of Debiasing on the Performance of Language Models in Downstream Tasks is Underestimated	null	2,023	cc-by	4,685	Abstract Pre-trained language models trained on large- scale data have learned serious levels of so- cial biases. Consequently, various methods have been proposed to debias pre-trained mod- els. Debiasing methods need to mitigate only discriminatory bias information from the pre- trained models, while retaining information that is useful for the downstream tasks. In previous research, whether useful information is retained has been confirmed by the perfor- mance of downstream tasks in debiased pre- trained models. On the other hand, it is not clear whether these benchmarks consist of data pertaining to social biases and are appropriate for investigating the impact of debiasing. For example in gender-related social biases, data containing female words (e.g. “she, female, woman”), male words (e.g. “he, male, man”), and stereotypical words (e.g. “nurse, doctor, professor”) are considered to be the most af- fected by debiasing. If there is not much data containing these words in a benchmark dataset for a target task, there is the possibility of erro- neously evaluating the effects of debiasing. In this study, we compare the impact of debiasing on performance across multiple downstream tasks using a wide-range of benchmark datasets that containing female, male, and stereotypical words. Experiments show that the effects of de- biasing are consistently underestimated across all tasks. Moreover, the effects of debiasing could be reliably evaluated by separately con- sidering instances containing female, male, and stereotypical words than all of the instances in a benchmark dataset. Table 1: The total number of instances containing fe- male, male, and occupational (Occ.) words in the GLUE development data. RoBERTa (Liu et al., 2019) easily learn discrimina- tory social biases expressed in human-written texts in massive datasets (Kurita et al., 2019; Zhou et al., 2022; Kaneko et al., 2022). For example, if a model is given “[MASK] is a nurse.” as the input, a gender biased PLM would predict “She” with a higer likeli- hood score than for “He” when filling the [MASK]. Various debiasing methods have been proposed to mitigate social biases in PLMs. Zhao et al. (2019); Webster et al. (2020) proposed a debiasing method by swapping the gender of female and male words in the training data. Kaneko and Bollegala (2021) proposed a method for debiasing by orthogonal- ising the vectors representing gender information with the hidden layer of a language model given a sentence containing a stereotypical word. Webster et al. Proceedings of the 13th International Joint Conference on Natural Language Processing and the 3rd Conference of the Asia-Paciﬁc Chapter of the Association for Computational Linguistics (Volume 2: Short Papers), pages 29–36 November 1–4, 2023. ©2023 Association for Computational Linguistics Abstract (2020) showed that dropout regularization can reduce overfitting to gender information, thereby can be used for debiasing PLMs. The debiasing method should mitigate only dis- criminatory information, while pre-trained useful information should be retained in the model. Evalu- ations in downstream tasks often employ the GLEU benchmark (Wang et al., 2018), which measures the ability to understand language (Kaneko and Bollegala, 2021; Guo et al., 2022; Meade et al., 2022). The data for downstream tasks are not se- lected in terms of whether they reflect the impact of The Impact of Debiasing on the Performance of Language Models in Downstream Tasks is Underestimated Masahiro Kaneko1,2 Danushka Bollegala3,4∗ Naoaki Okazaki2 1MBZUAI 2Tokyo Institute of Technology 3University of Liverpool 4Amazon Masahiro.Kaneko@mbzuai.ac.ae danushka@liverpool.ac.uk okazaki@c.titech.ac.jp The Impact of Debiasing on the Performance of Language Models in Downstream Tasks is Underestimated All Female Male Occ. CoLA 1,043 174 722 96 MNLI 9,832 3,467 8,875 1,415 MRPC 408 101 391 96 QNLI 5,463 2,149 5,371 1,066 QQP 40,430 7,415 29,638 3,331 RTE 277 113 269 94 SST-2 872 187 691 75 STS-B 1,500 513 1,277 151 WNLI 71 27 71 6 Table 1: The total number of instances containing fe- male, male, and occupational (Occ.) words in the GLUE development data. The Impact of Debiasing on the Performance of Language Models in Downstream Tasks is Underestimated Masahiro Kaneko1,2 Danushka Bollegala3,4∗ Naoaki Okazaki2 1MBZUAI 2Tokyo Institute of Technology 3University of Liverpool 4Amazon Masahiro.Kaneko@mbzuai.ac.ae danushka@liverpool.ac.uk okazaki@c.titech.ac.jp The Impact of Debiasing on the Performance of Language Models in Downstream Tasks is Underestimated Masahiro Kaneko1,2 Danushka Bollegala3,4∗ Naoaki Okazaki2 1MBZUAI 2Tokyo Institute of Technology 3University of Liverpool 4Amazon Masahiro.Kaneko@mbzuai.ac.ae danushka@liverpool.ac.uk okazaki@c.titech.ac.jp 1 Introduction Unfortunately, Pre-trained Language Models (PLMs) such as BERT (Devlin et al., 2019) and ∗Danushka Bollegala holds concurrent appointments as a Professor at University of Liverpool and as an Amazon Scholar. This paper describes work performed at the Univer- sity of Liverpool and is not associated with Amazon. 29 debiasing. To mitigate gender bias, data containing female words such as “she” and “woman”, male words such as “he” and “man”, and stereotypi- cal words such as “doctor” and “nurse” would be most affected by debiasing. Counterfactual Data Augmentation (CDA) de- biasing: CDA debiasing (Webster et al., 2020) swaps the gender of gender words in the training data. For example, “She is a nurse” is swapped to “He is a nurse”, and the swapped version is appended to the training dataset. This enables to learn a less biased model because the frequency of female and male words will be the same in the augmented dataset. Table 1 shows the total number of instances containing female, male, and occupational (Occ.) words in the development data in the GLUE benchmark suite (Wang et al., 2019), which is widely recognised as a standard evaluation bench- mark for LLMs. Occupational words have been used for probing LLMs for stereotypical social bi- ases (Bolukbasi et al., 2016). From Table 1, we see that the GLEU benchmark has little data related to females and occupations. Therefore, the impact of debiasing on data related to females and occu- pations may be potentially underestimated when LLMs are evaluated on GLUE. Dropout debiasing: Webster et al. (2020) intro- duced dropout regularisation as a method to miti- gate biases. They enhanced the dropout parameters for the attention weights and hidden activations of PLMs. Their research demonstrated that intensi- fied dropout regularisation diminishes gender bias in these PLMs. They showed that dropout inter- fers with the attention mechanism in PLMs, and prevents undesirable associations between words. However, it is also possible that the model may no longer be able to learn desirable associations. We first extract instances containing female words, data containing male words, and data con- taining stereotypical words from the benchmarks. We then calculated the performance difference be- tween the original model and the debiased model for each category and compared it to the perfor- mance difference using the entire benchmark. 2 Experiments We use the following nine downstream tasks from the GLEU benchmark: CoLA (Warstadt et al., 2019), MNLI (Williams et al., 2018), MRPC (Dolan and Brockett, 2005), QNLI (Ra- 2.2 Settings Although we use BERT (bert-base-cased1) (De- vlin et al., 2019) as our PML here as it has been the focus of much prior work on bias evalua- tions (Kaneko and Bollegala, 2021; Guo et al., 2022; Meade et al., 2022), the evaluation proto- col we use can be applied to any PLM. We used the word lists2 proposed by Kaneko and Bollegala (2021) as female words, male words, and occu- pational words for extracting data instances and debiasing. 1 Introduction The results showed that the debiased model performed worse than the original model on data related to females and occupations compared to the original model when evaluated on the entire dataset. There- fore, existing evaluations underestimate the impact of debiasing on the performance of the downstream task. Context debiasing: Kaneko and Bollegala (2021) proposed a method to debias MLMs through fine-tuning. It preserves semantic information while removing gender-related biases using orthog- onal projections at token- or sentence-level. This method targets male and female words and oc- cupational words in the text for debiasing. This method can be applied various MLMs, indepen- dent of the model architectures and pre-training methods. Token-level debiasing across all layers produces the best performance. It is important to be able to compare how well the effects of debiasing are captured in the datasets related to females, males, and occupations. We pro- pose a method to control the degree of debiasing of PLMs and investigate whether the performance difference between original and debiased models widens as the degree of debiasing increases. Ex- perimental results showed that the proportion of female, male and occupational words in the dataset is related to the susceptibility of the dataset to de- biasing. 2https://github.com/kanekomasahiro/ context-debias 1https://huggingface.co/bert-base-cased 1https://huggingface.co/bert-base-cased 2https://github.com/kanekomasahiro/ context-debias 2.1 Debiasing Methods We use the following three commonly used debias- ing methods in our experiments. We apply these de- biasing methods during fine-tuning in downstream tasks. 30 CDA Dropout Context All Female Male Occ. All Female Male Occ. All Female Male Occ. CoLA -1.36 -3.42 -2.01 -1.45 0.42 -0.14 -0.21 -0.07 -0.32 -0.86 -0.71 -0.55 MNLI -0.55 -0.90 -0.71 -0.63 0.23 0.13 0.01 0.05 -0.05 -0.47 -0.43 -0.32 MRPC -0.96 -1.28 -1.31 -1.03 -0.82 -1.12 -1.02 -1.04 -0.88 -1.01 -1.06 -0.92 QNLI -1.13 -1.42 -1.19 -1.27 -1.01 -1.11 -1.07 -1.21 0.25 -0.19 -0.06 -0.04 QQP -0.21 -0.69 -0.32 -0.25 0.53 0.13 0.47 0.30 0.14 -0.12 0.03 -0.05 RTE -1.16 -1.21 -1.02 -1.13 -1.01 -1.24 -0.96 -1.13 -0.43 -0.65 -0.51 -0.73 SST-2 -0.11 -0.81 -0.34 -0.25 0.45 0.20 0.12 0.23 0.22 -0.15 -0.02 -0.12 STS-B -1.01 -1.95 -1.34 -1.10 0.21 0.09 -0.03 -0.11 -0.08 -0.31 -0.38 -0.34 WNLI -2.82 -3.07 -2.82 -2.71 -2.01 -2.21 -2.01 -2.33 -1.52 -1.88 -1.52 -1.61 Table 2: Performance difference between the original model and debiased model for each dataset. Bolded values indicate the largest drop in performance of the debiased model. Table 2: Performance difference between the original model and debiased model for each dataset. Bolded values indicate the largest drop in performance of the debiased model. jpurkar et al., 2016), QQP3, RTE (Dagan et al., 2006; Haim et al., 2006; Giampiccolo et al., 2007; Bentivogli et al., 2009), SST-2 (Socher et al., 2013), STS-B (Cer et al., 2017), and WNLI (Levesque et al., 2012). Hyperparameters for debiasing fol- low previous studies (Kaneko and Bollegala, 2021; Webster et al., 2020), and we used the default val- ues of huggingface for downstream task hyperpa- rameters.4 For fine-tuning we use the entire train- ing dataset for each corresponding task, without splitting into male, female and occupational in- stances. We evaluate the performance on all tasks using the official development data. From the results in Table 2, it can be seen that the performance difference between the original model and the debiased model is larger for the Female, Male, and Occ. instances compared to that when using all instances. In particular, instances related to females exhibit a significant decrease in performance after debiasing. It can be seen that different word lists used for debiasing have different effects on the performance degradation in downstream tasks due to debiasing. Context debiasing uses occupational words for de- biasing, while CDA debiasing does not. 2.1 Debiasing Methods Therefore, in CDA debiasing, Occ. does not have a large performance difference compared to female- and male-related instances. Therefore, in CDA debi- asing, the performance difference for occupation- related instances is smaller than that for the female and male-related instances. On the other hand, in Context debiasing, occupation-related instances has the largest performance difference as well as female- and male-related instances. Dropout debi- asing does not use word lists for debiasing. There- fore, unlike CDA and context debiasing, we see large drops in performance for female, male and Occ. across tasks with Dropout debiasing. 2.3 Performance of Original vs. Debiased Models We extract instances containing female words, male words, and stereotypical words from each of the datasets. We then calculate the performance differ- ence between the original model and the debiased model for each dataset, and compare against the performance differences obtained when using all instances. If the performance difference for all in- stances is smaller than that when evaluated for the female, male, and occupational instances, it would indicate that the effect of debiasing is underesti- mated when evaluated on the entire dataset. 3https://quoradata.quora.com/ First-Quora-Dataset-Release-Question-Pairs 4https://github.com/huggingface/transformers/ tree/main/examples/pytorch/text-classification 2.4 Debias Controlled Method Table 2 shows the performance differences be- tween the original model and the debiased model for each dataset/task in the GLEU benchmark. All, Female, Male, and Occ. are the performance dif- ferences when evaluated on the entire task dataset, instances containing female words, instances con- taining male words, and instances containing occu- pational words, respectively. To understand how debiasing of an PLM affects the performance of individual downstream benchmark datasets, following the probing technique proposed by Kaneko et al. (2023), we apply different levels of debiasing to bert-base-cased PLM and measure the difference in performance with respect to its origi- nal (non-debiased) version. For this purpose we use CDA as the debiasing method, where we swap the gender-related pronouns in r ∈[0, 1] fraction of the total N instances of a dataset (i.e.the total num- ber of gender swapped instances in a dataset will be 31 (a) QQP. (b) MNLI. (c) QNLI. Figure 1: Performance difference between original and debiased models by debias rate r. The vertical axis shows the performance difference between the original and debiased models, and the horizontal axis shows the debias rate. (a) QQP. r × N). r = 0 corresponds to not swapping gender in any training instances of the dataset, whereas r = 1 swaps the gender in all instances. We in- crement r in step size 0.1 to obtain increasingly debiased version of the PLM, which is then fine- tuned for the downstream task5. Figure 1 shows the difference in performance between the original vs. debiased versions of the PLM for QQP, MNLI, and QNLI, which have the largest numbers of instances in the GLEU benchmark. (a) QQP. Note that CDA debiasing reverses gender with- out considering the context, as in “He gets preg- nant” for “She gets pregnant”. This is probleman- tic because it eliminates even useful gender-related information learnt by the PLM via co-occurring contexts. Therefore, CDA debiasing has a neg- ative impact on the performance of downstream tasks (Zmigrod et al., 2019) as shown by all three subplots in Figure 1. In fact, Table 2 shows that the performance difference of CDA debiasing is larger than that of dropout debiasing and context debiasing. Therefore, the larger r is for CDA, the more gender instances is balanced and debiased, but the performance is unfortunately degraded. (b) MNLI. (c) QNLI. (b) MNLI. 5In Appendix A, we show that the debias controlled method is able to debias the model according to r. 2.4 Debias Controlled Method If the dataset of the downstream task is sensitive to the effect of debiasing, the performance differ- ence between the original model and the debiased model widens as r increases. On the other hand, if the data set is insensitive to the effect of debiasing, the performance difference between the original model and the debiased model is unlikely to in- crease with the value of r. We find that the performance differences for the female, male, and occupational instances in the QQP, MNLI, and QNLI datasets increase with the value of r. On the other hand, for QQP and MNLI, there is a rise and fall in the performance differ- ence when all data are used. These results indicate that All, which includes instances related to non- gender, are less sensitive to the effect of debiasing compared to Female, Male, and Occupational in- stances. Figure 1: Performance difference between original and debiased models by debias rate r. The vertical axis shows the performance difference between the original and debiased models, and the horizontal axis shows the debias rate. References Luisa Bentivogli, Peter Clark, Ido Dagan, and Danilo Giampiccolo. 2009. The fifth pascal recognizing textual entailment challenge. In TAC. Citeseer. 5 Limitations Many previous studies have shown that various so- cial biases other than gender bias are learned in PLMs. This study targets only gender bias. While existing studies (Webster et al., 2020; Zhao et al., 2019) have debiasing various PLMs, we have exper- imented only with bert-base-cased. Furthermore, although this study targets only English, which is a morphologically limited language. On the other hand, various types of social biases are also learned in the PLMs across many languages (Kaneko et al., 2022; Névéol et al., 2022). Therefore, if the pro- posed method is to be used with other social bi- ases and PLMs, it is necessary to properly verify its effectiveness in languages other than English. Moreover, we have not verified the use of debias controlled methods in languages such as Spanish and Russian, where gender swapping is not easy from a grammatical point of view (Zmigrod et al., 2019). Sunipa Dev, Tao Li, Jeff M. Phillips, and Vivek Sriku- mar. 2020. On measuring and mitigating biased infer- ences of word embeddings. Proceedings of the AAAI Conference on Artificial Intelligence, 34(05):7659– 7666. Sunipa Dev, Masoud Monajatipoor, Anaelia Ovalle, Ar- jun Subramonian, Jeff Phillips, and Kai-Wei Chang. 2021. Harms of gender exclusivity and challenges in non-binary representation in language technologies. In Proceedings of the 2021 Conference on Empiri- cal Methods in Natural Language Processing, pages 1968–1994, Online and Punta Cana, Dominican Re- public. Association for Computational Linguistics. Jacob Devlin, Ming-Wei Chang, Kenton Lee, and Kristina Toutanova. 2019. BERT: Pre-training of deep bidirectional transformers for language under- standing. In Proceedings of the 2019 Conference of the North American Chapter of the Association for Computational Linguistics: Human Language Tech- nologies, Volume 1 (Long and Short Papers), pages 4171–4186, Minneapolis, Minnesota. Association for Computational Linguistics. 3 Conclusion This study focused on gender-related social biases and the presence of female, male, and stereotypi- cal words in benchmark datasets. Prior work had used the performance on downstream tasks to prove the usefulness of debiasing methods, overlooking the fact that only a small fraction of those down- stream benchmark datasets contain gender-related instances. On the contrary, we found that the effects of debiasing an PLM were consistently underesti- mated across all tasks. We recommend that the On the other hand, for QNLI, All has a small rise and fall in the performance difference. As seen from Table 1, QNLI contains more gender-related instances than QQP and MNLI. Therefore, it is likely that the performance decreases with r even for All. All and Male instances have a similar trend in performance difference with r. 32 References evaluation of debiasing effects must be more reli- ably conducted by considering instances containing specific gender-related words separately rather than evaluating all instances in a benchmark dataset. 4 Ethical Considerations Tolga Bolukbasi, Kai-Wei Chang, James Y Zou, Venkatesh Saligrama, and Adam T Kalai. 2016. Man is to computer programmer as woman is to home- maker? debiasing word embeddings. Advances in neural information processing systems, 29. This study uses existing methods and datasets for experiments and does not propose a debiasing method or create a new dataset for social bias. 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Association for Computational Linguistics. Ran Zmigrod, Sabrina J. Mielke, Hanna Wallach, and Ryan Cotterell. 2019. Counterfactual data augmenta- tion for mitigating gender stereotypes in languages with rich morphology. In Proceedings of the 57th Annual Meeting of the Association for Computational Linguistics, pages 1651–1661, Florence, Italy. Asso- ciation for Computational Linguistics. 35 35 Figure 2: Evaluation of debias controlled models using FN evaluation method. The vertical axis shows the bias score, and the horizontal axis shows r. Figure 2: Evaluation of debias controlled models using FN evaluation method. The vertical axis shows the bias score, and the horizontal axis shows r. A Bias Evaluation in NLI task We show that the debias controlled method is appro- priately debiasing according to r. We use Fraction Neutra (FN; Dev et al., 2020) as the bias evaluation method. The FN method evaluates bias in the NLI by considering the percentage of neutral labels pre- dicted by the model for the premise sentence (e.g. The driver owns a cabinet.) and the hypothesis sentence (e.g. The man owns a cabinet.) generated with the template. The FN method indicates that the lower the score, the more bias there is in the model. We evaluate PLMs trained on MNLI with FN method. Figure 2 shows the bias scores of FN method for each debias controlled model. It can be seen that the bias of the model is decreasing with r. Therefore, the debias controlled method is able to debias the models according to r. 36
https://openalex.org/W4232869083	https://www.qeios.com/read/FTAS61/pdf	English	null	RGS5 Gene	Definitions	2,020	cc-by	70	Qeios · Definition, February 7, 2020 Open Peer Review on Qeios Open Peer Review on Qeios RGS5 Gene National Cancer Institute National Cancer Institute Qeios ID: FTAS61 · https://doi.org/10.32388/FTAS61 Open Peer Review on Qeios Source National Cancer Institute. RGS5 Gene. NCI Thesaurus. Code C74517. National Cancer Institute. RGS5 Gene. NCI Thesaurus. Code C74517. This gene plays a role in the regulation of signal transduction. Qeios ID: FTAS61 · https://doi.org/10.32388/FTAS61 1/1
https://openalex.org/W2132256952	https://europepmc.org/articles/pmc3059277?pdf=render	English	null	Prevention of hypertension in patients with pre-hypertension: protocol for the PREVER-prevention trial	Trials	2,011	cc-by	4,526	* Correspondence: ffuchs@hcpa.ufrgs.br 1Hospital de Clínicas de Porto Alegre, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil Full list of author information is available at the end of the article Open Access Open Access Prevention of hypertension in patients with pre-hypertension: protocol for the PREVER-prevention trial Flávio D Fuchs1*, Sandra C Fuchs1, Leila B Moreira1, Miguel Gus1, Antônio C Nóbrega2, Carlos E Poli-de-Figueiredo3, Décio Mion4, Luiz Bortoloto5, Fernanda Consolim-Colombo5, Fernando Nobre6, Eduardo Barbosa Coelho6, José F Vilela-Martin7, Heitor Moreno Jr8, Evandro José Cesarino9, Roberto Franco10, Andréa Araujo Brandão11, Marcos R de Sousa12, Antônio Luiz Pinho Ribeiro12, Paulo Cesar Jardim13, Abrahão Afiune Neto14, Luiz César N Scala15, Marco Mota16, Hilton Chaves17, João Guilherme Alves18, Dario C Sobral Filho19, Ricardo Pereira e Silva20, José A Figueiredo Neto21, Maria Cláudia Irigoyen22, Iran Castro22, André Avelino Steffens23, Rosane Schlatter1, Renato Bandeira de Mello1, Francisca Mosele1, Flávia Ghizzoni1, Otávio Berwanger24 © 2011 Fuchs 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. TRIALS TRIALS Fuchs et al. Trials 2011, 12:65 http://www.trialsjournal.com/content/12/1/65 TRIALS Low effectiveness of non-drug interventions in patients with hypertension and pre-hypertension A meta-analysis of 61 cohort studies, with more than one million of subjects (12.7 million persons-year at risk), pre- senting more than 56,000 fatal cardiovascular events, demonstrated that the risk for cardiovascular events starts with systolic blood pressure higher than 115 mmHg or diastolic blood pressure higher than 75 mmHg, doubling at each 20 mmHg in the first case or 10 mmHg in the second [1]. The risks of pre-hypertension and high normal blood pressure have been confirmed in other cohorts [5,6]. The efficacy of blood pressure-lowering drugs to reduce such risks, with a magnitude anticipated by the cohort studies, corroborated in the experimental setting the estimation of risks [7]. The risk factors for hypertension, such as the excessive dietary sodium intake, low potassium consumption, excess of adiposity, insulin resistance, abuse of alcoholic beverages, and low consumption of fruits and vegetables are well-known. Many nutritional and behavioral inter- ventions are efficacious to reduce blood pressure, but outside the strict experimental conditions their effective- ness has been inconsistent. The mean weight of popula- tions is not stable but has increased in recent years worldwide. Randomized controlled trials with long fol- low-up periods have shown that the efficacy of weight control interventions is lost with time. In the TOHP-II trial, weight, salt consumption and blood pressure returned to the baseline values after 36 months of inter- vention [15]. The DASH diet, which was highly effica- cious in a strictly controlled trial [16], was only marginally efficacious in the PREMIER study [17], where differences in blood pressure emerged only when com- pared with the advice group. The efficacy of low-salt diets was negligible in trials that lasted more than six months [18]. In a clinical context, only the recommen- dation to lose weight had a short-term effect on blood pressure [19]. Physiopathological basis for early intervention The raising of blood pressure with age is not inexor- able and does not occur in populations that do not consume large amounts of salt. Under the contempor- ary and unnatural overload of dietary sodium, kidneys had to reset their primary sodium handling function from retention to excretion. Subjects with familial pre- disposition to hypertension require higher renal flow and consequently higher blood pressure to eliminate the sodium overload, resulting in extracellular volume expansion, increase in cardiac output and peripheral resistance. Low effectiveness of non-drug interventions in patients with hypertension and pre-hypertension With long-standing high blood pressure, loss of glomeruli and renal arterioles may further shift pressure natriuresis and exacerbate blood pressure ele- vation. The recurrence of this phenomenon along the years leads to arteriolar hypertrophy and sustained blood pressure rise [8-11]. After a long period of high peripheral resistance and diastolic blood pressure, stiff- ness of large vessels arises, with consequent rise of sys- tolic blood pressure. This deleterious natural history of blood pressure rising with ageing could be aborted in the very beginning by a low-salt diet or by increasing natriuresis, which could be accomplished by very low doses of diuretics or other drugs that enhance the renal capacity of excreting sodium. Background Incidence of hypertension in patients with pre- hypertension High blood pressure is the major risk factor for cardio- vascular disease. The risks start at blood pressure values as lower as 115/75 mmHg, but increase exponentially and confer increased absolute risks with blood pressure higher than 140/90 mmHg [1,2]. Cardiovascular disease is already the leading cause of death in Brazil. The pre- valence of hypertension in Brazil ranges from 22.3 to 44% of adults [3]. Therefore, interventions aiming to prevent or treat high blood pressure are highly needed. The rationale for precocious drug intervention to pre- vent hypertension was recently presented [4] and is summarized below. The worldwide prevalence of hypertension in individuals older than 70 years was 70% of women and 59% of men [12]. The incidence of hypertension increases with age until the fifth decade, particularly among individuals with high-normal blood pressure [13]. Four out of five individuals with pre-hypertension aged 40 to 49 years developed hypertension in 10 years in a population- based cohort study conducted in Porto Alegre, Brazil [14]. Therefore, pre-hypertension is not only a risk by itself but it identifies individuals at higher risk for the development of full hypertension in a short period of time. Abstract Background: Blood pressure (BP) within pre-hypertensive levels confers higher cardiovascular risk and is an intermediate stage for full hypertension, which develops in an annual rate of 7 out of 100 individuals with 40 to 50 years of age. Non-drug interventions to prevent hypertension have had low effectiveness. In individuals with previous cardiovascular disease or diabetes, the use of BP-lowering agents reduces the incidence of major cardiovascular events. In the absence of higher baseline risk, the use of BP agents reduces the incidence of hypertension. The PREVER-prevention trial aims to investigate the efficacy, safety and feasibility of a population- based intervention to prevent the incidence of hypertension and the development of target-organ damage. Methods: This is a randomized, double-blind, placebo-controlled clinical trial, with participants aged 30 to 70 years, with pre-hypertension. The trial arms will be chlorthalidone 12.5 mg plus amiloride 2.5 mg or identical placebo. The primary outcomes will be the incidence of hypertension, adverse events and development or worsening of microalbuminuria and of left ventricular hypertrophy in the EKG. The secondary outcomes will be fatal or non-fatal cardiovascular events: myocardial infarction, stroke, heart failure, evidence of new sub-clinical atherosclerosis, and sudden death. The study will last 18 months. The sample size was calculated on the basis of an incidence of hypertension of 14% in the control group, a size effect of 40%, power of 85% and P alpha of 5%, resulting in 625 participants per group. The project was approved by the Ethics committee of each participating institution. Discussion: The early use of blood pressure-lowering drugs, particularly diuretics, which act on the main mechanism of blood pressure rising with age, may prevent cardiovascular events and the incidence of hypertension in individuals with hypertension. If this intervention shows to be effective and safe in a population- based perspective, it could be the basis for an innovative public health program to prevent hypertension in Brazil. Trial Registration: Clinical Trials NCT00970931. Fuchs et al. Trials 2011, 12:65 http://www.trialsjournal.com/content/12/1/65 Fuchs et al. Trials 2011, 12:65 http://www.trialsjournal.com/content/12/1/65 Page 2 of 7 Fuchs et al. Trials 2011, 12:65 http://www.trialsjournal.com/content/12/1/65 Page 2 of 7 Research questions 1. Does an association of low doses of chlorthalidone and amiloride reduce the incidence of hypertension in individuals with pre-hypertension? Prevention of cardiovascular events in patients with blood pressure within normal values 2. Does an association of low doses of chlorthalidone and amiloride reduce the incidence of target-organ damage in patients with pre-hypertension? The expectation that BP agents have beneficial effects independent of the blood pressure-lowering one (pleio- tropic effects) is still deep-rooted in physician’s minds, but it has been repeatedly dismissed by independent clinical trials and by a large meta-analysis [7] The effi- cacy of beta-blockers in patients recovering from a myo- cardial infarction and in patients with heart failure, of ACE inhibitors in patients with heart failure, coronary heart disease, other evidences of atherosclerotic vascular disease or diabetes, and patients recovered from a stroke (with a diuretic) can be almost fully explained by the blood pressure-lowering effect [22]. At least part of the evidences supporting the existence of such effects may have resulted from biases in the planning and interpre- tation of clinical trials funded by pharmaceutical compa- nies [23]. 3. Does an association of low doses of chlorthalidone and amiloride reduce the incidence of cardiovascular events in patients with pre-hypertension? 4. Is the association of low doses of chlorthalidone and amiloride safe to be used on a population-based perspective? 4. Is the association of low doses of chlorthalidone and amiloride safe to be used on a population-based perspective? The efficacy of drugs to prevent hypertension in patients with pre-hypertension Two large clinical trials showed that the prevention of hypertension by drug treatment is feasible and well tol- erated. In the TROPHY study [20], 772 individuals with systolic blood pressure between 130 and 139 mmHg or diastolic blood pressure between 85 and 89 mmHg were randomized to candesartan, 16 mg daily or placebo, beside recommendations to change lifestyle. After two years, the incidence of hypertension was 66% lower in individuals treated with candesartan (relative risk 0.34, 95% CI: 0.25-0.44), corresponding to a NNT of 4, which means that four individuals should be treated to prevent Fuchs et al. Trials 2011, 12:65 http://www.trialsjournal.com/content/12/1/65 Fuchs et al. Trials 2011, 12:65 http://www.trialsjournal.com/content/12/1/65 Page 3 of 7 this association could be the basis for an innovative public health program to prevent hypertension in Brazil. one case of hypertension. After two years the treatment was interrupted and blood pressure tended to return to the levels of the control group. The treatment was well tolerated. Similar but not so intense efficacy was observed in the study PHARAO [21], which compared ramipril with placebo. Methods Design randomized, double-blind, clinical trial, controlled by placebo. Primary: Primary: 1. Incidence of hypertension. 2. Adverse events. 3. Development or worsening of microalbuminuria and of left ventricular hypertrophy in the EKG. Secondary: fatal or non-fatal cardiovascular events. Secondary: fatal or non-fatal cardiovascular events. Eligible participants individuals with 30 to 70 years of age with pre- hypertension. Interventions Interventions Chlorthalidone 12.5 mg plus amiloride 2.5 mg or identi- cal placebo. Figure 1 shows flow-chart of selection of participants and interventions. Trial rationale h l The early use of blood pressure-lowering drugs may prevent cardiovascular events and the incidence of hypertension. The very low absolute risk of pre-hyper- tension in young individuals free of diabetes or cardio- vascular disease precludes the launching of clinical trials with the aim to prevent hard outcomes. The blood pres- sure-lowering effect and the prevention of the incidence of full hypertension may be valid surrogate endpoints that could be investigated in feasible randomized clinical trials. The PREVER prevention trial study will test if a low dose of an association of chlorthalidone with amiloride prevents hypertension at an acceptable safety in a nationwide large sample of individuals with pre- hypertension (Clinical trials registration number: NCT00970931). Other intermediate outcomes, such as microalbuminuria and left ventricular hypertrophy, are going to be investigated. Diuretics were chosen in face of their cost-effectiveness, since they are at least as effi- cacious as other blood pressure agents [24], at very low frequency of adverse effects, and the fact that they are the cheapest agents. Chlorthalidone was chosen in face of its higher blood pressure-lowering efficacy and better performance in the prevention of major cardiovascular events than hydrochlorothiazide [24]. The association with amiloride aims to prevent the deleterious hypokale- mia induced by diuretics [25,26]. If the intervention shows to be effective and well-tolerated, the early use of low life expectancy, other indications for the use of diuretics, such as cardiovascular disease, intolerance to the study drugs, pregnancy. Random allocation will be generated by a computer, using a validated soft- ware (Random allocator), with variable block sizes and stratified by center. before the randomization and will be maintained throughout the trial. An extended follow-up is planned pending on additional funding. fabrication by Micromed Biotecnologia Ltda, Brasília, Brazil) ≥140/90 mmHg. The diagnosis will be confirmed by a new visit soon afterwards, with the average of blood pressure measurements of both consultations within Consent form Males/ Females, 30 - 70 years old POTENTIALLY ELIGIBLE (Time 0) Males/ Females, 30 70 years old Office average blood pressure: 120-139/80-89 mmHg If diabetes mellitus: Systolic BP:120-130 mmHg LIFESTYLE MODIFICATION (Months 1 to 3) Weight control Dash diet like Low sodium Stop smoking ( ) p g Physical activity Consent form RANDOMIZATION (Month 3) Prehypertension at office blood pressure Blood tests Urine analysis ECG Re-assessment Office blood pressure FOLLOW-UP VISITS (Months 6 to 15) p Side-effects Hypertension OUTCOMES (Month 18) yp Adverse events Target-organ damage Cardiovascular disease Figure 1 Flow chart of the PREVER-prevention trial, describing selection, randomization and follow-up process. before the randomization and will be maintained throughout the trial. An extended follow-up is planned pending on additional funding. before the randomization and will be maintained throughout the trial. An extended follow-up is planned pending on additional funding. fabrication by Micromed Biotecnologia Ltda, Brasília, Brazil) ≥140/90 mmHg. The diagnosis will be confirmed by a new visit soon afterwards, with the average of blood pressure measurements of both consultations within hypertensive levels. Follow-up and duration of the study consultations for evaluation and enrollment and there- after consultations at the third, 6th, 9th, 12th and 18th months. Figure 2 shows the summary of key practical aspects of the trial. Lifestyle interventions will be applied Page 4 of 7 Fuchs et al. Trials 2011, 12:65 http://www.trialsjournal.com/content/12/1/65 Assessment of outcomes Adverse events: adverse events will be investigated by open questions and by a semi-structured questionnaire including general symptoms and the presumed adverse effects of the drugs used in the trial. The incidence of Incidence of hypertension: the diagnosis of hypertension will be signaled at any of the follow-up visits by average blood pressure (two measurements by an automatic electronic device Microlife BP 3BTO-A, licensed for Page 5 of 7 Fuchs et al. Trials 2011, 12:65 http://www.trialsjournal.com/content/12/1/65 Males or females, 30 to 70 years-old, prehypertension (office blood pressure); no antihypertensive treatment, without allergy to chlortalidone and amiloride, and no previous CHD, allergy to chlortalidone and amiloride, and no previous CHD, severe chronic disease, or pregnant women Lifestyle modification Prehypertension R Chlortalidone and Amiloride Placebo 3 m 6 m 3 m 6 m 6 m 9 m 12 m 6 m 9 m 12 m 12 m 15 m 12 m 15 m Hypertension Adverse effects Target-organ damage Hypertension Adverse effects Target-organ damage Figure 2 Summary of the PREVER-Prevention trial key practical aspects. Males or females, 30 to 70 years-old, prehypertension (office blood pressure); no antihypertensive treatment, without allergy to chlortalidone and amiloride, and no previous CHD, allergy to chlortalidone and amiloride, and no previous CHD, severe chronic disease, or pregnant women Lifestyle modification Prehypertension Chlortalidone and Amiloride Placebo 6 m 6 m 12 m 12 m 12 m 12 m 15 m 15 m Hypertension Adverse effects Target-organ damage Hypertension Adverse effects Target-organ damage Figure 2 Summary of the PREVER-Prevention trial key practical aspects. be adjudicated on the basis of interview, hospital charts and exams, death certificates and verbal autopsy with next-of-kin, by members of the outcome committee. adverse effects will be determined by the incidence of adverse events more frequent in the drug arm than in the placebo arm. Laboratory adverse events, such as hypokalemia, hyperuricemia and diabetes (glycated hemoglobin and fasting glucose) will be investigated at the final visit of the participants. Sample size calculation assuming an incidence of hypertension of 14% in two years in the control group, a size effect of 40% of reduc- tion in the incidence in the treated group, for a power of 85% and P alpha of 5%, it will be necessary to study 568 individuals per group, rounded to 625 per group to compensate for losses in follow-up. Target-organ damage: Microalbuminuria will be deter- mined by nephelometry. Left ventricular hypertrophy will be investigated by electrocardiogram, using the Sokolow-Lyon voltage and the Cornell voltage-duration product. These examinations will be carried out at the baseline evaluation and the final visit. Statistics Cardiovascular outcomes: from a statistical point of view they will be secondary events, in face of the sample power calculated for the trial. Figure 3 presents the main outcomes that will be investigated. The cases will the cumulative incidence of hypertension and adverse events will be tested by Chi-square. Survival curves (Kaplan Meyer) will be compared by log-rank test, and Page 6 of 7 Fuchs et al. Trials 2011, 12:65 http://www.trialsjournal.com/content/12/1/65 accredited by the Office of Human Research Protections as an Institutional Review Board, and by the Ethic Com- mittees of all participating centers. All participants will sign an informed consent to participate. Authors’ contributions FDF conceived the study; all authors participated in the trial design and methodological considerations, contributed to the draft of this manuscript for intellectual content and approved its final version. They are the coordinators of the clinical centers that will enroll the trial participants. Competing interests The authors declare that they have no competing interests. The authors declare that they have no competing interests. Received: 8 December 2010 Accepted: 5 March 2011 Published: 5 March 2011 Received: 8 December 2010 Accepted: 5 March 2011 Published: 5 March 2011 Discussion The early use of blood pressure-lowering drugs in indivi- duals with pre-hypertension may prevent cardiovascular events, target-organ damage and the incidence of full hypertension. Non-drug treatment (life-style changing) has had low long-term effectiveness and blood pressure drugs can circumvent such limitation. Diuretics may be particu- larly efficacious in this regard, since they act on the main mechanism of blood pressure rising with ageing. The asso- ciation of chlorthalidone with amiloride combines the effi- cacy of chlorthalidone with the potassium-sparing effect of amiloride, preventing electrolyte and metabolic abnormal- ities induced by chlorthalidone. If this association shows to be effective and well-tolerated on a population-based perspective, it could be the basis for an innovative public health program to prevent hypertension in Brazil. Grants the Ministry of Health, Division of Science and Technol- ogy (DECIT), and Ministry of Science and Technology, FINEP and CNPq, Brazil, funded the study. yp q , ( p ) 4. Fuchs FD: Prehypertension: the rationale for early drug therapy. Cardiovasc Ther 2010, 28:339-43. 5. Hsia J, Margolis KL, Eaton CB, Wenger NK, Allison M, Wu L, LaCroix AZ, Black HR, Women’s Health Initiative Investigators: Pre-hypertension and cardiovascular disease risk in the Women’s Health Initiative. Circulation 2007, 115:855-60. Logistics this is a nation-based trial, with 24 clinical centers dis- tributed in nine States. A Coordinating Committee was responsible for the elaboration of this proposal and for the main decisions regarding the trial. The organiza- tional study chart will include an executive Committee, a safety committee, outcome committee, data, lab and EKG centers, and the research units References 1. Prospective Studies Collaboration: Age-specific relevance of usual blood pressure to vascular mortality: a meta-analysis of individual data for one million adults in 61 prospective studies. Lancet 2002, 360:1903-1913. 1. Prospective Studies Collaboration: Age-specific relevance of usual blood pressure to vascular mortality: a meta-analysis of individual data for one million adults in 61 prospective studies. Lancet 2002, 360:1903-1913. 2. World Health Report 2002: Reducing risks, promoting healthy life. Geneva, Switzerland: World Health Organization; 2002 [http://www.who.int/whr/ 2002], Accessed October 27, 2010. 3. Sociedade Brasileira de Cardiologia/Sociedade Brasileira de Hipertensão/ Sociedade Brasileira de Nefrologia. VI Brazilian Guidelines of Hypertension. Arq Bras Cardiol 2010, 95(1 supl 1):1-51. Author details 1 1Hospital de Clínicas de Porto Alegre, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil. 2Hospital Universitário Antônio Pedro, Universidade Federal Fluminense, Niterói, Brazil. 3Hospital São Lucas, Pontifícia Universidade Católica do Rio Grande do Sul, Porto Alegre, Brazil. 4Hospital das Clinicas, Universidade de São Paulo, São Paulo, Brazil. 5Instituto do Coração, Universidade de São Paulo, São Paulo, Brazil. 6Faculdade de Medicina de Ribeirão Preto - Universidade de São Paulo, Ribeirão Preto, Brazil. 7Faculdade de Medicina São José do Rio Preto, São José do Rio Preto, Brazil. 8Faculdade de Ciências Médicas, Universidade de Campinas, Campinas, Brazil. 9Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, Ribeirão Preto, Brazil. 10Faculdade de Medicina de Botucatu, Universidade Estadual de São Paulo, Botucatu, Brazil. 11Universidade do Estado do Rio de Janeiro, Rio de Janeiro, Brazil. 12Hospital das Clínicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil. 13Hospital das Clínicas de Goiânia, Universidade Federal de Goiás, Goiânia, Brazil. 14Anis Rassi Hospital, Goiânia, Brazil. 15Hospital Universitário Júlio Muller, Universidade Federal de Mato Grosso, Cuiabá, Brazil. 16Faculdade de Medicina, Universidade de Ciências da Saúde Alagoas, Maceió, Brazil. 17Faculdade de Medicina, Universidade Federal de Pernambuco, Recife, Brazil. 18Instituto de Medicina Integral Prof Fernando Figueira, Recife, Brazil. 19Hospital Universitário Oswaldo Cruz/PROCAPE, Universidade de Pernambuco, Recife, Brazil. 20Hospital Universitário Valter Cantídio, Universidade Federal do Ceará, Fortaleza, Brazil. 21Hospital Universitário, Universidade Federal Maranhão, São Luiz, Brazil. 22Instituto de Cardiologia, Fundação Universitária de Cardiologia, Porto Alegre, Brazil. 23Faculdade de Medicina, Universidade Federal de Pelotas, Pelotas, Brazil. 24Hospital do Coração, São Paulo, Brazil. Figure 3 PREVER-prevention trial cardiovascular outcomes. Cox hazard regression models. Blood pressure will be compared by ANOVA for multiple repeated measure- ments and factors, testing for the time-group interaction. Myocardial infarction Myocardial infarction Unstable angina requiring hospitalization hospitalization Stroke and Ischemic Transitory Attack Heart failure requiring hospitalization Heart failure requiring hospitalization Peripheral vascular disease requiring hospitalization hospitalization New subclinical evidence of vascular atherosclerotic disease atherosclerotic disease Sudden death Figure 3 PREVER-prevention trial cardiovascular outcomes. List of abbreviations BP: blood pressure; NNT: number needed to treat; ACE: Angiotensin converting inhibitors; EKG: Electrocardiogram. List of abbreviations BP: blood pressure; NNT: number needed to treat; ACE: Angiotensin converting inhibitors; EKG: Electrocardiogram. Fuchs et al. Trials 2011, 12:65 http://www.trialsjournal.com/content/12/1/65 Fuchs et al. Trials 2011, 12:65 http://www.trialsjournal.com/content/12/1/65 7. Law MR, Morris JK, Wald NJ: Use of blood pressure lowering drugs in the prevention of cardiovascular disease: meta-analysis of 147 randomised trials in the context of expectations from prospective epidemiological studies. BMJ 2009, 338:b1665. 8. Guyton AC, Coleman TG, Cowley AV Jr, Scheel KW, Manning RD Jr, Norman RA Jr: Arterial pressure regulation. Overriding dominance of the kidneys in long-term regulation and in hypertension. Am J Med 1972, 52:584-94. 9. Tobian L: Salt and hypertension. Lessons from animal models that relate to human hypertension. Hypertension 1991, 17(1 Suppl):I52-8. y y 10. Dahl LK: Salt intake and hypertension. In Genest. Edited by: Koiw, Kuchel. Hypertension. New York, McGraw Hill; 1977:548-559. 11. Folkow B, Hallback M, Lundgreen Y, Sivertsson R, Weiss L: Importance of adaptative changes in vascular design for establishment of primary hypertension, studied in man and in spontaneously hypertensive rats. Circ Res 1973, 32(Suppl I):2-16. 12. Kearney PM, Whelton M, Reynolds K, Muntner P, Whelton PK, He J: Global burden of hypertension: analysis of worldwide data. Lancet 2005, 365:217-23. 13. Leitschuh M, Cupples LA, Kannel W, Gagnon D, Chobanian A: High-normal blood pressure progression to hypertension in the Framingham Heart Study. Hypertension 1991, 17:22-27. 14. Moreira LB, Fuchs SC, Wiehe M, Gus M, Moraes RS, Fuchs FD: Incidence of hypertension in Porto Alegre, Brazil: a population-based study. J Hum Hypertens 2008, 22:48-50. 15. The Trials of Hypertension Prevention Collaborative Research Group: Effects of weight loss and sodium reduction intervention on blood pressure and hypertension incidence in overweight people with high-normal blood pressure. The Trials of Hypertension Prevention, phase II. Arch Intern Med 1997, 157:657-667. 16. Appel LJ, Moore TJ, Obarzanek E, Vollmer WM, Svetkey LP, Sacks FM, et al: A clinical trial of the effects of dietary patterns on blood pressure. DASH Collaborative Research Group. N Engl J Med 1997, 336:1117-1124. 17. Premier Collaborative Research Group: Effects of comprehensive lifestyle modification on blood pressure control. JAMA 2003, 289:2083-2093. 18. Hooper L, Bartlett C, Davey Smith G, Ebrahim S: Systematic review of long term effects of advice to reduce dietary salt in adults. BMJ 2002, 325:628. 19. Fuchs FD, Gus M, Moreira WD, Moreira LB, Moraes RS, Rosito GA, et al: Blood pressure effects of antyhypertensive drugs and lifestyle modification in a Brazilian hypertensive cohort. J Hypert 1997, 15:783-792. 20. Ethical approval The project was approved by the Ethics committee of the Hospital de Clínicas de Porto Alegre, which is . Russell LB, Valiyeva E, Carson JL: Effects of pre-hypertension on admissions and deaths: a simulation. Arch Intern Med 2004, 164:2119-24. Page 7 of 7 Page 7 of 7 Page 7 of 7 doi:10.1186/1745-6215-12-65 Cite this article as: Fuchs et al.: Prevention of hypertension in patients with pre-hypertension: protocol for the PREVER-prevention trial. Trials 2011 12:65. Fuchs et al. Trials 2011, 12:65 http://www.trialsjournal.com/content/12/1/65 Julius S, Nesbitt S, Egan B, Weber MA, Michelson EL, Kaciroti N, et al: Feasibility of treating prehypertension with an angiotensin-receptor blocker. N Engl J Med 2006, 354:1685-97. 21. Lüders S, Schrader J, Berger J, Unger T, Zidek W, Böhm M, et al: The PHARAO study: prevention of hypertension with the angiotensin- converting enzyme inhibitor ramipril in patients with high-normal blood pressure - a prospective, randomized, controlled prevention trial of the German Hypertension League. Journal of Hypertension 2008, 26:1487-1496. 22. Fuchs FD: Blood pressure-lowering drugs: essential therapy for some patients with normal blood pressure. Expert Rev Cardiovasc Ther 2004, 2:771-5. 23. Fuchs FD: The corporate bias and the molding of prescription practices: the case of hypertension. Braz J Med Biol Res 2009, 42:224-8. 23. Fuchs FD: The corporate bias and the molding of prescription practices: the case of hypertension. Braz J Med Biol Res 2009, 42:224-8. yp , 24. Fuchs FD: Diuretics: still essential drugs for the management of hypertension. Expert Rev Cardiovasc Ther 2009, 7:591-8. 24. Fuchs FD: Diuretics: still essential drugs for the management of hypertension. Expert Rev Cardiovasc Ther 2009, 7:591-8. 25. Franse LV, Pahor M, Di Bari M, Somes GW, Cushman WC, Applegate WB: Hypokalemia associated with diuretic use and cardiovascular events in the Systolic Hypertension in the Elderly Program. Hypertension 2000, 35:1025-30. 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Trials 2011 12:65. doi:10.1186/1745-6215-12-65 Cite this article as: Fuchs et al.: Prevention of hypertension in patients with pre-hypertension: protocol for the PREVER-prevention trial. Trials 2011 12:65.
https://openalex.org/W2044207779	https://journals.iucr.org/e/issues/2009/03/00/wk2097/wk2097.pdf	English	null	Construction of a dinuclear silver(I) coordination complex with a Schiff base containing 4-amino-1,2,4-triazole ligands	Acta crystallographica. Section E	2,009	cc-by	4,684	Qiaozhen Sun,* Feng Zheng, Xiaodan Sun and Wei Wang Department of Materials Chemistry, School of Materials Science and Engineering, Key Laboratory of Non-ferrous Metals of the Ministry of Education, Central South University, Changsha 410083, People’s Republic of China Correspondence e-mail: rosesunqz@yahoo.com.cn Experimental Crystal data [Ag2(C13H10N4O)4](NO3)2H2O Mr = 1310.78 Triclinic, P1 a = 9.8594 (15) A˚ b = 10.7081 (15) A˚ c = 12.8567 (19) A˚ = 82.391 (2) = 81.155 (2) = 77.626 (2) V = 1303.1 (3) A˚ 3 Z = 1 Mo K radiation = 0.83 mm1 T = 293 K 0.20 0.18 0.16 mm Data collection Bruker APEX CCD area-detector diffractometer Absorption correction: multi-scan (SADABS; Bruker, 2000) Tmin = 0.826, Tmax = 0.887 6610 measured reﬂections 4536 independent reﬂections 3137 reﬂections with I > 2(I) Rint = 0.118 Reﬁnement R[F 2 > 2(F 2)] = 0.055 wR(F 2) = 0.133 S = 1.00 4536 reﬂections 379 parameters H-atom parameters constrained max = 0.85 e A˚ 3 min = 0.84 e A˚ 3 Experimental Crystal data [Ag2(C13H10N4O)4](NO3)2H2O Mr = 1310.78 Triclinic, P1 a = 9.8594 (15) A˚ b = 10.7081 (15) A˚ c = 12.8567 (19) A˚ = 82.391 (2) = 81.155 (2) = 77.626 (2) V = 1303.1 (3) A˚ 3 Z = 1 Mo K radiation = 0.83 mm1 T = 293 K 0.20 0.18 0.16 mm Data collection Bruker APEX CCD area-detector diffractometer Absorption correction: multi-scan (SADABS; Bruker, 2000) Tmin = 0.826, Tmax = 0.887 6610 measured reﬂections 4536 independent reﬂections 3137 reﬂections with I > 2(I) Rint = 0.118 Reﬁnement R[F 2 > 2(F 2)] = 0.055 wR(F 2) = 0.133 S = 1.00 4536 reﬂections 379 parameters H-atom parameters constrained max = 0.85 e A˚ 3 min = 0.84 e A˚ 3 Received 1 November 2008; accepted 10 February 2009 Received 1 November 2008; accepted 10 February 2009 Key indicators: single-crystal X-ray study; T = 293 K; mean (C–C) = 0.008 A˚; disorder in solvent or counterion; R factor = 0.055; wR factor = 0.133; data-to- parameter ratio = 12.0. = 77.626 (2) V = 1303.1 (3) A˚ 3 Z = 1 Mo K radiation = 0.83 mm1 T = 293 K 0.20 0.18 0.16 mm The new ligand 1-(1,2,4-triazol-4-yliminomethyl)-2-naphthol (L) and the title silver(I) complex, namely bis[-1-(1,2,4- triazol-4-yliminomethyl)-2-naphthol]bis{[1-(1,2,4-triazol-4-yl- iminomethyl)-2-naphthol]silver(I)} dinitrate monohydrate, [Ag2(C13H10N4O)4](NO3)2H2O, were synthesized. Qiaozhen Sun,* Feng Zheng, Xiaodan Sun and Wei Wang Each silver center in the dimeric complex (related by an inversion centre) is coordinated by two bridging L ligands and one additional L ligand in a monodentate fashion, exhibiting a distorted trigonal-planar coordination. The discrete dimers are further linked through O—H O hydrogen bonds and weak – stacking interactions [the shortest atom–atom separation is ca 3.46 A˚ between the parallel stacking pairs]. Intramolecular O—H N hydrogen bonds are also present. metal-organic compounds Acta Crystallographica Section E Structure Reports Online ISSN 1600-5368 Acta Crystallographica Section E Structure Reports Online ISSN 1600-5368 Related literature Table 1 Hydrogen-bond geometry (A˚ , ). metal-organic compounds Han, W., Yi, L., Liu, Z.-Q., Gu, W., Yan, S.-P., Cheng, P., Liao, D.-Z. & Jiang, Z.-H. (2004). Eur. J. Inorg. Chem. pp. 2130–2136. Supplementary data and ﬁgures for this paper are available from the IUCr electronic archives (Reference: WK2097). ( ) pp ingele, M. H. & Brooker, S. (2003). Coord. Chem. Rev. 241, 1 g ( ) Liu, J. C., Guo, G. C., Huang, J. S. & You, X. Z. (2003). Inorg. Chem. 42, 235– 243. Liu, B., Xu, L., Guo, G.-C. & Huang, J.-S. (2006). Inorg. Chem. Commun. 9, 687–690. Table 1 For the structures of other triazole Schiff base compounds, see: Beckmann & Brooker (2003); Drabent et al. (2003, 2004); Garcia et al. (1997); Klingele & Brooker (2003); Liu et al. (2003, 2006); Wang et al. (2006); Yi et al. (2004); Zhai et al. (2006). For related literature, see: Han et al. (2004). D—H A D—H H A D A D—H A O1—H1A N1 0.82 1.83 2.548 (4) 145 O2—H2B N5 0.82 1.87 2.588 (5) 145 O1W—H1WA O3i 0.85 1.85 2.594 (15) 145 Symmetry code: (i) x; y 1; z. Data collection: SMART (Bruker, 2000); cell reﬁnement: SAINT (Bruker, 2000); data reduction: SAINT; program(s) used to solve structure: SHELXTL (Sheldrick, 2008); program(s) used to reﬁne structure: SHELXTL; molecular graphics: SHELXTL; software used to prepare material for publication: SHELXTL. The authors acknowledge the ﬁnancial support from the Innovation Program for College Students of Central South University (grant No. 081053308). Sun and Zheng m283 Acta Cryst. (2009). E65, m283–m284 doi:10.1107/S1600536809004760 Acta Cryst. (2009). E65, m283–m284 m284 Sun and Zheng [Ag2(C13H10N4O)4](NO3)2H2O ( ) g pp Klingele, M. H. & Brooker, S. (2003). Coord. Chem. Rev. 241, 119–132. Han, W., Yi, L., Liu, Z.-Q., Gu, W., Yan, S.-P., Cheng, P., Liao, D.-Z. & Jiang, Z.-H. (2004). Eur. J. Inorg. Chem. pp. 2130–2136. S1. Comment Sheets formed through C-H···O hydrogen bonds are further aggregated into three- dimensions by weak π-π stacking interactions between the naphthyl rings of the neighbouring dimers in different sheets and the shortest atom···atom separation is ca 3.46 Å between the parallel stacking pairs. The anions and water molecules interact with one another through O—H···N, O—H···O hydrogen bonds. References Beckmann, U. & Brooker, S. (2003). Coord. Chem. Rev. 245, 17–29. Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122. Wang, Y., Yi, L., Yang, X., Ding, B., Cheng, P., Liao, D.-Z. & Yan, S.-P. (2006). Inorg. Chem. 45, 5822–5829. Bruker (2000). SMART, SAINT and SADABS. Bruker AXS Inc., Madison, Wisconsin, USA. Yi, L., Ding, B., Zhao, B., Cheng, P., Liao, D.-Z., Yan, S.-P. & Jiang, Z.-H. (2004). Inorg. Chem. 43, 33–43. Drabent, K., Białon´ska, A. & Ciunik, Z. (2004). Inorg. Chem. Commun. 7, 224–227. Zhai, Q.-G., Wu, X.-Y., Chen, S.-M., Lu, C.-Z. & Yang, W.-B. (2006). Cryst. Growth Des. 6, 2126–2135. Drabent, K., Ciunik, Z. & Chmielewski, P. J. (2003). Eur. J. Inorg. Chem. pp. 1548–1554. Garcia, Y., van Koningsbruggen, P. J., Bravic, G., Guionneau, P., Chasseau, D., Cascarano, G. L., Moscovici, J., Lambert, K., Michalowicz, A. & Kahn, O. (1997). Inorg. Chem. 36, 6357–6365. m284 m284 Sun and Zheng [Ag2(C13H10N4O)4](NO3)2H2O Acta Cryst. (2009). E65, m283–m284 supporting information supporting information Acta Cryst. (2009). E65, m283–m284 [doi:10.1107/S1600536809004760] Acta Cryst. (2009). E65, m283–m284 [doi:10.1107/S1600536809004760] S1. Comment 1,2,4-triazoles and their derivatives are interesting bridging ligands. 1,2,4-triazoles can coordinate with metals by bridging two adjacent nitrogen atoms (N1 and N2) or via the 4-positioned one (N4). It is also a readily available and inexpensive resource. In the past two decades, a variety of coordination compounds containing 1,2,4-triazoles, N4 substituted 1,2,4-triazoles and their derivatives as ligands coordinated to metal ions have been reported (Beckmann & Brooker, 2003; Garcia et al., 1997; Klingele & Brooker, 2003; Liu et al., 2006; Liu et al., 2003; Yi et al., 2004; Zhai et al., 2006). Relatively few structurally characterized compounds based on 4-amido-1,2,4-triazoles Schiff base ligands have been reported (Drabent et al., 2004 and 2003; Wang et al., 2006). Here we describe the synthesis of the Ag(I) metal complex with a Schiff-base containing triazole ligand. The molecular structure of complex 1 is shown in Figure 1. It consists of a discrete binuclear complex of Ag(I) bridged by two N1,N2-coordinated triazole ligands and an additional triazole ligand is bound to the Ag(I) ion in a monodentate fashion. This coordination mode results in a trigonal planar coordination environment (the sum of the angles around Ag metal atom is equal to 360 °). The Ag—Ag distance is equal to 3.81 Å, which is over the summed van der Waals radii of two Ag(I) atoms (3.44 Å) (Han et al., 2004). The Ag—N bond distances are in the range of 2.18–2.33 Å. The six- membered Ag-[N—N]2-Ag rings remain almost planar (the mean plane deviation is 0.06 Å) from planarity, which is similar withdinuclear Cu(I) complex (Drabent et al., 2004). The Ag—N—N—Ag dihedral angle is 15.1 °. In this complex all the ligands L are coordinated in almost planar E configuration and the resulting binuclear units can be described as X-shaped dimers. Sheets formed through C-H···O hydrogen bonds are further aggregated into three- dimensions by weak π-π stacking interactions between the naphthyl rings of the neighbouring dimers in different sheets and the shortest atom···atom separation is ca 3.46 Å between the parallel stacking pairs. The anions and water molecules interact with one another through O—H···N, O—H···O hydrogen bonds. In this complex all the ligands L are coordinated in almost planar E configuration and the resulting binuclear units can be described as X-shaped dimers. S3. Refinement All of the non-hydrogen atoms were refined with anisotropic thermal displacement coefficients. The positions of hydrogen atoms were fixed geometrically at calculated distances and allowed to ride on the parent non-hydrogen atoms. The water molecule was refined as disordered with the s.o.f. being fixed at 0.5 and its hydrogen atoms located in the difference Fourier maps and fixed at calculated distances from the parent oxygen atom. All of the non-hydrogen atoms were refined with anisotropic thermal displacement coefficients. The positions of hydrogen atoms were fixed geometrically at calculated distances and allowed to ride on the parent non-hydrogen atoms. y g g y p y g The water molecule was refined as disordered with the s.o.f. being fixed at 0.5 and its hydrogen atoms located in the difference Fourier maps and fixed at calculated distances from the parent oxygen atom. Figure 1 A perspective view of the molecular structure showing the atom-numbering scheme. Displacement ellipsoids are drawn at the 30% probability level. [symmetry code: (A) 2 - x, -y, 2 - z.] S2. Experimental Preparation of complex 1: The ligand L (0.1 mmol, 0.024 g) and AgNO3 (0.1 mmol, 0.017 g) were mixed in acetonitrile and stirred at room temperature for one hour, the yellow solution was filtered and evaporated at room temperature. A few days later orange block crystals were obtained. Preparation of the ligand L: An ethanolic solution (20 ml) of 2-hydroxy-1-napthaldehyde (1.72 g, 10 mmol) was added to a warm ethanolic solution (10 ml) of 4-amino- 1,2,4-triazole (0.84 g, 10 mmol) and the resulting solution was refluxed for four hours. The reaction mixture was then cooled to room temperature. Upon standing overnight the resultant yellow solid was filtered off, washed with diethyl ether and dried under vacuum. Yield: 90%. 1H NMR (500 MHz, DMSO, 298 K): 9.66 (s, 1H), 9.34 (s, 2H), 8.84–8.86 (d, 1H), 8.05–8.07 (d, 1H), 7.90–7.92 (d, 1H), 7.61–7.64 (t, 1H), 7.43–7.46 (t, 1H), 7.28–7.30 (d, 1H). sup-1 Acta Cryst. (2009). E65, m283–m284 supporting information Acta Cryst. (2009). E65, m283–m284 Figure 1 Figure 1 A perspective view of the molecular structure showing the atom-numbering scheme. Displacement ellipsoids are drawn at the 30% probability level. [symmetry code: (A) 2 - x, -y, 2 - z.] g A perspective view of the molecular structure showing the atom-numbering scheme. Displacement ellipsoids are drawn at the 30% probability level. [symmetry code: (A) 2 - x, -y, 2 - z.] g A perspective view of the molecular structure showing the atom-numbering scheme. Displacement ellipsoids are drawn at the 30% probability level. [symmetry code: (A) 2 - x, -y, 2 - z.] sup-2 Acta Cryst. (2009). E65, m283–m284 supporting information Figure 2 A ki di f th t l l b i th NO - H O d h d t itt d f l it Refinement Refinement Refinement on F2 Least-squares matrix: full R[F2 > 2σ(F2)] = 0.055 wR(F2) = 0.133 S = 1.00 4536 reflections 379 parameters 0 restraints Primary atom site location: structure-invariant direct methods Secondary atom site location: difference Fourier map Hydrogen site location: geom, H2O from difmap H-atom parameters constrained w = 1/[σ2(Fo2) + (0.0601P)2] where P = (Fo2 + 2Fc2)/3 (Δ/σ)max = 0.001 Δρmax = 0.85 e Å−3 Δρmin = −0.84 e Å−3 Secondary atom site location: difference Fourier map Hydrogen site location: geom, H2O from difmap H-atom parameters constrained w = 1/[σ2(Fo2) + (0.0601P)2] where P = (Fo2 + 2Fc2)/3 (Δ/σ)max = 0.001 Δρmax = 0.85 e Å−3 Δρmin = −0.84 e Å−3 Secondary atom site location: difference Fourier map Hydrogen site location: geom, H2O from difmap H-atom parameters constrained w = 1/[σ2(Fo2) + (0.0601P)2] where P = (Fo2 + 2Fc2)/3 (Δ/σ)max = 0.001 Δρmax = 0.85 e Å−3 Δρmin = −0.84 e Å−3 Figure 2 A packing diagram for the crystal along b axis, the NO3-, H2O and hydrogen atoms are omitted for clarity. bis[µ-1-(1,2,4-triazol-4-yliminomethyl)-2-naphthol]bis{[1-(1,2,4-triazol-4- yliminomethyl)-2-naphthol]silver(I)} dinitrate monohydrate bis[µ-1-(1,2,4-triazol-4-yliminomethyl)-2-naphthol]bis{[1-(1,2,4-triazol-4- yliminomethyl)-2-naphthol]silver(I)} bis[µ-1-(1,2,4-triazol-4-yliminomethyl)-2-naphthol]bis{[1-(1,2,4-triazol-4- yliminomethyl)-2-naphthol]silver(I)} dinitrate monohydrate Crystal data [Ag2(C13H10N4O)4](NO3)2·H2O Mr = 1310.78 Triclinic, P1 Hall symbol: -P 1 a = 9.8594 (15) Å b = 10.7081 (15) Å c = 12.8567 (19) Å α = 82.391 (2)° β = 81.155 (2)° γ = 77.626 (2)° V = 1303.1 (3) Å3 Z = 1 F(000) = 662 Dx = 1.670 Mg m−3 Mo Kα radiation, λ = 0.71073 Å Cell parameters from 2250 reflections θ = 2.4–25.4° µ = 0.83 mm−1 T = 293 K Block, orange 0.2 × 0.18 × 0.16 mm Data collection Bruker APEX CCD area-detector diffractometer Radiation source: fine-focus sealed tube Graphite monochromator φ and ω scans Absorption correction: multi-scan (SADABS; Bruker, 2000) Tmin = 0.826, Tmax = 0.887 6610 measured reflections 4536 independent reflections Z = 1 F(000) = 662 Dx = 1.670 Mg m−3 Mo Kα radiation, λ = 0.71073 Å Cell parameters from 2250 reflections θ = 2.4–25.4° µ = 0.83 mm−1 T = 293 K Block, orange 0.2 × 0.18 × 0.16 mm Absorption correction: multi-scan (SADABS; Bruker, 2000) Tmin = 0.826, Tmax = 0.887 6610 measured reflections 4536 independent reflections sup-3 Acta Cryst. (2009). E65, m283–m284 supporting information h = −10→11 k = −12→8 l = −15→14 3137 reflections with I > 2σ(I) Rint = 0.118 θmax = 25.0°, θmin = 1.6° 3137 reflections with I > 2σ(I) Rint = 0.118 θmax = 25.0°, θmin = 1.6° h = −10→11 k = −12→8 l = −15→14 Refinement Refinement on F2 Least-squares matrix: full R[F2 > 2σ(F2)] = 0.055 wR(F2) = 0.133 S = 1.00 4536 reflections 379 parameters 0 restraints Primary atom site location: structure-invariant direct methods Secondary atom site location: difference Fourier map Hydrogen site location: geom, H2O from difmap H-atom parameters constrained w = 1/[σ2(Fo2) + (0.0601P)2] where P = (Fo2 + 2Fc2)/3 (Δ/σ)max = 0.001 Δρmax = 0.85 e Å−3 Δρmin = −0.84 e Å−3 Special details Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell e.s.d.'s are taken into account individually in the estimation of e.s.d.'s in distances, angles and torsion angles; correlations between e.s.d.'s in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s. planes. Refinement. Refinement of F2 against ALL reflections. The weighted R-factor wR and goodness of fit S are based on F2, conventional R-factors R are based on F, with F set to zero for negative F2. The threshold expression of F2 > σ(F2) is used only for calculating R-factors(gt) etc. and is not relevant to the choice of reflections for refinement. R-factors based on F2 are statistically about twice as large as those based on F, and R- factors based on ALL data will be even larger. Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) x y z Uiso/Ueq Occ. (<1) Ag1 0.84563 (4) 0.12190 (4) 0.95202 (3) 0.0613 (2) O1 0.3147 (4) 0.5935 (3) 0.5781 (2) 0.0564 (9) H1A 0.3862 0.5514 0.6002 0.085 O2 1.2327 (4) 0.0054 (4) 0.4026 (3) 0.0701 (11) H2B 1.2018 0.0029 0.4656 0.105* N1 0.4657 (4) 0.4732 (4) 0.7179 (3) 0.0488 (10) N2 0.5888 (4) 0.3908 (4) 0.7472 (3) 0.0447 (10) N3 0.7966 (4) 0.2720 (4) 0.7236 (3) 0.0590 (12) N4 0.7427 (4) 0.2564 (4) 0.8288 (3) 0.0554 (11) N5 1.2436 (4) −0.0497 (4) 0.6037 (3) 0.0453 (9) N6 1.1907 (4) −0.0406 (3) 0.7095 (3) 0.0412 (9) N7 1.0373 (4) 0.0221 (4) 0.8420 (3) 0.0489 (10) N8 1.1554 (4) −0.0524 (4) 0.8806 (3) 0.0447 (10) C1 0.1290 (5) 0.6319 (4) 0.8481 (4) 0.0423 (11) C2 0.1404 (6) 0.6048 (5) 0.9573 (4) 0.0530 (13) H2A 0.2232 0.5561 0.9789 0.064* C3 0.0338 (6) 0.6481 (6) 1.0306 (4) 0.0633 (15) H3A 0.0449 0.6287 1.1019 0.076* C4 −0.0937 (6) 0.7218 (6) 1.0028 (4) 0.0663 (15) H4A −0.1664 0.7506 1.0545 0.080* C5 −0.1082 (6) 0.7501 (5) 0.8987 (5) 0.0650 (15) H5A −0.1922 0.7993 0.8794 0.078* Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) x y z Uiso/Ueq l atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) sup-4 Acta Cryst. (2009). Acta Cryst. (2009). E65, m283–m284 Special details E65, m283–m284 supporting information C6 −0.0002 (5) 0.7072 (5) 0.8189 (4) 0.0514 (12) C7 −0.0158 (6) 0.7374 (5) 0.7109 (4) 0.0582 (14) H7A −0.1003 0.7859 0.6921 0.070 C8 0.0863 (6) 0.6988 (5) 0.6352 (4) 0.0552 (14) H8A 0.0722 0.7200 0.5646 0.066* C9 0.2163 (5) 0.6255 (4) 0.6608 (4) 0.0442 (11) C10 0.2382 (5) 0.5910 (4) 0.7651 (4) 0.0404 (11) C11 0.3702 (5) 0.5102 (4) 0.7907 (4) 0.0419 (11) H11A 0.3840 0.4858 0.8611 0.050* C12 0.6178 (5) 0.3277 (5) 0.8406 (4) 0.0515 (13) H12A 0.5582 0.3338 0.9040 0.062* C13 0.7004 (5) 0.3530 (5) 0.6776 (4) 0.0548 (14) H13A 0.7082 0.3809 0.6057 0.066* C14 1.5717 (5) −0.2019 (5) 0.4532 (4) 0.0459 (11) C15 1.6506 (5) −0.2714 (5) 0.5336 (4) 0.0570 (13) H15A 1.6110 −0.2711 0.6041 0.068* C16 1.7841 (6) −0.3386 (6) 0.5089 (5) 0.0684 (16) H16A 1.8327 −0.3846 0.5629 0.082* C17 1.8483 (7) −0.3395 (6) 0.4050 (6) 0.083 (2) H17A 1.9405 −0.3821 0.3897 0.099* C18 1.7744 (7) −0.2771 (6) 0.3256 (5) 0.0719 (17) H18A 1.8153 −0.2810 0.2556 0.086* C19 1.6372 (6) −0.2068 (5) 0.3479 (4) 0.0577 (14) C20 1.5622 (7) −0.1409 (6) 0.2656 (4) 0.0679 (17) H20A 1.6040 −0.1457 0.1959 0.082* C21 1.4303 (7) −0.0703 (6) 0.2851 (4) 0.0673 (17) H21A 1.3840 −0.0259 0.2294 0.081* C22 1.3641 (6) −0.0650 (5) 0.3907 (4) 0.0526 (13) C23 1.4329 (5) −0.1275 (4) 0.4742 (3) 0.0435 (11) C24 1.3675 (5) −0.1161 (5) 0.5824 (3) 0.0441 (11) H24A 1.4163 −0.1578 0.6377 0.053* C25 1.0619 (5) 0.0279 (5) 0.7388 (4) 0.0477 (12) H25A 1.0000 0.0725 0.6926 0.057* C26 1.2458 (5) −0.0883 (4) 0.7999 (3) 0.0432 (11) H26A 1.3346 −0.1390 0.8039 0.052* N9 0.5609 (6) 0.7008 (7) 0.8823 (5) 0.0847 (17) O3 0.5165 (10) 0.7921 (8) 0.9419 (6) 0.183 (4) O4 0.5557 (6) 0.7361 (6) 0.7950 (4) 0.125 (2) O5 0.5869 (6) 0.5986 (5) 0.9295 (5) 0.116 (2) O1W 0.6044 (13) −0.0162 (13) 0.9945 (10) 0.143 (5) 0.50 H1WA 0.6068 −0.0783 0.9589 0.171* 0.50 H1WB 0.5600 −0.0207 1.0566 0.171* 0.50 Atomic displacement parameters (Å2) sup-5 Acta Cryst. (2009). Acta Cryst. (2009). E65, m283–m284 Special details E65, m283–m284 Atomic displacement parameters (Å2) U11 U22 U33 U12 U13 U23 Ag1 0.0486 (3) 0.0810 (4) 0.0381 (2) 0.0158 (2) −0.00257 (16) 0.00200 (18) O1 0.053 (2) 0.067 (2) 0.0399 (17) 0.0063 (18) −0.0092 (16) 0.0002 (16) Atomic displacement parameters (Å2) supporting information O2 0.076 (3) 0.077 (3) 0.0453 (19) 0.013 (2) −0.0091 (18) −0.0055 (18) N1 0.041 (2) 0.054 (3) 0.046 (2) 0.005 (2) −0.0109 (19) −0.0009 (19) N2 0.037 (2) 0.051 (2) 0.039 (2) 0.0072 (18) −0.0074 (17) −0.0035 (17) N3 0.046 (3) 0.076 (3) 0.041 (2) 0.011 (2) −0.0012 (19) −0.001 (2) N4 0.048 (3) 0.073 (3) 0.037 (2) 0.009 (2) −0.0116 (19) −0.001 (2) N5 0.047 (2) 0.046 (2) 0.0352 (19) 0.0018 (19) 0.0022 (17) −0.0045 (16) N6 0.038 (2) 0.041 (2) 0.0361 (19) 0.0033 (18) 0.0052 (16) −0.0051 (16) N7 0.038 (2) 0.054 (3) 0.043 (2) 0.0105 (19) −0.0008 (17) −0.0038 (18) N8 0.037 (2) 0.053 (3) 0.036 (2) 0.0062 (18) −0.0033 (17) −0.0029 (17) C1 0.036 (3) 0.039 (3) 0.051 (3) −0.003 (2) −0.010 (2) −0.004 (2) C2 0.045 (3) 0.061 (3) 0.052 (3) −0.008 (3) −0.011 (2) −0.003 (2) C3 0.058 (4) 0.087 (4) 0.044 (3) −0.017 (3) 0.006 (2) −0.013 (3) C4 0.049 (3) 0.082 (4) 0.063 (3) −0.004 (3) 0.009 (3) −0.022 (3) C5 0.041 (3) 0.060 (4) 0.089 (4) 0.001 (3) −0.005 (3) −0.012 (3) C6 0.035 (3) 0.051 (3) 0.066 (3) −0.002 (2) −0.009 (2) −0.008 (2) C7 0.043 (3) 0.058 (3) 0.070 (4) 0.005 (3) −0.024 (3) −0.001 (3) C8 0.056 (3) 0.056 (3) 0.049 (3) 0.006 (3) −0.022 (3) 0.000 (2) C9 0.046 (3) 0.041 (3) 0.045 (3) −0.003 (2) −0.011 (2) −0.001 (2) C10 0.034 (3) 0.036 (3) 0.051 (3) −0.003 (2) −0.011 (2) −0.005 (2) C11 0.037 (3) 0.046 (3) 0.039 (2) 0.000 (2) −0.007 (2) −0.005 (2) C12 0.042 (3) 0.067 (3) 0.035 (2) 0.014 (2) −0.008 (2) −0.006 (2) C13 0.045 (3) 0.071 (4) 0.036 (2) 0.009 (3) −0.001 (2) 0.002 (2) C14 0.048 (3) 0.043 (3) 0.048 (3) −0.014 (2) 0.005 (2) −0.014 (2) C15 0.048 (3) 0.058 (3) 0.062 (3) −0.004 (3) 0.006 (3) −0.019 (3) C16 0.041 (3) 0.076 (4) 0.086 (4) −0.004 (3) −0.001 (3) −0.022 (3) C17 0.053 (4) 0.079 (5) 0.109 (5) −0.007 (3) 0.025 (4) −0.040 (4) C18 0.068 (4) 0.076 (4) 0.069 (4) −0.024 (3) 0.031 (3) −0.029 (3) C19 0.062 (4) 0.056 (3) 0.055 (3) −0.022 (3) 0.018 (3) −0.020 (3) C20 0.085 (5) 0.074 (4) 0.042 (3) −0.027 (4) 0.022 (3) −0.014 (3) C21 0.097 (5) 0.068 (4) 0.033 (3) −0.014 (4) −0.002 (3) −0.002 (2) C22 0.063 (4) 0.048 (3) 0.043 (3) −0.008 (3) 0.003 (2) −0.008 (2) C23 0.049 (3) 0.040 (3) 0.039 (2) −0.010 (2) 0.005 (2) −0.008 (2) C24 0.042 (3) 0.048 (3) 0.039 (2) −0.006 (2) 0.003 (2) −0.003 (2) C25 0.038 (3) 0.055 (3) 0.039 (2) 0.007 (2) −0.002 (2) 0.001 (2) C26 0.041 (3) 0.047 (3) 0.036 (2) 0.002 (2) −0.005 (2) −0.004 (2) N9 0.076 (4) 0.099 (5) 0.081 (4) −0.028 (4) −0.029 (3) 0.021 (4) O3 0.216 (9) 0.163 (7) 0.149 (6) −0.041 (6) 0.080 (6) −0.057 (6) O4 0.101 (4) 0.177 (6) 0.076 (3) −0.002 (4) −0.015 (3) 0.031 (4) O5 0.132 (5) 0.090 (4) 0.118 (4) −0.007 (3) −0.041 (4) 0.027 (3) O1W 0.115 (10) 0.181 (12) 0.125 (9) −0.003 (9) 0.005 (8) −0.057 (8) G i (Å º) Geometric parameters (Å, º) Ag1—N8i 2.182 (3) C7—H7A 0.9300 Ag1—N4 2.209 (4) C8—C9 1.413 (6) Ag1—N7 2.329 (4) C8—H8A 0.9300 O1—C9 1.351 (6) C9—C10 1.381 (6) O1—H1A 0.8200 C10—C11 1.458 (6) sup-6 supporting information supporting information supporting information C25—N7—N8 106.9 (4) C17—C16—H16A 119.3 C25—N7—Ag1 130.5 (3) C18—C17—C16 119.1 (6) N8—N7—Ag1 122.5 (3) C18—C17—H17A 120.4 C26—N8—N7 107.8 (3) C16—C17—H17A 120.4 C26—N8—Ag1i 129.5 (3) C17—C18—C19 121.0 (6) N7—N8—Ag1i 121.5 (3) C17—C18—H18A 119.5 C2—C1—C6 117.2 (4) C19—C18—H18A 119.5 C2—C1—C10 124.7 (4) C18—C19—C20 120.7 (5) C6—C1—C10 118.1 (4) C18—C19—C14 120.6 (6) C3—C2—C1 121.2 (5) C20—C19—C14 118.7 (5) C3—C2—H2A 119.4 C21—C20—C19 121.9 (5) C1—C2—H2A 119.4 C21—C20—H20A 119.1 C2—C3—C4 121.9 (5) C19—C20—H20A 119.1 C2—C3—H3A 119.0 C20—C21—C22 119.6 (5) C4—C3—H3A 119.0 C20—C21—H21A 120.2 C5—C4—C3 118.5 (5) C22—C21—H21A 120.2 C5—C4—H4A 120.8 O2—C22—C23 123.6 (5) C3—C4—H4A 120.8 O2—C22—C21 115.6 (5) C4—C5—C6 121.9 (5) C23—C22—C21 120.8 (5) C4—C5—H5A 119.0 C22—C23—C14 119.4 (4) C6—C5—H5A 119.0 C22—C23—C24 120.6 (5) C5—C6—C7 121.6 (5) C14—C23—C24 120.0 (4) C5—C6—C1 119.2 (5) N5—C24—C23 121.5 (4) C7—C6—C1 119.2 (5) N5—C24—H24A 119.3 C8—C7—C6 121.9 (5) C23—C24—H24A 119.3 C8—C7—H7A 119.0 N7—C25—N6 109.7 (4) C6—C7—H7A 119.0 N7—C25—H25A 125.2 C7—C8—C9 120.6 (4) N6—C25—H25A 125.2 C7—C8—H8A 119.7 N8—C26—N6 109.3 (4) C9—C8—H8A 119.7 N8—C26—H26A 125.3 O1—C9—C10 123.3 (4) N6—C26—H26A 125.3 O1—C9—C8 116.2 (4) O4—N9—O5 134.0 (9) C10—C9—C8 120.5 (5) O4—N9—O3 112.1 (8) C9—C10—C1 119.7 (4) O5—N9—O3 113.6 (7) C9—C10—C11 120.1 (4) H1WA—O1W—H1WB 115.9 C1—C10—C11 120.2 (4) C11—N1—N2—C13 −175.1 (5) C1—C10—C11—N1 179.3 (4) C11—N1—N2—C12 10.7 (8) N3—N4—C12—N2 −0.9 (6) C13—N3—N4—C12 0.2 (6) Ag1—N4—C12—N2 −176.5 (3) C13—N3—N4—Ag1 175.9 (4) C13—N2—C12—N4 1.1 (6) N8i—Ag1—N4—C12 −11.8 (6) N1—N2—C12—N4 176.1 (5) N7—Ag1—N4—C12 170.0 (5) N4—N3—C13—N2 0.5 (6) N8i—Ag1—N4—N3 173.4 (3) C12—N2—C13—N3 −1.0 (6) N7—Ag1—N4—N3 −4.9 (4) N1—N2—C13—N3 −176.5 (4) C24—N5—N6—C25 179.6 (4) C19—C14—C15—C16 0.5 (8) C24—N5—N6—C26 1.0 (7) C23—C14—C15—C16 −178.6 (5) N8i—Ag1—N7—C25 170.2 (4) C14—C15—C16—C17 1.3 (9) 106.9 (4) C17—C16—H16A 119.3 130.5 (3) C18—C17—C16 119.1 (6) 122.5 (3) C18—C17—H17A 120.4 107.8 (3) C16—C17—H17A 120.4 129.5 (3) C17—C18—C19 121.0 (6) 121.5 (3) C17—C18—H18A 119.5 117.2 (4) C19—C18—H18A 119.5 124.7 (4) C18—C19—C20 120.7 (5) 118.1 (4) C18—C19—C14 120.6 (6) 121.2 (5) C20—C19—C14 118.7 (5) 119.4 C21—C20—C19 121.9 (5) 119.4 C21—C20—H20A 119.1 121.9 (5) C19—C20—H20A 119.1 119.0 C20—C21—C22 119.6 (5) 119.0 C20—C21—H21A 120.2 118.5 (5) C22—C21—H21A 120.2 120.8 O2—C22—C23 123.6 (5) 120.8 O2—C22—C21 115.6 (5) 121.9 (5) C23—C22—C21 120.8 (5) 119.0 C22—C23—C14 119.4 (4) 119.0 C22—C23—C24 120.6 (5) 121.6 (5) C14—C23—C24 120.0 (4) 119.2 (5) N5—C24—C23 121.5 (4) 119.2 (5) N5—C24—H24A 119.3 121.9 (5) C23—C24—H24A 119.3 119.0 N7—C25—N6 109.7 (4) 119.0 N7—C25—H25A 125.2 120.6 (4) N6—C25—H25A 125.2 119.7 N8—C26—N6 109.3 (4) 119.7 N8—C26—H26A 125.3 123.3 (4) N6—C26—H26A 125.3 116.2 (4) O4—N9—O5 134.0 (9) 120.5 (5) O4—N9—O3 112.1 (8) 119.7 (4) O5—N9—O3 113.6 (7) 120.1 (4) H1WA—O1W—H1WB 115.9 120.2 (4) −175.1 (5) C1—C10—C11—N1 179.3 (4) 10.7 (8) N3—N4—C12—N2 −0.9 (6) 0.2 (6) Ag1—N4—C12—N2 −176.5 (3) 175.9 (4) C13—N2—C12—N4 1.1 (6) −11.8 (6) N1—N2—C12—N4 176.1 (5) 170.0 (5) N4—N3—C13—N2 0.5 (6) 173.4 (3) C12—N2—C13—N3 −1.0 (6) −4.9 (4) N1—N2—C13—N3 −176.5 (4) 179.6 (4) C19—C14—C15—C16 0.5 (8) 1.0 (7) C23—C14—C15—C16 −178.6 (5) 170.2 (4) C14—C15—C16—C17 1.3 (9) sup-8 Acta Cryst. supporting information supporting informat O2—C22 1.349 (7) C11—H11A 0.9300 O2—H2B 0.8200 C12—H12A 0.9300 N1—C11 1.259 (6) C13—H13A 0.9300 N1—N2 1.410 (5) C14—C19 1.410 (6) N2—C13 1.333 (6) C14—C15 1.422 (7) N2—C12 1.338 (5) C14—C23 1.434 (7) N3—C13 1.298 (6) C15—C16 1.370 (8) N3—N4 1.377 (5) C15—H15A 0.9300 N4—C12 1.300 (6) C16—C17 1.388 (9) N5—C24 1.284 (6) C16—H16A 0.9300 N5—N6 1.389 (5) C17—C18 1.363 (9) N6—C25 1.349 (6) C17—H17A 0.9300 N6—C26 1.351 (5) C18—C19 1.407 (8) N7—C25 1.307 (6) C18—H18A 0.9300 N7—N8 1.383 (5) C19—C20 1.409 (8) N8—C26 1.301 (6) C20—C21 1.362 (9) N8—Ag1i 2.182 (3) C20—H20A 0.9300 C1—C2 1.412 (6) C21—C22 1.416 (7) C1—C6 1.428 (6) C21—H21A 0.9300 C1—C10 1.435 (6) C22—C23 1.378 (7) C2—C3 1.346 (8) C23—C24 1.450 (6) C2—H2A 0.9300 C24—H24A 0.9300 C3—C4 1.403 (8) C25—H25A 0.9300 C3—H3A 0.9300 C26—H26A 0.9300 C4—C5 1.355 (8) N9—O4 1.141 (6) C4—H4A 0.9300 N9—O5 1.176 (7) C5—C6 1.406 (7) N9—O3 1.284 (9) C5—H5A 0.9300 O1W—H1WA 0.8499 C6—C7 1.408 (7) O1W—H1WB 0.8500 C7—C8 1.326 (8) N8i—Ag1—N4 147.87 (16) N1—C11—C10 120.2 (4) N8i—Ag1—N7 114.48 (13) N1—C11—H11A 119.9 N4—Ag1—N7 97.64 (14) C10—C11—H11A 119.9 C9—O1—H1A 109.5 N4—C12—N2 109.3 (4) C22—O2—H2B 109.5 N4—C12—H12A 125.4 C11—N1—N2 117.8 (4) N2—C12—H12A 125.4 C13—N2—C12 106.0 (4) N3—C13—N2 111.0 (4) C13—N2—N1 123.0 (4) N3—C13—H13A 124.5 C12—N2—N1 130.8 (4) N2—C13—H13A 124.5 C13—N3—N4 105.8 (4) C19—C14—C15 116.7 (5) C12—N4—N3 108.0 (4) C19—C14—C23 119.6 (5) C12—N4—Ag1 126.5 (3) C15—C14—C23 123.7 (4) N3—N4—Ag1 125.4 (3) C16—C15—C14 121.1 (5) C24—N5—N6 117.6 (4) C16—C15—H15A 119.5 C25—N6—C26 106.3 (4) C14—C15—H15A 119.5 C25—N6—N5 121.5 (4) C15—C16—C17 121.4 (6) C26—N6—N5 132.2 (4) C15—C16—H16A 119.3 sup-7 Acta Cryst. (2009). E65, m283–m284 Acta Cryst. (2009). E65, m283–m284 supporting information (2009). E65, m283–m284 supporting information N4—Ag1—N7—C25 −10.8 (5) C15—C16—C17—C18 −3.2 (10) N8i—Ag1—N7—N8 −14.1 (4) C16—C17—C18—C19 3.3 (9) N4—Ag1—N7—N8 164.8 (3) C17—C18—C19—C20 179.0 (6) C25—N7—N8—C26 0.0 (5) C17—C18—C19—C14 −1.5 (9) Ag1—N7—N8—C26 −176.6 (3) C15—C14—C19—C18 −0.3 (7) C25—N7—N8—Ag1i −168.3 (3) C23—C14—C19—C18 178.7 (5) Ag1—N7—N8—Ag1i 15.1 (5) C15—C14—C19—C20 179.1 (5) C6—C1—C2—C3 0.0 (7) C23—C14—C19—C20 −1.8 (7) C10—C1—C2—C3 179.0 (5) C18—C19—C20—C21 −178.8 (6) C1—C2—C3—C4 0.1 (9) C14—C19—C20—C21 1.7 (9) C2—C3—C4—C5 −0.4 (9) C19—C20—C21—C22 −1.7 (9) C3—C4—C5—C6 0.4 (9) C20—C21—C22—O2 −178.9 (5) C4—C5—C6—C7 −179.7 (5) C20—C21—C22—C23 1.8 (9) C4—C5—C6—C1 −0.2 (8) O2—C22—C23—C14 178.8 (5) C2—C1—C6—C5 0.0 (7) C21—C22—C23—C14 −1.9 (8) C10—C1—C6—C5 −179.1 (5) O2—C22—C23—C24 −2.5 (8) C2—C1—C6—C7 179.5 (5) C21—C22—C23—C24 176.7 (5) C10—C1—C6—C7 0.5 (7) C19—C14—C23—C22 2.0 (7) C5—C6—C7—C8 179.1 (5) C15—C14—C23—C22 −179.0 (5) C1—C6—C7—C8 −0.5 (8) C19—C14—C23—C24 −176.7 (4) C6—C7—C8—C9 −0.3 (9) C15—C14—C23—C24 2.3 (7) C7—C8—C9—O1 −178.4 (5) N6—N5—C24—C23 −178.0 (4) C7—C8—C9—C10 1.2 (8) C22—C23—C24—N5 1.1 (7) O1—C9—C10—C1 178.4 (4) C14—C23—C24—N5 179.7 (5) C8—C9—C10—C1 −1.2 (7) N8—N7—C25—N6 0.4 (6) O1—C9—C10—C11 −3.4 (7) Ag1—N7—C25—N6 176.6 (3) C8—C9—C10—C11 177.0 (4) C26—N6—C25—N7 −0.7 (6) C2—C1—C10—C9 −178.6 (5) N5—N6—C25—N7 −179.5 (4) C6—C1—C10—C9 0.3 (7) N7—N8—C26—N6 −0.4 (5) C2—C1—C10—C11 3.2 (7) Ag1i—N8—C26—N6 166.7 (3) C6—C1—C10—C11 −177.8 (4) C25—N6—C26—N8 0.7 (6) N2—N1—C11—C10 −177.4 (4) N5—N6—C26—N8 179.4 (4) C9—C10—C11—N1 1.2 (7) Hydrogen-bond geometry (Å, º) D—H···A D—H H···A D···A D—H···A O1—H1A···N1 0.82 1.83 2.548 (4) 145 O2—H2B···N5 0.82 1.87 2.588 (5) 145 O1W—H1WA···O3ii 0.85 1.85 2.594 (15) 145 Symmetry code: (ii) x, y−1, z. sup-9 Acta Cryst. (2009). E65, m283–m284
https://openalex.org/W4205990741	https://journal.transpl.ru/vtio/article/download/1407/1224	Russian	null	Lung cancer in solid organ transplant recipients	Vestnik transplantologii i iskusstvennyh organov	2,021	cc-by	5,321	Ключевые слова: реципиенты солидных органов, иммуносупрессивная терапия, злокачественны опухоли, рак легких. Lung cancer in solid organ transplant recipients A.V. Nikulin, I.V. Pashkov, Ya.S. Yakunin Shumakov National Medical Research Center of Transplantology and Artificial Organs, Moscow, Russian Federation Lung cancer remains the leading cause of cancer mortality worldwide. Solid organ transplant recipients are at risk of developing malignant tumors, including lung cancer, due to long-term use of immunosuppressive drugs. Development of cancer, including lung cancer, in this patient cohort, has a number of peculiarities. Moreover, malignant tumors in these patients are difficult to treat and have a poorer prognosis. This review presents a study of the issues concerning the mechanisms of lung cancer development, screening methods and treatment in solid organ transplant recipients. Трансплантация солидных органов является единст венным и до настоящего времени незаменимым ме- тодом лечения терминальных стадий заболеваний, когда другие средства лечения бессильны. В мире год от года растет количество трансплантаций со- лидных органов. 2020 год стал исключением из-за пандемии Covid-19. По данным международного общества донорства и трансплантации, в мире вы- полнено 113 363 трансплантации солидных органов. Реципиенты солидных органов находятся в группе риска развития злокачественных новообразований, включая рак легкого, за счет длительного приема иммуносупрессивных препаратов. Кроме того, зло- качественные новообразования у реципиентов со- лидных органов трудно поддаются лечению и имеют худший прогноз [2–8, 52]. Тел. (495) 190 35 62. E mail: nikulin5642@gmail.com Corresponding author: Andrey Nikulin. Address: 1, Shchukinskaya str., Moscow, 123182, Russian Federation. Phone: (495) 190-35-62. E-mail: nikulin5642@gmail.com Клиническая трансплантология Клиническая трансплантология ак легкого у реципиентов солидных органо А.В. Никулин, И.В. Пашков, Я.С. Якунин ФГБУ «Национальный медицинский исследовательский центр трансплантологии и искусственных органов имени академика В.И. Шумакова» Минздрава России, Москва, Российская Федерация Рак легкого остается ведущей причиной смерти в популяции среди всех злокачественных новообразований. Реципиенты солидных органов находятся в группе риска развития злокачественных новообразований, вклю- чая рак легкого, за счет длительного приема иммуносупрессивных препаратов. Развитие онкологических заболеваний, включая рак легкого, у этой категории больных отличается рядом особенностей. Кроме того, злокачественные новообразования у реципиентов солидных органов трудно поддаются лечению и имеют худший прогноз. Изучению вопросов, касающихся механизмов развития, методов скрининга и лечения рака легкого у реципиентов солидных органов, посвящен данный обзор. Ключевые слова: реципиенты солидных органов, иммуносупрессивная терапия, злокачественные опухоли, рак легких. Для корреспонденции: Никулин Андрей Владимирович. Адрес: 123182, Москва, ул. Щукинская, д. 1. Тел. (495) 190-35-62. E-mail: nikulin5642@gmail.com Corresponding author: Andrey Nikulin. Address: 1, Shchukinskaya str., Moscow, 123182, Russian Federation. Phone: (495) 190-35-62. E-mail: nikulin5642@gmail.com Для корреспонденции: Никулин Андрей Владимирович. Адрес: 123182, Москва, ул. Щукинская, д. 1. Тел. (495) 190-35-62. E-mail: nikulin5642@gmail.com DOI: 10.15825/1995-1191-2021-4-19-25 DOI: 10.15825/1995-1191-2021-4-19-25 Патогенез и факторы риска Во время наблюдения было диа- гностировано 23 (3,63%) случая заболевания раком легкого. У 5 пациентов рак легкого был случайной находкой в эксплантированном легком реципиента. У 18 пациентов рак развился de novo у реципиентов ВЕСТНИК ТРАНСПЛАНТОЛОГИИ И ИСКУССТВЕННЫХ ОРГАНОВ ВЕСТНИК ТРАНСПЛАНТОЛОГИИ И ИСКУССТВЕННЫХ ОРГАНОВ том XXIII № 4–2021 Заболеваемость У 18 пациентов рак развился de novo у реципиентов Общая заболеваемость злокачественными но- вообразованиями у реципиентов солидных орга- нов зависит от страны проживания, особенностей питания, привычек, состояния окружающей среды и других факторов. Мировая статистика приводит разные данные о заболеваемости злокачественными новообразованиями у реципиентов солидных орга- нов в зависимости от группы, возраста исследуемых и трансплантированного органа. Среднее значение заболеваемости составляет 2–6% [9, 10]. Наиболее часто встречаемыми злокачественными новообра- зованиями являются лимфопролиферативные забо- левания и рак кожи. Риск развития рака легкого у реципиентов солидных органов составляет от 0,3 до 0,85%, что аналогично заболеваемости населения в целом [9, 10]. Отмечается увеличение заболеваемос- ти раком легкого у реципиентов легких и сердца, в сравнении с реципиентами печени и почек (соот- ношение составило 5,5; 2,9; 2 и 1 соответственно). А у реципиентов комплекса «сердце–легкие» риск развития рака легкого в 9,3 раза выше по сравнению с общей популяцией. Авторы пришли к выводу, что это связано не только с иммуносупрессивной тера- пией, но также с возрастом и длительным стажем курения [9, 11]. По данным A.-M. Noone et al., cре- ди 221 962 исследованных реципиентов солидных органов у 15 012 развились злокачественные ново- образования (6,76%). Смертность от рака легкого составила 3,1%, от неходжкинской лимфомы – 1,9%, от колоректального рака – 0,7%, от рака почки – 0,5%. Заболеваемость однолегочного трансплантата (в 12 случаях в натив- ном легком и в 6 случаях в донорском легком) [14]. Заболеваемость Общая заболеваемость злокачественными но- вообразованиями у реципиентов солидных орга- нов зависит от страны проживания, особенностей питания, привычек, состояния окружающей среды и других факторов. Мировая статистика приводит разные данные о заболеваемости злокачественными новообразованиями у реципиентов солидных орга- нов в зависимости от группы, возраста исследуемых и трансплантированного органа. Среднее значение заболеваемости составляет 2–6% [9, 10]. Наиболее часто встречаемыми злокачественными новообра- зованиями являются лимфопролиферативные забо- левания и рак кожи. Риск развития рака легкого у реципиентов солидных органов составляет от 0,3 до 0,85%, что аналогично заболеваемости населения в целом [9, 10]. Отмечается увеличение заболеваемос- ти раком легкого у реципиентов легких и сердца, в сравнении с реципиентами печени и почек (соот- ношение составило 5,5; 2,9; 2 и 1 соответственно). А у реципиентов комплекса «сердце–легкие» риск развития рака легкого в 9,3 раза выше по сравнению с общей популяцией. Авторы пришли к выводу, что это связано не только с иммуносупрессивной тера- пией, но также с возрастом и длительным стажем курения [9, 11]. По данным A.-M. Noone et al., cре- ди 221 962 исследованных реципиентов солидных органов у 15 012 развились злокачественные ново- образования (6,76%). Смертность от рака легкого составила 3,1%, от неходжкинской лимфомы – 1,9%, от колоректального рака – 0,7%, от рака почки – 0,5%. Неходжкинская лимфома была самой частой причиной смерти у детей (4,1%), а рак легких – у реципиентов солидных органов в возрасте ≥50 лет (3,7–4,3%). Авторы пришли к выводу, что смерт ность от онкозаболеваний увеличивается с воз- растом и временем после трансплантации. Таким образом, по мере увеличения продолжительности жизни реципиентов солидных органов смертность от рака различной локализации будет становиться все более актуальной проблемой [12]. В исследовании E. Yanik et al. среди 187 384 реципиентов солидных органов, из которых реципиенты почки составили 58%, печени – 22%, сердца – 10% и легких – 4%, было выявлено 9323 случая возникновения злокачест- венных новообразований (4,97%). Наиболее распро- страненным был рак легкого (n = 1993; 1,06%) [13]. D. Pérez-Callejo et al. проанализировали истории бо- лезни 633 пациентов после трансплантации легких. Наиболее частыми причинами трансплантации были идиопатический фиброз легких (47,8%) и эмфизе- ма легких (43,4%). Во время наблюдения было диа- гностировано 23 (3,63%) случая заболевания раком легкого. У 5 пациентов рак легкого был случайной находкой в эксплантированном легком реципиента. Патогенез и факторы риска Существует несколько механизмов возникнове- ния рака легкого у реципиентов солидных органов: de novo в нативном легком (в случае однолегочной трансплантации), в донорском легком или как про- грессирование ранее существовавшей в экспланти- рованном легком опухоли. Сообщения о выявлении рака легкого в эксплантированном легком реципи- ента не так редки, что наталкивает на мысль о более тщательном обследовании реципиентов солидных органов перед операцией. Например, Y. Jun Choi et al. из 247 реципиентов легких у 6 (2,4%) диагностирова- ли рак легкого как случайную находку в эксплантиро- ванном легком [15]. Вероятность передачи опухоли с донорским легким крайне мала, однако такой меха- низм передачи существует, и необходимо в обязатель- ном порядке проводить компьютерную томографию потенциальному донору солидных органов. Это в большей степени относится к трансплантации лег- ких, однако в литературе описаны случаи передачи рака легкого, например, с трансплантированной пе- ченью (в данном наблюдении в печени был выявлен метастаз рака легкого из невыявленного очага) [16]. Среди факторов риска развития рака легкого по- мимо курения, которое большинство авторов считает основным, также выделяют вирус Эпштейна–Барр и прогрессирование посттрансплантационных лим- фопролиферативных заболеваний [14]. Кроме того, на развитие рака легкого могут оказывать влияние неблагоприятные условия окружающей среды, та- кие как воздействие диоксида кремния и асбеста. Некоторые терминальные стадии заболеваний, по поводу которых осуществляется трансплантация лег- ких, например хроническая обструктивная болезнь легких (ХОБЛ) и фиброз легких, также предполагают повышенный риск развития рака легкого при одноле- гочной трансплантации в сравнении с двулегочной. Большинство авторов говорит о возрастании риска развития рака легкого почти вдвое после 60 лет [8, 14, 17–21]. р р , , р , Неходжкинская лимфома была самой частой причиной смерти у детей (4,1%), а рак легких – у реципиентов солидных органов в возрасте ≥50 лет (3,7–4,3%). Авторы пришли к выводу, что смерт ность от онкозаболеваний увеличивается с воз- растом и временем после трансплантации. Таким образом, по мере увеличения продолжительности жизни реципиентов солидных органов смертность от рака различной локализации будет становиться все более актуальной проблемой [12]. В исследовании E. Yanik et al. среди 187 384 реципиентов солидных органов, из которых реципиенты почки составили 58%, печени – 22%, сердца – 10% и легких – 4%, было выявлено 9323 случая возникновения злокачест- венных новообразований (4,97%). Наиболее распро- страненным был рак легкого (n = 1993; 1,06%) [13]. D. Pérez-Callejo et al. проанализировали истории бо- лезни 633 пациентов после трансплантации легких. Наиболее частыми причинами трансплантации были идиопатический фиброз легких (47,8%) и эмфизе- ма легких (43,4%). ВВЕДЕНИЕ Несмотря на достижения последних лет в облас- ти иммунологии, генетики, фармакологии и других наук, рак легкого остается ведущей причиной смер- ти среди всех злокачественных новообразований. В мире с ростом числа курильщиков растет и забо- леваемость раком легкого [1]. По оценке международного агентства по изуче- нию рака легкого, в 2020 году выявлено 2 206 771 но- вых случаев заболевания раком легкого, что состав- ляет 11,4% от общего количества онкологических заболеваний. Смертность составила 1 796 144, что составляет 18% общей смертности от онкологиче ских заболеваний в 2020 году. У мужчин по уров- ню смертности он занимает первое место (21,5% в структуре общей смертности), а у женщин – второе место после рака молочной железы (13,7%) [51]. 19 Скрининг Возможность раннего выявления и своевремен- ного лечения злокачественных новообразований у реципиентов солидных органов напрямую зависит от периодических скрининговых обследований [25, 28]. Хотя ранняя диагностика рака легкого может улуч- шить результаты лечения у этой категории больных, вызывает недоумение позиция некоторых авторов, высказывающих сомнения в целесообразности про- ведения скрининга у реципиентов солидных органов с опасными для жизни сопутствующими заболева- ниями или с ожидаемой продолжительностью жиз- ни менее 5–10 лет [29]. Современные англоязычные рекомендации по скринингу рака различных локали- заций, включая рак легкого, для реципиентов солид- ных органов основаны на экстраполяции результатов скрининговых исследований в общей популяции, а также на понимании высокого риска развития рака легкого у этой категории больных [29]. У реципиен- тов легких с длительным стажем курения в анамнезе, несмотря на отказ от курения, тщательное наблюде- ние является обязательным условием ранней диаг ностики рака легкого [9]. Результаты скрининговой программы по выяв- лению рака легкого у населения в США показали, что использование низкодозовой мультиспиральной компьютерной томографии в сравнении с рентге- нографией снижает смертность от рака легких на 20% [20, 21]. Использование системы оценки данных «Lung-RADS» (от англ. Lung Imaging Reporting and Data System) для интерпретации выявленных при скрининге изменений позволяет стандартизировать описание компьютерной томографии (КТ), а также выработать четкие рекомендации по определению тактики лечения (табл.) [30, 31]. Таким образом, использование такой системы оценки данных для диагностики рака легкого у реципиентов солидных органов представляется перспективным. Видеоторакоскопические (ВАТС) анатомические резекции все чаще применяются в мире для лече- ния различных заболеваний, включая первичный рак легких [40]. По мере накопления хирургами практи- ческого опыта и совершенствования хирургической техники расширяется спектр применения ВАТС-опе- раций в различных сферах хирургии органов грудной полости [40]. Подавляющее большинство торакаль- ных операций, ранее традиционно выполняемых из торакотомии, могут быть выполнены при помощи эндоскопического оборудования из небольших раз- резов [41]. В специализированных торакальных от- делениях количество ВАТС-лобэктомий зачастую превосходит количество выполняемых открытых лобэктомий [41]. Постепенный отказ от использова- Иммуносупрессивная терапия – специфический фактор риска рака легкого Иммунотерапия в настоящее время выходит на первое место в ряде случаев при различных типах опухолей (высокая экс- прессия PD-L1 и мутационной нагрузки опухоли), однако вмешательство в иммунную систему может иметь катастрофические последствия у пациентов, находящихся на иммуносупрессивной терапии, т. к. остается неизученным вопрос одновременного при- ема иммуноонкологических и иммуносупрессивных препаратов [35]. Хирургическая операция является «золотым стандартом» при I и II стадии немелко клеточного рака легкого (НМРЛ). Вместе с тем сте- реотаксическая абляционная лучевая терапия (СЛТ) (от англ. SABR – Stereotactic ablative radiotherapy) является методом выбора у неоперабельных из-за соматического статуса пациентов с НМРЛ I стадии [36–38]. Однако безопасность СЛТ не была оцене- на у реципиентов солидных органов, что является серьезным недостатком данного метода. По мнению G. Drevet et al., хирургический метод лечения более предпочтителен в лечении рака легких II и IIIА ста- дий в случае резектабельной опухоли и операбель- ности пациентов. Традиционная лучевая терапия и химиотерапия рекомендуются для лечения неопе- рабельного рака легких II стадии и местнораспро- страненного рака легких III стадии. У реципиентов солидных органов следует соблюдать особую осто- рожность при назначении лучевой или химиотерапии в связи с иммуносупрессивной терапией, сопутству- ющими заболеваниями, частым наличием почечной недостаточности [39]. Иммуносупрессивная терапия – специфический фактор риска рака легкого Утрата иммунологического надзора в связи со снижением противоопухолевого иммунитета, в осо- бенности у пациентов с легочным фиброзом, риски заболеваемости раком легкого у которых примерно в 7 раз выше в сравнении с популяцией, активирование проонкогенных вирусов, непосредственное канцеро- генное действие иммуносупрессивных препаратов – специфические факторы риска развития рака легкого 20 Клиническая трансплантология у реципиентов солидных органов в сравнении с по- пуляцией [4–6, 12, 22–27]. рамках комплексного лечения часто не может быть проведена в полном объеме из-за сопутствующих заболеваний и опасности отторжения трансплантата, вызываемой снижением дозировок иммуносупрес- сивных препаратов [3]. В англоязычной литературе по настоящее время нет рекомендаций по изменению схемы иммуносупрессивной терапии у реципиен- тов солидных органов после диагностированного рака легкого, хотя при проведении химиотерапии обычно происходит снижение интенсивности им- муносупрессивной терапии [33, 34]. Иммунотерапия в настоящее время выходит на первое место в ряде случаев при различных типах опухолей (высокая экс- прессия PD-L1 и мутационной нагрузки опухоли), однако вмешательство в иммунную систему может иметь катастрофические последствия у пациентов, находящихся на иммуносупрессивной терапии, т. к. остается неизученным вопрос одновременного при- ема иммуноонкологических и иммуносупрессивных препаратов [35]. Хирургическая операция является «золотым стандартом» при I и II стадии немелко клеточного рака легкого (НМРЛ). Вместе с тем сте- реотаксическая абляционная лучевая терапия (СЛТ) (от англ. SABR – Stereotactic ablative radiotherapy) является методом выбора у неоперабельных из-за соматического статуса пациентов с НМРЛ I стадии [36–38]. Однако безопасность СЛТ не была оцене- на у реципиентов солидных органов, что является серьезным недостатком данного метода. По мнению G. Drevet et al., хирургический метод лечения более предпочтителен в лечении рака легких II и IIIА ста- дий в случае резектабельной опухоли и операбель- ности пациентов. Традиционная лучевая терапия и химиотерапия рекомендуются для лечения неопе- рабельного рака легких II стадии и местнораспро- страненного рака легких III стадии. У реципиентов солидных органов следует соблюдать особую осто- рожность при назначении лучевой или химиотерапии в связи с иммуносупрессивной терапией, сопутству- ющими заболеваниями, частым наличием почечной недостаточности [39]. Видеоторакоскопические (ВАТС) анатомические рамках комплексного лечения часто не может быть проведена в полном объеме из-за сопутствующих заболеваний и опасности отторжения трансплантата, вызываемой снижением дозировок иммуносупрес- сивных препаратов [3]. В англоязычной литературе по настоящее время нет рекомендаций по изменению схемы иммуносупрессивной терапии у реципиен- тов солидных органов после диагностированного рака легкого, хотя при проведении химиотерапии обычно происходит снижение интенсивности им- муносупрессивной терапии [33, 34]. Профилактика и лечение Основополагающим методом профилактики раз- вития рака легкого является «онкологическая насто- роженность» на всех этапах оказания медицинской помощи и динамического наблюдения. К методам профилактики помимо полного отказа от курения также относится и «разумная» минимизация имму- носупрессии [13, 25]. Лечение рака легкого у реципиентов солидных органов не отличается от такового в популяции. Стра- тегия лечения зависит от стадии, гистологического строения опухоли и наличия сопутствующих забо- леваний у реципиента [25, 32]. Особенностями этой категории больных является то, что химиотерапия в 21 ВЕСТНИК ТРАНСПЛАНТОЛОГИИ И ИСКУССТВЕННЫХ ОРГАНОВ том XXIII № 4–2021 Таблица «Lung-RADS» – система оценки изменений в легких, выявленных на МСКТ. Лечебная тактика и риски малигнизации [31] Lung-RADS, a system for assessing changes in the lungs detected by MSCT. Профилактика и лечение Treatment tactics and risks of malignancy [31] Категория Индекс Находка Тактика Риск малиг- низации Распростра- ненность в популяции Неполное иссле- дование 0 Нет данных для сравнения Дополнительное МСКТ – 1% Нет узелков / узелки досто- верно доброка- чественные 1 Нет узелков ИЛИ узелок(и) со специфическими кальцинатами: полные, центральные, в виде попкорна, концентриче ские кольца и жиросодержащие узелки МСКТ через 12 месяцев <1% 90% Доброкачест- венные (узелки с очень низкой вероятностью озлокачествле- ния из-за разме- ра или отсутст вия роста) 2 Перифиссуральный узелок(и) <10 мм Солидный узелок: <6 мм новый <4 мм Частично солидный узелок(и): <6 мм «Матовое стекло» (GGN): <30 мм ИЛИ ≥30 мм без динамики или с медленным ростом Категория 3 или 4 – узелки без изменений в течение ≥3 месяцев Вероятно доб- рокачественные изменения 3 Солидный узелок: от ≥6 до <8 мм ИЛИ новые от 4 мм до <6 мм Частично солидный узелок(и): ≥6 мм с солидным компонентом <6 мм ИЛИ новый <6 мм «Матовое стекло» (GGN): ≥30 мм МСКТ через 6 месяцев 1–2% 5% Подозритель- ные изменения 4А Солидный узелок: от ≥8 до <15 мм в исходном состоянии ИЛИ при росте <8 мм ИЛИ при росте от 6 до <8 мм Частично солидный узелок: ≥6 мм с солидным компонентом от ≥6 мм до <8 мм ИЛИ с новым или растущим солидным компонентом <4 мм Эндобронхиальный узелок МСКТ через 3 месяца / ПЭТ при наличии солидного компо- нента ≥8 мм 5–15% 2% Очень подозри- тельные изме- нения 4В Солидный узелок: ≥15 мм ИЛИ новые или растущие солидные узелки, и ≥8 мм Частично солидный узелок: с солидным компонентом ≥8 мм ИЛИ новый или растущий с солидным компонентом ≥4 мм МСКТ / ПЭТ и/или морфологическая верификация Для новых круп- ных узелков КТ, через 1 мес. для исключения воспалительных изменений >15% 2% 4X Узелки категории 3 или 4 с дополнительными призна- ками, повышающими онконастороженность (появление спикул, удвоение в размерах матового стекла за год, увеличенные лимфатические узлы и т. д.) Другие клини- Таблица «Lung-RADS» – система оценки изменений в легких, выявленных на МСКТ. Лечебная тактика и риски малигнизации [31] 22 Клиническая трансплантология ния торакотомии в пользу ВАТС привел к улучшению качества жизни пациентов при сохранении той же бе- зопасности выполнения хирургических манипуляций [42]. Профилактика и лечение И если на момент появления этой технологии еще были сомнения относительно радикальности выполняемых операций и долгосрочной выживае- мости пациентов с раком легкого, то в настоящее время общепризнано, что торакоскопический до- ступ при лобэктомиях по поводу немелкоклеточного рака легкого не приводит к ухудшению отдаленных результатов выживаемости больных в сравнении с традиционной торакотомией [40]. Вместе с тем ВАТС-операции обладают рядом преимуществ, а именно меньшим количеством осложнений в раннем и позднем послеоперационном периоде и меньши- ми сроками пребывания больного в стационаре [40, 43]. T. Demmy et al. подчеркивает, что у пожилых и ослабленных пациентов непосредственные и отда- ленные результаты при применении ВАТС-операций лучше в сравнении с торакотомией [41]. По данным P. Falcoz et al., смертность в раннем послеоперацион- ном периоде в группе ВАТС-лобэктомий у пациентов с немелкоклеточным раком легкого в сравнении в открытыми лобэктомиями была в два раза ниже [40]. Преимущества ВАТС-операций особенно отчетливо проявляются у пожилых пациентов (старше 70 лет), и дефицитом массы тела, и прогнозируемыми низкими функциональными показателями в послеопераци- онном периоде (ОФВ1 < 40%) [40]. Сообщения об использовании ВАТС-операций у реципиентов со- лидных органов редки. M. Al-Ameri et al., сравнивая непосредственные и отдаленные результаты одно- портового и многопортового доступов ВАТС у паци- ентов с различной патологией легких и средостения, приходят к выводу, что нет достоверных различий в количестве послеоперационных осложнений (6% в обеих группах), а также уровне смертности и общей выживаемости в течение года (0 и 97% при одно- портовом доступе и 0,5 и 98% при многопортовом, р = 0,71). Кроме того, автор сообщает о более быст- рой реабилитации и меньшем времени пребывания в стационаре при однопортовом доступе (76,2% против 62,1%, р = 0,008) [44]. Вместе с тем однопортовый доступ все еще не получил широкого распростране- ния согласно анкетированию членов Европейского общества торакальных хирургов (англ. European So- ciety of Thoracic Surgeons – ESTS) [45]. J. Seitlinger et al. отмечают, что процент конверсии, т. е. перехода из мини-доступа в обычную торакотомию, снижается по мере совершенствования хирургической техники и составляет менее 10% [46]. Вместе с тем пациен- ты, подвергшиеся конверсии, имеют более высокий риск развития осложнений в раннем и отдаленном послеоперационном периоде (40 9% против 16 8%) т. к. при всех своих преимуществах ВАТС-операции неприменимы в ситуации, когда невозможно создать адекватное рабочее пространство для безопасных манипуляций внутри грудной полости во время опе- рации (например, непереносимость однолегочной вентиляции) [41]. Интересно исследование H. Maeda et al., которые проанализировали 12 случаев ВАТС- операций у реципиентов почки [47]. Профилактика и лечение Авторы срав- нивали как лабораторные показатели, в частности уровень креатинина в сыворотке крови, так и оцени- вали скорость клубочковой фильтрации до и после операции и послеоперационные осложнения. Всего применялись: ВАТС-клиновидная резекция легких (n = 4), ВАТС-сегментэктомия (n = 4), ВАТС-лобэк- томия (n = 2), ВАТС-удаление опухоли средостения (n = 1) и ВАТС-резекция опухоли грудной стенки (n = 1). Все пациенты получали два-три иммуносуп- рессивных препарата, периоперационный гемодиа- лиз не проводился. Бронхолегочных осложнений в раннем послеоперационном периоде не было. Разли- чий между дооперационным и послеоперационным уровнем креатинина сыворотки крови и расчетной скоростью клубочковой фильтрации не наблюдалось. Авторы делают вывод о безопасности таких опера- ций у реципиентов, находящихся на иммуносупрес- сивной терапии [47]. ЗАКЛЮЧЕНИЕ 4. Rousseau-Gazaniol C et al. Lung cancer in renal trans- plant recipients: A case-control study. Lung Cancer El- sevier Ireland Ltd. 2017; 111: 96–100. doi: 10.1016/j. lungcan.2017.07.011. Несмотря на противоречивые данные различных авторов, данные литературы свидетельствуют о по- вышенном риске развития рака легкого у реципиен- тов солидных органов, в особенности реципиентов легких и комплекса «сердце–легкие». Учитывая воз- растающую роль злокачественных новообразований, включая рак легкого, в общей смертности реципиен- тов солидных органов, а также увеличение продол- жительности жизни реципиентов солидных органов, разработка методов скрининга и профилактики рака легкого у реципиентов солидных органов является своевременной и актуальной задачей. По нашему мнению, нужно критически относиться к рекомен- дациям некоторых авторов о нецелесообразности проведения скрининга у реципиентов солидных ор- ганов с опасными для жизни сопутствующими за- болеваниями и продолжительностью жизни менее 10 лет. Создание научно обоснованной скринигновой программы, направленной на раннее выявление рака легкого у реципиентов солидных органов (напри- мер системы оценки данных «Lung-RADS», отлично зарекомендовавшей себя в Соединенных Штатах), позволит начать лечение на ранних стадиях. Вопросы выбора оптимальной тактики лечения рака легкого у реципиентов солидных органов требуют дальней- шего изучения. По мере накопления данных можно будет сделать вывод о безопасности химиотерапии и иммунотерапии у этой категории больных. Хирур- гический метод лечения рака легкого у реципиентов солидных органов принципиально не отличается от такового в популяции, а ВАТС-операции не уступа- ют по эффективности открытым операциям, обла- дая вместе с этим рядом преимуществ. Внедрение малоинвазивных методов хирургического лечения рака легкого у этой категории больных позволит со- кратить сроки пребывания больного в стационаре, а также значительно ускорить реабилитацию за счет меньшего болевого синдрома и меньшей операци- онной травмы. 5. Acuna SA. Etiology of increased cancer incidence after solid organ transplantation. Transplant Rev Elsevier Inc. 2018; 32 (4): 218–224. doi: 10.1016/j.trre.2018.07.001. 6. Katabathina VS et al. Malignancy after solid organ transplantation: Comprehensive imaging review. Ra- diographics. 2016; 36 (5): 1390–1407. doi: 10.1148/ rg.2016150175. 7. Yanik EL et al. Cancer risk after pediatric solid organ transplantation. Pediatrics. 2017; 139 (5). doi: 10.1542/ peds.2016-3893. 8. Potaris K et al. Lung cancer after heart transplantation: A 17-year experience. Ann Thorac Surg. 2005; 79 (3): 980–983. doi: 10.1016/j.athoracsur.2004.05.021. 9. Drevet G et al. Lung cancer surgical treatment after so- lid organ transplantation: A single center 30-year expe- rience. Lung Cancer Elsevier. 2020; 139: 55–59. doi: 10.1016/j.lungcan.2019.10.023. 10. Robbins HY, Arcasoy SM. Malignancies Following Lung Transplantation. Clin Chest Med. 2011; 32 (2): 343–355. doi: 10.1016/j.ccm.2011.02.011. 11. Olland ABM et al. Прогноз Течение злокачественных новообразований раз- личных локализаций, в том числе и рака легкого, у реципиентов солидных органов более агрессивное, а прогноз и ожидаемая продолжительность жизни оп- ределяются стадией заболевания, наличием N2 ста- туса, драйверных мутаций, степенью патоморфоза, применяемой схемой лечения и так далее [20, 25, 48]. По данным G. Drevet et al., 5-летняя выживаемость резектабельным раком легкого после хирургического лечения составила 40,6%, что сопоставимо с выжи- ваемостью в популяции (40,7 до 50%) [9]. L. Nora Chen et al. сообщают, что средняя выживаемость ре- ципиентов легких после постановки диагноза «рак легкого» составила 32 месяца (IQR, 10–52 месяца), что значительно меньше в сравнении с общей попу- ляцией [48]. S. Zhang et al. сообщают о 17,9% общей 5-летней выживаемости у реципиентов почки после постановки диагноза «рак легкого» [49]. K. Sigel et al., исследовав 597 случаев выявления рака легкого у реципиентов солидных органов, сделали вывод, что выживаемость реципиентов солидных органов, не включая реципиентов легкого, хуже в сравнении с пациентами с раком легкого в популяции [50]. Не- обходимо с осторожностью относиться к данным литературы, т. к. нужно учитывать все вышеперечис- ленные прогностические факторы, в первую очередь поражение регионарных лимфоузлов. 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Cancer-attributable mortality among so- lid organ transplant recipients in the United States: 1987 through 2014. Cancer. 2019; 125 (15): 2647–2655. doi: 10.1002/cncr.32136. 13. Yanik EL et al. Comparison of cancer diagnoses between the US solid organ transplant registry and linked cen- tral cancer registries. Am J Transplant. 2016; 16 (10): 2986–2993. doi: 10.1111/ajt.13818. 14. Pérez-Callejo D et al. Lung cancer in lung transplanta- tion: Incidence and outcome. Postgrad Med J. 2018; 94 (1107): 15–19. doi: 10.1136/postgradmedj-2017-134868. 15. Choi YJ et al. Incidental lung cancer of explanted lungs from lung transplant recipients: Incidence, characteris- tics, and 5-year survival. Yonsei Med J. 2020; 61 (11): 958–964. doi: 10.3349/ymj.2020.61.11.958. 16. Sonbol MB et al. A Case of Donor-Transmitted Non- Small Cell Lung Cancer After Liver Transplantation: An Unwelcome Guest. Oncologist. 2019; 24 (6): 2018– 2020. doi: 10.1634/theoncologist.2018-0517. 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Cancer Risks in Solid Organ Transplant Recipients: Results from a Comprehensive Analysis of 72 Cohort Studies. Oncoimmunology. Taylor & Francis. 2020; 9 (1). doi: 10.1080/2162402X.2020.1848068. 41. Demmy T, Dexter E. Overview of minimally invasive thoracic surgery. UpToDate. 2019; 1–84. 42. Cheng X et al. Minimally Invasive Thoracic Surge- ry 3.0. Ann Surg. 2018; 267 (1): 37–38. doi: 10.1097/ SLA.0000000000002. 25. Brennan DC et al. Статья поступила в редакцию 19.08.2021 г. The article was submitted to the journal on 19.08.2021 Список литературы / References transproceed.2019.09.012. 50. Sigel K et al. Lung cancer prognosis in elderly solid organ transplant recipients. Transplantation. 2015; 99 (10): 2181–2189. doi: 10.1097/TP.0000000000000715. 34. Engels EA. Cancer in Solid Organ Transplant Recipients: There Is Still Much to Learn and Do. Am J Transplant. 2017; 17 (8): 1967–1969. doi: 10.1111/ajt.14140.i 51. https://gco.iarc.fr [Internet]. International Agency for Research on Cancer. Available from: https://gco.iarc.fr. 35. Wong K et al. Safety and Efficacy of Immune Checkpoint Inhibitors in Patients With Metastatic Cancer Post Solid Organ Transplantation: A Case Report and Review of the Literature. Transplant Proc Elsevier Inc. 2019; 51 (9): 3053–3058. doi: 10.1016/j.transproceed.2019.08.002. 52. http://www.transplant-observatory.org [Internet]. Global Observatory on Donation and Transplantation. Available from: http://www.transplant-observatory.org. Статья поступила в редакцию 19.08.2021 г. The article was submitted to the journal on 19.08.2021 36. Acuna SA et al. Cancer recurrence after solid organ transplantation: A systematic review and meta-analysis. 25
https://openalex.org/W2805408650	https://authors.library.caltech.edu/90492/1/e01171-18.full.pdf	English	null	Metabolic Capability and Phylogenetic Diversity of Mono Lake during a Bloom of the Eukaryotic Phototroph Picocystis sp. Strain ML	Applied and environmental microbiology	2,018	cc-by	11,463	Metabolic Capability and Phylogenetic Diversity of Mono Lake during a Bloom of the Eukaryotic Phototroph Picocystis sp. Strain ML Blake W. Stamps,a Heather S. Nunn,b Victoria A. Petryshyn,c Ronald S. Oremland,e Laurence G. Miller,e Michael R. Rosen,f Kohen W. Bauer,g Katharine J. Thompson,g Elise M. Tookmanian,h Anna R. Waldeck,i Sean J. Loyd,j Hope A. Johnson,k Bradley S. Stevenson,b William M. Berelson,d Frank A. Corsetti,d John R. Speara on October 30, 201 http://aem.asm.org/ Downloaded from on October 30, 2018 by guest http://aem.asm.org/ Downloaded from aDepartment of Civil and Environmental Engineering, Colorado School of Mines, Golden, Colorado, USA bDepartment of Microbiology and Plant Biology, University of Oklahoma, Norman, Oklahoma, USA cEnvironmental Studies Program, University of Southern California, Los Angeles, California, USA aDepartment of Civil and Environmental Engineering, Colorado School of Mines, Golden, Colorado, USA bDepartment of Microbiology and Plant Biology, University of Oklahoma, Norman, Oklahoma, USA cEnvironmental Studies Program, University of Southern California, Los Angeles, California, USA dDepartment of Earth Sciences, University of Southern California, Los Angeles, California, USA eU.S. Geological Survey, Menlo Park, California, USA on October 30, 2018 by guest http://aem.asm.org/ ded from dDepartment of Earth Sciences, University of Southern California, Los Angeles, California, USA fU.S. Geological Survey, Carson City, Nevada, USA gDepartment of Earth, Ocean and Atmospheric Sciences, University of British Columbia, Vancouver, British Columbia, Canada gDepartment of Earth, Ocean and Atmospheric Sciences, University of British Columbia, Vancouver, British Columbia, Canada hDivision of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California, USA iDepartment of Earth and Planetary Sciences, Harvard University, Cambridge, Massachusetts, USA jDepartment of Geological Sciences, California State University Fullerton, Fullerton, California, USA kDepartment of Biological Science, California State University Fullerton, Fullerton, California, USA hDivision of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California, USA iDepartment of Earth and Planetary Sciences, Harvard University, Cambridge, Massachusetts, USA jDepartment of Geological Sciences, California State University Fullerton, Fullerton, California, USA kDepartment of Biological Science, California State University Fullerton, Fullerton, California, USA ABSTRACT Algal blooms in lakes are often associated with anthropogenic eutrophica- tion; however, they can occur without the human introduction of nutrients to a lake. A rare bloom of the alga Picocystis sp. strain ML occurred in the spring of 2016 at Mono Lake, a hyperalkaline lake in California, which was also at the apex of a multiyear-long drought. These conditions presented a unique sampling opportunity to investigate mi- crobiological dynamics and potential metabolic function during an intense natural algal bloom. ENVIRONMENTAL MICROBIOLOGY ENVIRONMENTAL MICROBIOLOGY Metabolic Capability and Phylogenetic Diversity of Mono Lake during a Bloom of the Eukaryotic Phototroph Picocystis sp. Strain ML We conducted a comprehensive molecular analysis along a depth transect near the center of the lake from the surface to a depth of 25 m in June 2016. Across sam- pled depths, rRNA gene sequencing revealed that Picocystis-associated chloroplasts were found at 40 to 50% relative abundance, greater than values recorded previously. Despite high relative abundances of the photosynthetic oxygenic algal genus Picocystis, oxygen declined below detectable limits below a depth of 15 m, corresponding with an increase in microorganisms known to be anaerobic. In contrast to previously sampled years, both metagenomic and metatranscriptomic data suggested a depletion of anaerobic sulfate- reducing microorganisms throughout the lake’s water column. Transcripts associated with photosystem I and II were expressed at both 2 m and 25 m, suggesting that lim- ited oxygen production could occur at extremely low light levels at depth within the lake. Blooms of Picocystis appear to correspond with a loss of microbial activity such as sulfate reduction within Mono Lake, yet microorganisms may survive within the sedi- ment to repopulate the lake water column as the bloom subsides. Received 29 May 2018 Accepted 4 August 2018 Accepted manuscript posted online 17 August 2018 Citation Stamps BW, Nunn HS, Petryshyn VA, Oremland RS, Miller LG, Rosen MR, Bauer KW, Thompson KJ, Tookmanian EM, Waldeck AR, Loyd SJ, Johnson HA, Stevenson BS, Berelson WM, Corsetti FA, Spear JR. 2018. Metabolic capability and phylogenetic diversity of Mono Lake during a bloom of the eukaryotic phototroph Picocystis sp. strain ML. Appl Environ Microbiol 84:e01171-18. https://doi .org/10.1128/AEM.01171-18. Editor Frank E. Löfﬂer, University of Tennessee and Oak Ridge National Laboratory Copyright © 2018 Stamps et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to John R. Spear, jspear@mines.edu. aem.asm.org 1 Received 29 May 2018 Accepted 4 August 2018 Accepted manuscript posted online 17 August 2018 Accepted manuscript posted online 17 August 2018 Citation Stamps BW, Nunn HS, Petryshyn VA, Oremland RS, Miller LG, Rosen MR, Bauer KW, Thompson KJ, Tookmanian EM, Waldeck AR, Loyd SJ, Johnson HA, Stevenson BS, Berelson WM, Corsetti FA, Spear JR. 2018. Metabolic capability and phylogenetic diversity of Mono Lake during a bloom of the eukaryotic phototroph Picocystis sp. strain ML. Appl Environ Microbiol 84:e01171-18. https://doi .org/10.1128/AEM.01171-18. Editor Frank E. Löfﬂer, University of Tennessee and Oak Ridge National Laboratory Copyright © 2018 Stamps et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. IMPORTANCE Mono Lake, California, provides a habitat to a unique ecological com- munity that is heavily stressed due to recent human water diversions and a period of extended drought. To date, no baseline information exists from Mono Lake to un- derstand how the microbial community responds to human-inﬂuenced drought or algal bloom or what metabolisms are lost in the water column as a consequence of such environmental pressures. While previously identiﬁed anaerobic members of the microbial community disappear from the water column during drought and bloom, Address correspondence to John R. Spear, jspear@mines.edu. November 2018 Volume 84 Issue 21 e01171-18 Applied and Environmental Microbiology aem.asm.org 1 Applied and Environmental Microbiology Stamps et al. sediment samples suggest that these microorganisms survive at the lake bottom or in the subsurface. Thus, the sediments may represent a type of seed bank that could restore the microbial community as a bloom subsides. Our work sheds light on the potential photosynthetic activity of the halotolerant alga Picocystis sp. strain ML and how the function and activity of the remainder of the microbial community re- sponds during a bloom at Mono Lake. KEYWORDS Mono Lake, algal bloom, alkaline lake, geomicrobiology, metagenomics, transcriptomics M ono Lake is a large hypersaline alkaline lake with a maximum depth of 50 m in the Mono Basin near the eastern foothills of the Sierra Nevada Mountains, California (Fig. 1). It formed from the remnant of Paleolake Russell (a Pleistocene glacial lake) and has existed as a closed basin for at least 50,000 years (1). Accepted manuscript posted online 17 August 2018 Diversion of tributary streams to Mono Lake by the city of Los Angeles began in 1941 and resulted in a drop of over 13 m in lake level by 1978 (2), with a corresponding increase in water salinity from 48 g/liter to 81 g/liter by the 1990s (3) and a current alkalinity of 30,400 ppm HCO3 (4). The steep decline in lake level also resulted in increasing concentrations of other solutes (including arsenic), resulting in unusual lake geochemistry and an ab- sence of large macrofauna (e.g., ﬁsh) (5). Mono Lake is home to a photosynthetic eukaryotic alga, Picocystis (6), that is the primary food source of a brine shrimp endemic to the lake, Artemia monica (7). In turn, Artemia is a crucial food source for birds along the North American Paciﬁc ﬂyway (8, 9) where Mono Lake’s microbial/eukaryotic ecosystem serves a unique, multicompartment, interlinked ecosystem role. M on October 30, 2018 by guest http://aem.asm.org/ Downloaded from Picocystis is a genus of phototrophic alga, previously characterized in other saline or alkaline environments (10, 11). Picocystis sp. strain ML, identiﬁed in Mono Lake (6), is a near relative of Picocystis salinarium, isolated from the San Francisco Salt Works in a high-salinity (85‰) pond (10). In addition to P. salinarium, other near relatives have been identiﬁed from hypersaline environments in inner Mongolia (12). Picocystis sp. strain ML at Mono Lake is responsible for 100 mmol of C m2 day1 of the primary productivity in the lake (6). The population density of Picocystis varies throughout the year, often reaching a maximum in early spring before falling as Artemia shrimp graze on the algal cells, reproduce, and greatly reduce their number as measured by cell count (5). Although Picocystis is nitrogen limited, if sufﬁcient concentrations of ammo- nia are present during lake mixing and turnover (5), a bloom can occur often coinciding with periods of lake anoxia (6). Elevated concentrations of chlorophyll a and Picocystis are commonly detected below the oxycline (6), but it is unknown if Picocystis is actively producing photosynthetic pigments and photosynthesizing under low-light conditions in situ at depth. If Picocystis is capable of phototrophic growth below the oxycline, particularly during a bloom when it is found in high densities at all depths, localized production of oxygen may disrupt anaerobic microbial communities in the bottom waters of Mono Lake. November 2018 Volume 84 Issue 21 e01171-18 Accepted manuscript posted online 17 August 2018 How the bacterial and archaeal communities of Mono Lake respond during periods of algal bloom remains an open question. Prior to 2017, microbial community surveys of the lake during meromixis were carried out using only 16S rRNA gene clone library sequencing or denaturing gradient gel electrophoresis (DGGE) (13) that provide limited coverage (14). More recent work using 454 pyrosequencing (15) at Mono Lake provided additional diversity information for the lake during the onset of meromixis; however, the PCR primers chosen for previous high-throughput and clone library-based amplicon surveys at Mono Lake were potentially biased against certain bacterial phyla and lacked the ability to concurrently amplify the eukaryotes (16, 17). With more recent primers for Illumina-based amplicon sequencing (17), a more accurate representation of the lake microbial community is now possible. Furthermore, community distribution and pro- ﬁling within Mono Lake during monomixis, or mixing of lake waters during a single time in a year, has yet to occur. Metagenomic and transcriptomic sequencing can also provide additional insight into the genetic activity of Mono Lake. Recently, genes aem.asm.org 2 Applied and Environmental Microbiology Metabolism and Diversity of Mono Lake in Algal Bloom FIG 1 Overview of northeastern California, with Mono Lake inset. The approximate sampling location is shown by the large white circle/cross. Secondary sampling sites shown are sediment (1), well water (2), and Mill (3) and Wilson (4) creeks. Rush and Lee Vining creeks are not visible on the map. The overview image was captured from Google Earth/Landsat. The inset image was modiﬁed from U.S. Geological Survey Miscellaneous Field Studies map MF-2393 (56). on October 30, 2018 by guest http://aem.asm.org/ Downloaded from on October 30, 2018 by guest http://aem.asm.org/ Downloaded from on October 30, 2018 by guest http://aem.asm.org/ aded from FIG 1 Overview of northeastern California, with Mono Lake inset. The approximate sampling location is shown by the large white circle/cross. Secondary sampling sites shown are sediment (1), well water (2), and Mill (3) and Wilson (4) creeks. Rush and Lee Vining creeks are not visible on the map. The overview image was captured from Google Earth/Landsat. The inset image was modiﬁed from U.S. Geological Survey Miscellaneous Field Studies map MF-2393 (56). associated with sulfate reduction were identiﬁed below the oxycline (15 m) while the lake was meromictic (i.e., stratiﬁed) (18). November 2018 Volume 84 Issue 21 e01171-18 Accepted manuscript posted online 17 August 2018 In that study (18), Edwardson and Hollibaugh conducted transcriptional proﬁling with the same samples sequenced for rRNA gene analyses in another recent study (15), which describes the microbial activity from the surface to below the oxycline. However, these analyses were heavily focused on the bacterial and archaeal components of Mono Lake, ignoring the eukarya. A molecular description of the eukaryote responsible for much of the primary productivity in Mono Lake remains lacking from recent research. A description of how the green alga responsible for this primary productivity, Picocystis sp. strain ML (phylum Chlorophyta), is distributed within Mono Lake during a bloom and its impact on the ecophysiology of the lake are of crucial importance to ensure that a critical food source for migratory macrofauna is not lost. Furthermore, there is an outstanding question of whether or not sulfate-reducing microorganisms previously identiﬁed alongside strictly anaerobic Clostridia at a depth of 15 m below the oxycline (18) remain during a bloom of phototrophic alga that produces oxygen throughout the water column. g yg g Mono Lake entered into a period of monomixis in 2012 corresponding to the onset of a near-record drought in the eastern Sierra. This resulted in a subsequent bloom of Picocystis in 2013 that failed to subside over the next 3 years (19) and corresponded with a near-record low of Artemia present within the lake in 2015. Lake clarity was at near-record lows, and measured chlorophyll a concentrations were high in 2016 (19). Here, we describe the effects of an algal bloom during a period of intense drought within the Mono Lake watershed during the summer of 2016 on the distribution and abundance of the bacterial, archaeal, and eukaryotic planktonic microbial communities and compare the results to those of previously sampled years within the lake (15, 18). This analysis was conducted via a combination of 16S/18S rRNA gene sequencing, metagenomics, and metatranscriptomic sequencing coupled to measurements of wa- ter chemistry and other physical measurements within the lake. The possibility that the November 2018 Volume 84 Issue 21 e01171-18 aem.asm.org 3 Applied and Environmental Microbiology Stamps et al. FIG 2 (A) CTD measurements taken during 2016 sampling with salinity (squares), ﬂuorescence (circles), and PAR (crosses) shown on the upper axis; temperature (triangles) and dissolved oxygen (diamonds) are shown on the lower axis. Points are half-meter averages, with standard deviations shown. For clarity, lines connecting temper- ature and PAR are dashed. Accepted manuscript posted online 17 August 2018 PSU, practical salinity units. (B) Quantiﬁcation of 16S (ﬁlled circles) and 18S (ﬁlled squares) rRNA gene copy numbers at discrete sampling depths of 2, 10, 20, and 25 m. Error bars represent the mean standard deviation of triplicate biological and triplicate technical replicates. on October 30, 2018 by guest http://aem.asm.org/ Downloaded from FIG 2 (A) CTD measurements taken during 2016 sampling with salinity (squares), ﬂuorescence (circles), and PAR (crosses) shown on the upper axis; temperature (triangles) and dissolved oxygen (diamonds) are shown on the lower axis. Points are half-meter averages, with standard deviations shown. For clarity, lines connecting temper- ature and PAR are dashed. PSU, practical salinity units. (B) Quantiﬁcation of 16S (ﬁlled circles) and 18S (ﬁlled squares) rRNA gene copy numbers at discrete sampling depths of 2, 10, 20, and 25 m. Error bars represent the mean standard deviation of triplicate biological and triplicate technical replicates. microbial community within the lake could be repopulated by the sediment, ground- water, and the inﬂuent streams that feed Mono Lake is also addressed by 16S/18S rRNA gene sequencing. Finally, we sought to determine if Picocystis sp. strain ML is tran- scriptionally active under extremely low light levels in the lake. November 2018 Volume 84 Issue 21 e01171-18 RESULTS BDL, below detectable limit; NA, not available (sample not measured). bMeasured using ICP-AES. cMeasured using ion chromatography. dDissolved inorganic carbon (DIC) reported from a single sample per site. eMeasurement from one or two samples. No standard deviation was calculated. TABLE 1 Measured geochemical parameters from the water column, as well as nearby streams and well water, representing subsurface water below Mono Lake on October 30, 201 http://aem.asm.org/ Downloaded from on October 30, 2018 by guest http://aem.asm.org/ Downloaded from on October 30, 2018 by guest http://aem.asm.org/ ded from dDissolved inorganic carbon (DIC) reported from a single sample per site. eMeasurement from one or two samples. No standard deviation was calculated. minimal differences in anion or cation concentrations were detected within Mono Lake. Nitrate, nitrite, and sulfate were elevated at 10 m relative to levels at 2, 20, and 25 m. No phosphate was detectable by ion chromatography (IC) from 2 to 25 m within Mono Lake though surface water taken near shore had an average value of 0.02 mM (Table 1). Average total dissolved phosphorus (potentially including phosphate and organo- phosphorus) measured by inductively coupled plasma atomic emission spectroscopy (ICP-AES) ranged from 0.59 to 0.63 mM from the surface to a depth of 25m, respectively (Table 1). Most major anions and cations and dissolved inorganic carbon were below detectable limits in the sampled inﬂuent stream water and nearby well water, with the exception of calcium, which was elevated relative to levels in Mono Lake water samples (Table 1). Individual replicate results for ICP-AES and IC are shown in Table S1 in the supplemental material. Bacterial and eukaryotic microbial communities of Mono Lake, sediment, and inﬂuent streams. Water and sediment samples, as well as water samples from inﬂuent streams and groundwater, were PCR ampliﬁed to determine their bacterial, archaeal, and eukaryotic community composition. After quality control, a total of 694,948 16S rRNA gene sequence reads were obtained for an average of 13,364 reads per sample, clustering into 831 operational taxonomic units (OTUs); summary statistics are found in Table S2. Chloroplast sequences were abundant across all lake water samples (Fig. S1) and were removed from analysis to more clearly visualize the remaining bacterial and archaeal microbial communities (Fig. 3A). Both the bacterial and archaeal communities differed in structure above and below the oxycline at 10 m (Fig. 3A). RESULTS Major ion chemistry and microbial rRNA gene copy number within Mono Lake. Water was sampled along a continuous transect for temperature, photosynthetically active radiation (PAR), dissolved oxygen (DO), and ﬂuorescence (Fig. 1). At depths between 5 and 15 m, the water temperature decreased from 15 to 7°C. DO and PAR declined rapidly within the ﬁrst 10 m, yet ﬂuorescence was above detectable limits throughout the sampled depths (Fig. 2A). Discrete samples were also taken for micro- bial density and water chemistry at the surface and at depths of 2, 10, 20, and 25 m. Microbial density estimated by bacterial and archaeal 16S rRNA gene copy number varied by less than 10% from 2 to 25 m. In contrast, a eukaryotic 18S rRNA gene copy number maximum was present at 20 and 25 m (Fig. 2B). Major anions, including sodium (Na), were consistent, and levels were near previously reported values (Table 1). RESULTS Only aem.asm.org 4 November 2018 Volume 84 Issue 21 e01171-18 Metabolism and Diversity of Mono Lake in Algal Bloom Applied and Environmental Microbiology TABLE 1 Measured geochemical parameters from the water column, as well as nearby streams and well water, representing subsurface water below Mono Lake Analyte Value by sample depth (mM)a Value by sample source (mM)a Surface 2 m 10 m 20 m 25 m Well Lee Vining Mill Rush Wilson Asb 0.19 0.01 0.18 0.00 0.19 0.01 0.17 0.00 0.20 0.03 BDL BDL BDL BDL BDL Brb 0.76 0.09 0.98 0.00 1.10 0.05 1.97e 0.96 0.01 BDL BDL BDL BDL BDL Cab BDL BDL BDL BDL BDL 3.2 3.4 2.7e 1.1 1.7 4.5 4.0 1.6 2.4 Clc 57 4.1 588 1.9 695 35 585 7.6 577 7.3 0.24 0.01 0.02 0.00 0.01 0.00 0.07 0.00 0.01 0.00 Fc 2.7 0.34 3.6 0.02 4.1 0.17 3.5 0.03 3.5 0.06 0.02 0.00 BDL 0.01e BDL BDL Feb 0.01 0.01 BDL BDL BDL 0.02 BDL BDL BDL 0.01 Kb 37 7.1 39 2.0 38 4.0 37 5.5 41 3.8 0.24 0.21 BDL BDL BDL BDL Mgb 1.9 1.4 1.7 1.2 1.0 0.02 1.0 0.10 1.4 0.30 2.8 4.2 1.5e 0.14e 0.34e 5.1e Nab 1,030 10 874 13 866 74 696 286 892 59 4.2 0.31 0.29 0.26 0.33e 0.37 0.17 0.55 0.60 NO2c 0.37e BDL 0.77e BDL BDL BDL BDL BDL BDL BDL NO3c 0.01e 0.03e 0.15e 0.03e 0.09e 0.01 0.00 0.01 0.00 BDL BDL BDL Pb 0.59 0.08 0.62 0.05 0.61 0.03 0.59 0.04 0.63 0.07 BDL BDL BDL BDL BDL PO4c 0.02e BDL BDL BDL BDL BDL BDL BDL BDL BDL Sb 111 13 125 1.2 124.68 2.21 120.48 3.6 130 4.7 0.29 0.11 0.05 0.04 0.17 0.06 0.16 0.12 0.25 0.10 SO4c 122 1.1 113 0.71 136.73 9.30 113.62 1.7 112 1.1 0.25 0.01 0.05 0.00 0.13 0.00 0.05 0.00 0.15 0.00 DICd NA 313 300 322 318 5.19 NA NA NA NA aValues are the average of triplicate samples, unless otherwise noted. November 2018 Volume 84 Issue 21 e01171-18 RESULTS Samples taken from sediment were distinct in bacterial, archaeal, and eukaryotic community structures from those in the sampled water column. Two OTUs most closely related to genera within the order Bacteroidetes decreased in relative abundance steadily with depth (Psychroﬂexus and ML602M-17), whereas unclassiﬁed Bacteroidetes remained relatively constant in abundance throughout the water column (Fig. 3A). An OTU most closely related to the genus Thioalkalivibrio increased in abundance as depth increased. Unique to the sediment were Euryarchaeota (Thermoplasmata, DHVEG-1) and the bacterial genus Desulfonatronobacter. An increase in the relative abundance of chloro- plast sequence was noted at 20 m, increasing from 40% at the surface to 48% at 10 m and then to 62% relative abundance at 20 m (Fig. S1). Nearby well water taken to compare to lake water samples contained an abundant population of OTUs most November 2018 Volume 84 Issue 21 e01171-18 aem.asm.org 5 Stamps et al. Applied and Environmental Microbiology FIG 3 Heat map of the top 25 OTUs within the bacteria/archaea (A) and eukaryotes (B). OTUs are named by phyla and the most likely genera. on October 30, 2018 by guest http://aem.asm.org/ Downloaded from on October 30, 201 http://aem.asm.org/ Downloaded from on October 30, 2018 by guest http://aem.asm.org/ ded from FIG 3 Heat map of the top 25 OTUs within the bacteria/archaea (A) and eukaryotes (B). OTUs are named by p FIG 3 Heat map of the top 25 OTUs within the bacteria/archaea (A) and eukaryotes (B). OTUs are named by phyla and the most likely genera. he top 25 OTUs within the bacteria/archaea (A) and eukaryotes (B). OTUs are named by phyla and the most likely genera. closely related to sulfur-oxidizing Proteobacteria including Thiothrix and Thiobacillus, as well as Actinobacteria (Rhodococcus), and an abundant unclassiﬁed OTU within the Hydrogenophilaceae. Mono Lake inﬂuent stream water samples collected and examined from Rush, Mill, Lee Vining, and Wilson creeks were distinct from samples taken from the lake itself, with the Flavobacteria, Sediminibacterium, and the hgcI clade of the Actinobacteria being the most abundant OTUs across all stream samples (Fig. 3A). Mill samples were outliers to other stream water samples, lacking abundant populations of Actinobacteria (“Candidatus Planktophila” and hgcI clade) and having a lower abun- dance of the Sediminibacterium than samples from Lee Vining, Rush, and Wilson streams. RESULTS on October 30, 2018 by guest http://aem.asm.org/ Downloaded from g Metagenomic and transcriptomic proﬁling of Mono Lake and sediments. The abundance of sulfate (100 mM) and the lack of oxygen below the 10-m oxycline beg the question as to whether active sulfate reduction is occurring in the dissolved organic carbon (DOC)-rich waters of Mono Lake. No sulﬁte oxidase (sox) genes were identiﬁed; however, genes for the complete reduction of sulfate to sulﬁde were identiﬁed in the sediment metagenome, and genes for reverse-dissimilatory (or oxidative) sulﬁte reduc- tases (dsrA) were identiﬁed in water column metagenomes. Only reverse, or oxidative, dsrA genes were identiﬁed in the water column metagenomes. Dissimilatory sulﬁte reductase genes within the sediment metagenome had high (80%) homology to known deltaproteobacterial sulfate-reducing microorganisms. Reductive dsrA and dsrB genes were identiﬁed within the water column, putatively via BLAST, and were most closely related to known Thioalkalivibrio dsrA and dsrB genes. Sulﬁte reductase genes did not appear to be expressed within the metatranscriptome (Table S4). Nitrate and nitrite reductases were found at 20 and 25 m and within the sediment, while nitric oxide reductase (nor) was identiﬁed only within the sediment (Table S3). Genes associated with nitrogen ﬁxation, including nifH, nifD, and nifK, were found at 20 m within the water column and within the sediment metagenome. No genes associated with ammonium oxidation by bacteria or archaea (AOB/AOA) were identiﬁed. Formate- dependent nitrite reductases were identiﬁed as both genes and transcripts. A compre- hensive list of identiﬁed transcripts is available in Table S4 in the supplemental material. A summary of assembly statistics for sediment and water samples is available in Table S3. y y MAGs of Mono Lake and sediment. After reﬁnement binning, a metagenomic analysis identiﬁed 80 metagenome-assembled genomes (MAGs) with various levels of completion and contamination (Table S3). Of the 80 identiﬁed MAGs, 38 were greater than 50% complete and contained less than 10% contaminating DNA sequence. A total of 21 MAGs contained rRNA gene sequence, and a putative identiﬁcation was produced from these data (Fig. S2). As with the rRNA gene sequencing data, MAGs indicated that microbial community composition shifted by depth and correlated with the decline in oxygen at 10 m (Fig. 5). A large number of MAGs were unique to the sediment, including a euryarchaeon most closely related to the Thermoplasmatales (Fig. 5; see also Fig. S2 in the supplemental material). RESULTS Community membership and distribution in the lake water column proﬁle samples were signiﬁcantly inﬂuenced (P 0.002, R2 0.90) by depth, and the transition to anoxia was visualized by weighted UniFrac principal coordinate analysis (PCoA) ordination and a corresponding Adonis test (Fig. 4A). The eukaryotic community contained far fewer OTUs than the observed bacterial and archaeal communities. Within the water column at Mono Lake, an almost homog- enous distribution of OTUs most closely related to the genus Picocystis was observed at all depths, with a maximum of 97.9% relative abundance at a depth of 10 m (Fig. 3B) FIG 4 Principal coordinate analysis (PCoA) ordination of bacterial/archaeal (A) and eukaryotic (B) communities of water samples taken at Mono Lake. Ordination is based on a weighted UniFrac distance matrix. N b 2018 V l 84 I 21 01171 18 6 PCoA) ordination of bacterial/archaeal (A) and eukaryotic (B) communities of water samples taken at Mono Lake. Ordination tance matrix. FIG 4 Principal coordinate analysis (PCoA) ordination of bacterial/archaeal (A) and eukaryotic (B) communities of water samp is based on a weighted UniFrac distance matrix. FIG 4 Principal coordinate analysis (PCoA) ordination of bacterial/archaeal (A) and eukaryotic (B) communities of water samples taken at Mono Lake. Ordination is based on a weighted UniFrac distance matrix. November 2018 Volume 84 Issue 21 e01171-18 November 2018 Volume 84 Issue 21 e01171-18 aem.asm.org 6 Metabolism and Diversity of Mono Lake in Algal Bloom Applied and Environmental Microbiology during the sampled bloom event of 2016. Within the sediment, an OTU of unclassiﬁed Branchiopoda was most abundant, comprising 90.9% of all sediment eukaryotic se- quence. BLAST results of this OTU suggest that it is most likely Artemia monica, endemic to Mono Lake, although because of the short sequence read length of 250 bp the identiﬁcation is ambiguous. Inﬂuent stream water samples were distinct from samples of the water and sediment of Mono Lake, with few overlapping OTUs among the samples (Fig. 3B). Speciﬁcally, multiple OTUs most closely related to the Ochrophyta (heterokont alga), Ciliophora, and Chytridiomycota were unevenly distributed across the stream and well water sampled. Community membership and distribution within the water column in Mono Lake were signiﬁcantly inﬂuenced (P 0.017, R2 0.61) by depth although less signiﬁcantly than for the bacteria and archaeal communities (P 0.002, R2 0.90) (Fig. 4B). RESULTS Estimates of GC content, completeness (Compl.), and contamination (or redundancy [Redund.]) of each MAG are also given. Presence (black) of key genes related to sulfur, nitrogen, and carbon cycling, as well as respiration is also shown. fcc, sulﬁde dehydrogenase; sqr, sulﬁde-quinone reductase; sat, sulfate adenylyltransferase; apr, adenosine-5-phosphosulfate reductase; dsr, dissimilatory sulﬁte reductase; nap, periplasmic nitrate reductase; nar, nitrate reductase; nrf, nitrite reductase; nir, nitrite reductase; nor, nitric oxide reductase; nos, nitric oxide synthase; nifD, nitrogenase molybdenum-iron protein alpha chain; nifH, nitrogenase iron protein 1; nifK, nitrogenase molybdenum-iron protein beta chain; PSII, photosystem II genes psbA and psbB; cbb, ribulose 1,5-bisphosphate carboxylase/oxygenase; bic, bicarbonate transporter; acc, acetyl-coenzyme A carboxylase; pcc, propionyl-coenzyme A carboxylase; fad, long-chain fatty acid-coenzyme A ligase; fadE, acyl-coenzyme A dehydrogenase; cox, cytochrome c oxidase; hyd, hydrogenase I; hyf, hydrogenase-4; hoxS, bidirectional NiFe hydrogenase. identiﬁed by BLAST within the largest eukaryotic MAG as being closely related to that of known Picocystis sp. chloroplasts. Additionally, two 28S rRNA gene fragments were also identiﬁed. The putatively identiﬁed Picocystis MAG was estimated to be 49.40% complete, with a total length of 11.14 Mbp, and 2.41% redundant. Putative identiﬁca- tion of sequence by BLASTx within the MAG returned homology to other known algae. The annotated genome contained no genes related to sulfur cycling or other incom- plete metabolic cycles (Fig. S3) although completion estimates suggest that only half of its genome was identiﬁed. Summary assembly statistics, including completion and redundancy estimates of each putative eukaryotic MAG, are available in Table S3. Photosynthesis transcripts are detectable at 25 m. Assembly of transcriptomes from 2 m and 25 m resulted in 113,202 coding sequences and 111,709 annotated protein coding genes (Table S4). No transcripts were identiﬁed with homology to known dissimilatory sulﬁte reductases in contrast to results for previously sampled years (11). A total of 3,117 genes were differentially expressed (P 0.05, false discovery rate [FDR] corrected) between 2 m and 25 m (Table S4). Genes associated with photosystem I and II pathways were expressed at both sampled depths (Table 2; see also Table S4). Normalized expression values (reported as the number of transcripts per kilobase million, or TPM) for photosystem I and II, including those of psaA and psaB, psbA and psbB, and psbC transcripts, were at higher levels at a depth of 25 m than at 2 m (Table 2). RESULTS No archaea were found in abundance throughout the sampled water column, and no genes associated with the production of methane (archaeal methanogenesis) were identiﬁed. Multiple MAGs were recovered from uncul- tivated orders within the Actinobacteria, Gammaproteobacteria, and Bacteroidetes (Table S3), including MAGs with 16S rRNA gene sequences previously identiﬁed by rRNA gene clone library sequencing at Mono Lake, such as ML602J-51 (13). A summary of each genome is available in Table S3 and Fig. 5. No MAGs were identiﬁed with the genes required for sulfate reduction, with only reverse dsr genes for sulfur oxidation found in MAGs. Nitrogen ﬁxation genes (nifH, nifD, and nifK) were identiﬁed within three MAGs, two within the Gammaproteobacteria (bins 10 and 23) and a single unclassiﬁed bin (bin_11_2). One bin (bin 45) contained photosystem II-associated genes, identiﬁed within the Epsilonproteobacteria (Fig. 5 and Table S3). Three MAGs were identiﬁed in a separate ﬁltered metagenomic data set. A 1,469-bp 16S rRNA gene sequence was aem.asm.org 7 November 2018 Volume 84 Issue 21 e01171-18 Applied and Environmental Microbiology Stamps et al. FIG 5 Overview of detected MAGs across sampled metagenomes. An increase in the color intensity from gray to blue corresponds to an increase in the coverage of each MAG within each sample. Estimates of GC content, completeness (Compl.), and contamination (or redundancy [Redund.]) of each MAG are also given. Presence (black) of key genes related to sulfur, nitrogen, and carbon cycling, as well as respiration is also shown. fcc, sulﬁde dehydrogenase; sqr, sulﬁde-quinone reductase; sat, sulfate adenylyltransferase; apr, adenosine-5-phosphosulfate reductase; dsr, dissimilatory sulﬁte reductase; nap, periplasmic nitrate reductase; nar, nitrate reductase; nrf, nitrite reductase; nir, nitrite reductase; nor, nitric oxide reductase; nos, nitric oxide synthase; nifD, nitrogenase molybdenum-iron protein alpha chain; nifH, nitrogenase iron protein 1; nifK, nitrogenase molybdenum-iron protein beta chain; PSII, photosystem II genes psbA and psbB; cbb, ribulose 1,5-bisphosphate carboxylase/oxygenase; bic, bicarbonate transporter; acc, acetyl-coenzyme A carboxylase; pcc, propionyl-coenzyme A carboxylase; fad, long-chain fatty acid-coenzyme A ligase; fadE, acyl-coenzyme A dehydrogenase; cox, cytochrome c oxidase; hyd, hydrogenase I; hyf, hydrogenase-4; hoxS, bidirectional NiFe hydrogenase. on October 30, 201 http://aem.asm.org/ Downloaded from on October 30, 2018 by guest http://aem.asm.org/ Downloaded from on October 30, 2018 by guest http://aem.asm.org/ ded from FIG 5 Overview of detected MAGs across sampled metagenomes. An increase in the color intensity from gray to blue corresponds to an increase in the coverage of each MAG within each sample. RESULTS Transcripts for multiple housekeeping genes, including gyrA and gyrB, the glyceraldehyde- 3-phosphate dehydrogenase (GAPDH) gene, and the tubulin subunit alpha gene were either at similar numbers for the different depths or lower at 25 m, indicating similar or lower numbers of cells at depth, despite higher transcript levels (Table 2; see also Table S4). In addition, several light-independent protochlorophyllide reductase transcripts were differentially expressed at 25 m, while no transcripts related to chlorophyll production were differentially expressed at 2 m (Table S4). DISCUSSION Beginning in late 2012, Mono Lake exhibited signs of persistent Picocystis blooms that corresponded to developing drought conditions within the region. Subsequently, from 2013 to 2016, both lake clarity and Artemia abundance declined dramatically (19). aem.asm.org 8 November 2018 Volume 84 Issue 21 e01171-18 Metabolism and Diversity of Mono Lake in Algal Bloom Applied and Environmental Microbiology TABLE 2 Mean expression values of select genes identiﬁed associated with photosynthesis Genea Avg expression (TPM) at: Log2 fold change P value (FDR) 25 m 2 m psaA 155.4 80.4 1.0 0.008 psaA 209.7 86.8 1.3 0.0001 psaB 247.0 123.0 1.0 0.002 psbA 563.7 176.7 1.7 0.0001 psbB 241.1 97.4 1.3 0.0001 psbB 165.4 74.8 1.1 0.001 psbC 183.5 84.1 1.1 0.0001 psbY 23.5 18.4 0.3 0.05 TUBA gene 1.7 4.5 1.5 0.05 gyrAb 4.2 4.4 0.03 gyrBb 5.2 5.5 0.04 GAPDH geneb 5.2 9.4 0.86 aTUBA, tubulin alpha-1 chain; GAPDH, glyceraldehyde 3-phosphate dehydrogenase. bShown as an average expression value of all annotated transcripts at each sampled depth. TABLE 2 Mean expression values of select genes identiﬁed associated with photosynthesis on October 30, 2018 by guest http://aem.asm.org/ Downloaded from Surface concentrations of chlorophyll a had been relatively stable over the past 2 decades, with an average 3.8 M in July (1994 to 2013), yet in the 2016 algal bloom, concentrations were 10 times higher, 33.9 M (19). The elevated chlorophyll a con- centration and Secchi disk values (indicative of lake clarity) above 1 m are consistent with a bloom of Picocystis in Mono Lake. In addition, the relative abundances of microorganisms presented here and the well-mixed major ions of Mono Lake relative to levels reported in previous work (13, 18) indicated that Mono Lake was well within a bloom of the green alga Picocystis and a concurrent period of monomixis. Genes required for sulfate reduction to sulﬁde were detected only in the sequenced lake sediment, while both metagenomic and 16S rRNA gene sequencing indicated a nearly complete loss of the detectable anaerobic sulfate-reducing potential within the water column of Mono Lake. Instead, a mixed algal and facultatively anaerobic microbial community was present below the detectable oxycline at 10 m, more similar to the near-surface microbial community than previously reported (18). It is yet unknown how the microbial community of Mono Lake will rebound after such a signiﬁcant algal bloom and a decline in the population of Artemia within the lake. November 2018 Volume 84 Issue 21 e01171-18 DISCUSSION Our survey allowed a comprehensive evaluation of the genomic potential and expressed genes associated with metabolic processes throughout the water column. Dissimilatory nitrate reduction to ammonium (DNRA) appeared active, with formate- dependent cytochrome c nitrite reductases detected within the transcriptome (see Table S4 in the supplemental material) and formate-dependent nitrite reductase sub- units detected within the assembled metagenomes (Table S3). No genes associated with bacterial ammonium oxidation (AOB) were identiﬁed, in contrast to ﬁndings in previous years (20), in either the transcriptome or metagenome, suggesting that the ammonia produced within the lake was assimilated, likely by the dense population of growing Picocystis. In addition to nitrate reduction, another key anaerobic respiratory process, sulfate reduction, was largely absent from the water column. Previous work during meromixis/nonbloom intervals has shown that sulfate reduc- tion is a key respiratory process in the Mono Lake water column, supporting the growth of multiple species of sulﬁde-oxidizing aerobic microorganisms above the oxycline (18). We found that microorganisms capable of sulfate reduction were identiﬁed only in sediment metagenomic samples during the 2016 bloom. Dissimilatory-type reverse sulﬁte reductases associated with sulfur oxidizing gammaproteobacterial (21) taxa were identiﬁed at 20 and 25 m, but no reductive sulﬁte reductases were found in sequenced water samples. Taxa known to reduce sulfate were also identiﬁed only by 16S rRNA gene sequencing within the water column in stark contrast to previously sampled years (18, 22). Instead, the most abundant microorganisms with identiﬁable dsrA and dsrB aem.asm.org 9 November 2018 Volume 84 Issue 21 e01171-18 Applied and Environmental Microbiology Stamps et al. gene clusters were reverse-dsr type reductases identiﬁed previously in the gammapro- teobacterium genus Thioalkalivibrio (21). While lake sulfate reduction rates are typically very low (23), our data suggest that microorganisms known to reduce sulfate, as well as key genes in dissimilatory sulfate reduction, are lost in the water column during a bloom. It is also possible that rather than a total loss of this metabolic capability, there is instead a heavy reduction in the number of microorganisms that falls below the level of detection of our sequencing effort. It is likely that during a bloom, sulfate reduction is repressed as more oxidizing conditions are present throughout the water column due to an increased abundance of oxygenic photosynthetic algae. DISCUSSION Members of the Bacteroidetes were in high abundance throughout the water column, including OTUs most closely related to ML310M-34, a Bacteroidetes identiﬁed previously in Mono Lake (13), which remained abundant through the water column while Psychroﬂexus de- creased in abundance from 2 to 25 m as oxygen levels declined. The eukaryotic microbial community was more evenly distributed throughout the water column, with Picocystis detected in nearly equivalent relative abundances throughout the column (Fig. 3B), agreeing with reported chlorophyll levels (19) as well as ﬂuorescence values measured as a part of this study (Fig. 2A). on October 30, 2018 by guest http://aem.asm.org/ Downloaded from Eukaryotic 18S rRNA gene copy number was greater at 20 and 25 m than above the oxycline by approximately 40%. The results were similar to previous estimates of Picocystis biomass during bloom events in which the greatest concentrations of Pico- cystis were near the bottom of the lake (6). Artemia grazing pressure was unusually low during 2016, which then allowed for the increase in Picocystis abundance throughout the sampled water column and accounted for the similarly low visibility (Secchi disk) readings. Additional primary productivity in the lake could also account for the oxycline shallowing from a depth of 15 m in 2013 (13, 18) to a 10-m depth in July 2016. This expansion of anoxic waters likely limits Artemia populations from grazing on Picocystis, resulting in a lower total amount of biomass of Artemia available for migratory birds along the North American Paciﬁc ﬂyway. Lake temperature decreases at the surface relative to temperatures reported in previous studies may also slow the metabolism of Artemia, resulting in reduced fecundity and increased mortality (24, 25). A decline in the Artemia population could also impact bird mortality though this was outside the scope of this study and should be investigated at a later date. Evidence exists that Picocystis sp. strain ML appears capable of photosynthesis under very low light conditions near the bottom of Mono Lake. Previous work suggested that Picocystis sp. strain ML is capable of growth with less than 0.1% of incident summertime solar irradiance, which corresponded with increased concentrations of chlorophyll below 15 m at Mono Lake (6). In our work, chloroplast 16S rRNA gene sequence was most abundant at 20 m, corresponding to a peak in total 16S rRNA copy number (Fig. 2B). aem.asm.org 10 aem.asm.org 10 DISCUSSION 18S rRNA gene sequences identiﬁed as Picocystis sequences were most abundant at 10 m, yet chloroplast relative abundance peaked at 20 m, near previously recorded peak depths in other recorded bloom events (6). Despite the high relative abundance of Picocystis throughout the water column, the isolation and characterization of the Picocystis genome remain elusive. Metagenomic binning resulted in a partial MAG of 50% completion. Genome sequencing of Picocystis, recently isolated and sequenced twice independently (Ronald Oremland, personal communication), will allow for its genome to be removed from subsequent sequencing efforts, which will simplify assembly and enhance the resolution of bacterial and archaeal binning efforts in the future, yielding a better understanding of the microbial community responsible for the diverse metabolic potential in both the sediments and water of Mono Lake. Our de novo transcriptomic assembly was able to recover Picocystis chloroplast- associated transcripts in addition to other microbial transcripts. At a 25-m depth, differential expression of photosystem II was observed (Table 1; see also Table S4 in the supplemental material), suggesting active photosynthesis under extremely low light conditions. Recently, photosynthesis in a microbial mat was shown to be capable under extremely low light conditions although in a bacterial system (26). It is possible that Picocystis is sinking from 2 m, settling near 25 m, and artiﬁcially increasing the estimates aem.asm.org 10 November 2018 Volume 84 Issue 21 e01171-18 Metabolism and Diversity of Mono Lake in Algal Bloom Applied and Environmental Microbiology of eukarya and associated transcription estimates at depth. Housekeeping genes including GAPDH, known to work well in the normalization of algal transcriptome data (27), and also including gyrA and gyrB were expressed below a 1-log2-fold change. Another eukaryote-associated housekeeping gene, TUBA (27), was expressed above a 1.5-log2-fold change but was not signiﬁcantly differentially expressed. All housekeeping gene expression levels were far below those of the identiﬁed photosynthesis transcripts (Table 2), and genes identiﬁed as eukaryotic in origin were not differentially expressed (Table S4), suggesting that the numbers of transcriptionally active eukarya throughout the water column are similar. The differential transcription of chloroplast-related chlo- rophyll proteins is in line with previous research that showed a 10-fold increase in the amount of photopigment present at low light (6) and suggests that under extreme- low-light conditions, photosynthesis may still occur. DISCUSSION Still, the difference could be accounted for by falling biomass in such an extreme bloom rather than by an active photosynthetic metabolism as alga could remain metabolically active having just landed in the sediment zone. Further sampling during a bloom event, including rate measurements of oxygen production, photosynthesis, and pigments, may help to resolve this question. Additional laboratory examination of Picocystis in isolation under extremely low light conditions must also be carried out before this ﬁnding can be conﬁrmed. on October 30, 2018 by guest http://aem.asm.org/ Downloaded from During algal bloom, Mono Lake experiences signiﬁcant shifts in both the bacterial and archaeal microbial communities and in its metabolic potential from nonbloom years (15, 18). Picocystis was present throughout the water column with photosystem I and II transcripts identiﬁed, even at a 25-m depth. While Picocystis bloomed through- out Mono Lake, there was also a loss of detectable sulfate-reducing microorganisms. The lack of detectable sulfate reduction at and below 20 m within Mono Lake is in contrast to ﬁndings of previous work and is possibly linked to the intense drought experienced by Mono Lake from 2012 to 2016. During a multiyear drought, anaerobic microorganisms may survive within the underlying sediment even after they are killed off within the water column. By sequencing nearby sediment, we have shown that even if sulfate reduction is temporarily lost in the planktonic community of Mono Lake, the sediment may act as a seed bank or refugia for organisms capable of this and likely for other necessary metabolisms dependent upon overlying water/lake conditions (28). Alternatively, the sulfate-reducing microorganisms may ﬁnd a better reduced substrate or fewer inhibitors in the sedimentary environment. Furthermore, the recovery of microbial populations within Mono Lake must come from its sediment or underlying groundwater, not from the streams that feed it as no overlapping taxa exist from the inﬂuent streams (Fig. 2). While humans have not added signiﬁcant amounts of nutrients to Mono Lake, they have withdrawn large quantities of water, possibly exacerbating drought conditions over the years (2). Establishing if and how the chemistry and microbiota of Mono Lake recover after monomixis, drought, and algal bloom should be the focus of future work. DISCUSSION Such research can be compared against our metagenomic and transcriptomic data during bloom as well as to previous metatranscriptomic sequenc- ing (18) to better understand how or if the microbial community of Mono Lake returns to its previous state after extended periods of both monomixis and algal bloom. November 2018 Volume 84 Issue 21 e01171-18 MATERIALS AND METHODS Major cations were also measured using a Perkin-Elmer Optima 5300 DV inductively coupled plasma optical emission spectrometer (ICP-OES). Both ion chromatography (IC) and ICP were conducted in the Department of Chemistry at the Colorado School of Mines. All sediment samples were extracted for IC and ICP analysis according to Florida Department of Environmental Protection method NU-044-3.12. All ﬂuid samples were ﬁltered in the ﬁeld using 0.22-m-pore-size polyethersulfone (PES) ﬁlters. All ICP samples were acidiﬁed with trace metal-grade nitric acid as per a standard procedure to ensure stabilization of all metal cations. Environmental sampling, ﬁeld preservation, and DNA/RNA extraction of samples. Immediately after sampling concluded, water from Mono Lake and surrounding streams was ﬁltered onto 25-mm 0.22-m-pore-size polyethersulfone ﬁlters (Merck Millipore Corp., Billerica, MA) in triplicate. Separate triplicate ﬁlters were obtained from each water sample for DNA and RNA extraction. Filter volumes are available in Table S1 in the supplemental material. After ﬁltration, samples were immediately suspended in 750 l of DNA/RNA shield (Zymo Research Co., Irvine, CA) and homogenized on site using a custom-designed lysis head for 1 min using a reciprocating saw. Sediment samples were immediately preserved on site by adding sediment directly to DNA/RNA shield as above. Preserved samples were maintained on dry ice and then stored at 80°C (RNA) or 20°C (DNA) until extractions were performed. DNA extraction was carried out using a Zymo Xpedition DNA minikit (Zymo Research Co.), and samples were eluted into a ﬁnal volume of 100 l. RNA extraction was performed using a Zymo Quick-RNA Miniprep kit (Zymo Research Co.) according to the manufacturer’s instructions. on October 30, 2018 by guest http://aem.asm.org/ Downloaded from rRNA gene sequencing library preparation. Libraries of bacterial, archaeal, and eukaryotic small- subunit (SSU) rRNA gene fragments were ampliﬁed from each DNA extraction using PCR with primers (Integrated DNA Technologies Co., Coralville, IA) that spanned the rRNA gene V4 hypervariable region between position 515 and 926 (Escherichia coli numbering) that produced a 400-bp fragment for bacteria and archaea and a 600-bp fragment for the eukaryotes. These primers evenly represent a broad distribution of all three domains of life (17). The forward primer 515F-Y (GTAAAACGACGGCCAGCCGT GYCAGCMGCCGCGGTAA-3=) contains the M13 forward primer (in bold) fused to the SSU RNA gene- speciﬁc forward primer (underlined) while the reverse primer 926R (5=-CCGYCAATTYMTTTRAGTTT-3=) was unmodiﬁed from Parada et al. (17). MATERIALS AND METHODS 5 Prime Hot master mix (5 Prime, Inc., Gaithersburg, MD) was used for all reactions at a ﬁnal volume of 50 l. Reaction products were puriﬁed using AmpureXP paramag- netic beads (Beckman Coulter, Inc., Indianapolis, IN) at a ﬁnal concentration of 0.8 (vol/vol). After puriﬁcation, 4 l of PCR product was used in a barcoding reaction, cleaned, concentrated, and pooled in equimolar amounts as previously described (29). The pooled, prepared library was then submitted for sequencing on an Illumina MiSeq platform (Illumina, Inc., San Diego, CA) using V2 PE250 chemistry. Quantitative PCR. Total bacterial/archaeal and eukaryotic small-subunit (SSU) rRNA gene counts within the water column were obtained using two TaqMan-based probe assays as previously described (30, 31). Brieﬂy, both assays were carried out using 25-l reaction mixtures containing 1 the ﬁnal concentration of Platinum Quantitative PCR SuperMix-UDG with 6-carboxy-X-rhodamine (ROX) (Thermo Fisher Scientiﬁc, Inc.), 1.8 M each primer, and 225 nM bacterial/archaeal or eukaryotic probe. Further information related to the quantitative PCRs (qPCRs) carried out can be found in Table S5. SSU rRNA gene analysis. Sequence reads were demultiplexed in QIIME, version 1.9.1 (32), and ﬁltered at a minimum Q score of 20 prior to clustering. Sequence reads were ﬁrst denoised and then clustered into operational taxonomic units (OTUs) at 97% similarity using UPARSE (33). After clustering, OTUs were assigned taxonomy using mothur (34) against the SILVA database (release 128 [r128]) (35). Each OTU was then aligned against the SILVA r128 database using pyNAST (36) and ﬁltered to remove uninformative bases, and then a tree was created using the maximum likelihood method and the Jukes-Cantor evolutionary model within FastTree, version 2 (37). A BIOM-formatted ﬁle (38) was then produced for use in analyses downstream. To limit OTUs originating from contaminating microorganisms found in extraction and PCR reagents (39), all extraction blanks and PCR controls were processed separately, and a core microbiome was computed. Any OTU found in 95% of controls was ﬁltered from the overall data set. Differences in community composition were estimated using the weighted UniFrac index (40). The effect of depth was tested with Adonis using the R package Vegan (41) within QIIME. Taxon heat maps and ordination plots were generated using phyloseq (42) and AmpVis (43). A mapping ﬁle is available in Table S2. November 2018 Volume 84 Issue 21 e01171-18 MATERIALS AND METHODS Sampling. A vertical proﬁle of photosynthetic active radiation (PAR) (2 quantum sensor; wave- length, 400 to 700 nm [Li-Cor]; energy [E] per square meter per second), dissolved oxygen (SBE 43 sensor; milligrams/liter), and attenuation coefﬁcient (WetLabs Transmissometer; 600-nm wavelength light source, 10-cm path length; per meter) from surface (0 m) to 30 m was taken using a SeaBird SBE 19 conductivity, temperature, and depth (CTD) probe calibrated for use at Mono Lake. After measurements were obtained, water was pumped from depth to the surface at station 6 (lat 37.95739, long 119.0316) (Fig. 1) and sampled at 2 m, 10 m, 20 m, and 25 m the following day (due to unfavorable lake conditions) using a submersible well pump. Water was allowed to ﬂow from the measured depth for 1 to 2 min to clear any residual water from the lines prior to sampling. Artemia organisms were removed from water samples using clean cheese cloth prior to ﬁlling 1-liter sterile high-density polyethylene (HDPE) contain- ers. Samples were stored in a dark cooler until ﬁltration occurred. Sediment was sampled at a 10-m depth approximately 6.9 kilometers away from the water transect (lat 37.9800, long 119.1048) using a box-core sampling device. Well water (lat 38.0922, long 118.9919) was sampled by allowing the aem.asm.org 11 Applied and Environmental Microbiology Stamps et al. wellhead to ﬂow for approximately 5 min before a 5-liter HDPE container was completely ﬁlled. For inﬂuent stream water, 1 liter of water was taken from each location (Mill, lat 38.0230, long 119.1333; Rush, lat 37.8883, long 119.0936; Wilson, lat 38.0430, long 119.1191) into a sterile HDPE container. Lee Vining (lat 37.9422, long 119.1194) was sampled with the use of a submersible pump (as described above) into a sterile 1-liter HDPE container. wellhead to ﬂow for approximately 5 min before a 5-liter HDPE container was completely ﬁlled. For inﬂuent stream water, 1 liter of water was taken from each location (Mill, lat 38.0230, long 119.1333; Rush, lat 37.8883, long 119.0936; Wilson, lat 38.0430, long 119.1191) into a sterile HDPE container. Lee Vining (lat 37.9422, long 119.1194) was sampled with the use of a submersible pump (as described above) into a sterile 1-liter HDPE container. Geochemical water analysis. To characterize the water samples taken from 2 m to 25 m, major anions were measured using a Dionex ICS-90 ion chromatography system running an AS14A (4- by 250-mm) column. aem.asm.org 12 MATERIALS AND METHODS The mapping ﬁle, as well as BIOM ﬁles used for analyses, are available at https://zenodo.org/record/1247529, including an R Markdown notebook that lists the necessary steps to automate initial demultiplexing, quality ﬁltering, and OTU clustering, as well as to reproduce ﬁgures associated with the rRNA gene analyses. Metagenomic/transcriptomic sequencing. Metagenomic and metatranscriptomic samples were prepared using the Nextera XT library preparation protocol. Prior to library preparation, ﬁrst-strand cDNA synthesis was carried out using a ProtoScript cDNA synthesis kit (New England BioLabs, Ipswich, MA), followed by second-strand synthesis using an NEBNext mRNA second-strand synthesis module (New England BioLabs). A mixture of random hexamer and poly(A) primers was used during ﬁrst-strand synthesis. After conversion to cDNA, samples were quantiﬁed using a QuBit HS assay and then prepared for DNA sequencing. Brieﬂy, 1 ng of DNA or cDNA was used as input into the NexteraXT protocol aem.asm.org 12 November 2018 Volume 84 Issue 21 e01171-18 Applied and Environmental Microbiology Metabolism and Diversity of Mono Lake in Algal Bloom (Illumina, Inc.) according to the manufacturer’s instructions. After ampliﬁcation, libraries were cleaned using AmpureXP paramagnetic beads and normalized according to the NexteraXT protocol. All metag- enomic and transcriptomic samples were then sequenced on an Illumina NextSeq 500 Instrument using PE150 chemistry (Illumina, Inc.) at the Oklahoma Medical Research Foundation (Oklahoma City, OK). (Illumina, Inc.) according to the manufacturer’s instructions. After ampliﬁcation, libraries were cleaned using AmpureXP paramagnetic beads and normalized according to the NexteraXT protocol. All metag- enomic and transcriptomic samples were then sequenced on an Illumina NextSeq 500 Instrument using PE150 chemistry (Illumina Inc ) at the Oklahoma Medical Research Foundation (Oklahoma City OK) Metagenomic assembly and binning. Prior to assembly, metagenomic libraries were quality ﬁltered, and adapters were removed using PEAT (44). A coassembly was produced using MEGAHIT (45) with a minimum contig length of 5,000 base pairs. After assembly, quality-ﬁltered reads from triplicate individual samples concatenated to form single libraries per depth were mapped to the coassembly using Bowtie2 (42). Assembled contigs greater than 5 kb in length were ﬁrst ﬁltered to remove eukaryotic sequence using EukRep (46) and then binned into MAGs using CONCOCT (47) and reﬁned using Anvi’o (48) in an attempt to manually reduce potential contamination or redundancy within each bin. Finally, bin quality was assessed using CheckM (49). CheckM was also used to identify possible SSU rRNA gene fragments within each bacterial or archaeal bin. SUPPLEMENTAL MATERIAL Supplemental material for this article may be found at https://doi.org/10.1128/AEM .01171-18. SUPPLEMENTAL FILE 1, XLSX ﬁle, 0.1 MB. SUPPLEMENTAL FILE 2, XLSX ﬁle, 0.1 MB. SUPPLEMENTAL FILE 3, XLSX ﬁle, 0.1 MB. SUPPLEMENTAL FILE 4, PDF ﬁle, 1.0 MB. SUPPLEMENTAL FILE 5, XLSX ﬁle, 0.1 MB. MATERIALS AND METHODS Putatively identiﬁed SSU rRNA gene fragments were aligned against the SILVA 132 database (35) using SINA (50). After alignment, sequences were added to the SILVA tree by SINA, and near relatives were included to give a putative identiﬁcation of MAGs containing SSU sequence. Eukaryotic bins were checked for completeness with BUSCO (51). on October 30, 2018 by guest http://aem.asm.org/ Downloaded from Metatranscriptomic analysis. Metatranscriptome libraries were ﬁrst ﬁltered for quality and adapter removal using PEAT (44). After quality control, sequence ﬁles were concatenated into a single set of paired-end reads in FASTQ format and then assembled de novo using Trinity (52). Postassembly, the Trinotate package (https://trinotate.github.io/) was used to annotate assembled transcripts. After assem- bly, reads were quantiﬁed using Salmon (53), and differential signiﬁcance was assessed using DEseq2 (54). Expression values were normalized as the number of transcripts per million, or TPM. Additionally, multiple housekeeping genes, including DNA gyrase (gyrA and gyrB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and tubulin alpha were identiﬁed and used to determine if greater levels of overall transcription were identiﬁed at either sampled depth. Assembly, annotation, mapping, and statistical analyses were carried out using XSEDE compute resources (55). Accession number(s). Sequence data are available in the NCBI Sequence Read Archive under the BioProject accession number PRJNA387610. ACKNOWLEDGMENTS We thank all participants from the 2016 International GeoBiology Course and the Agouron Institute for course funding. Ann Close and Amber Brown of the University of Southern California were critical in logistics of the 2016 course and beyond. We thank Tom Crowe for access to his well and for transport on Mono Lake. Sequence data were generated by the Oklahoma Medical Research Foundation. The University of Oklahoma Supercomputing Center for Education and Research (OSCER) provided archival storage prior to sequence data submission to the NCBI Sequence Read Archive. This work used the Extreme Science and Engineering Discovery Environment (XSEDE), including the SDSC Comet and the TACC/IU Jetstream clusters under alloca- tion ID TG-BIO180010, which is supported by National Science Foundation grant number ACI-1548562. A California State Parks permit to the U.S. Geological Survey and Geobiology 2016 allowed us to conduct sampling on and around Mono Lake. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. November 2018 Volume 84 Issue 21 e01171-18 3. Jellison R, Melack JM. 2003. Meromixis in hypersaline Mono Lake, Cali- fornia. 1. Stratiﬁcation and vertical mixing during the onset, persistence, and breakdown of meromixis. Limnol Oceanogr 38:1008–1019. 4. Nielsen LC, DePaolo DJ. 2013. 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https://openalex.org/W2961378380	https://europepmc.org/articles/pmc6664683?pdf=render	English	null	Activation of Resolution Pathways to Prevent and Fight Chronic Inflammation: Lessons From Asthma and Inflammatory Bowel Disease	Frontiers in immunology	2,019	cc-by	18,316	REVIEW published: 23 July 2019 doi: 10.3389/ﬁmmu.2019.01699 published: 23 July 2019 doi: 10.3389/ﬁmmu.2019.01699 Activation of Resolution Pathways to Prevent and Fight Chronic Inﬂammation: Lessons From Asthma and Inﬂammatory Bowel Disease Cindy Barnig 1,2, Tjitske Bezema 3, Philip C. Calder 4,5, Anne Charloux 1,2, Nelly Frossard 6, Johan Garssen 7,8, Oliver Haworth 9, Ksenia Dilevskaya 10, Francesca Levi-Schaffer 11, Evelyne Lonsdorfer 1,2, Marca Wauben 12, Aletta D. Kraneveld 7,13 and Anje A. te Velde 14 Cindy Barnig 1,2, Tjitske Bezema 3, Philip C. Calder 4,5, Anne Charloux 1,2, Nelly Frossard 6, Johan Garssen 7,8, Oliver Haworth 9, Ksenia Dilevskaya 10, Francesca Levi-Schaffer 11, Evelyne Lonsdorfer 1,2, Marca Wauben 12, Aletta D. Kraneveld 7,13 and Anje A. te Velde 14 1 Department of Chest Disease, Strasbourg University Hospital, Strasbourg, France, 2 Equipe d’accueil 3072, University of Strasbourg, Strasbourg, France, 3 Immunowell Foundation, Utrecht, Netherlands, 4 Human Development and Health, Faculty of Medicine, University of Southampton, Southampton, United Kingdom, 5 National Institute for Health Research Southampton Biomedical Research Centre, University Hospital Southampton NHS Foundation Trust and University of Southampton, Southampton, United Kingdom, 6 UMR 7200 CNRS/Université de Strasbourg, Laboratoire d’Innovation Thérapeutique and LabEx MEDALIS, Faculté de Pharmacie, Strasbourg, France, 7 Division of Pharmacology, Utrecht Institute for Pharmaceutical Sciences, Faculty of Science, Utrecht University, Utrecht, Netherlands, 8 Nutricia Research, Utrecht, Netherlands, 9 Biochemical Pharmacology, William Harvey Research Institute, Bart’s School of Medicine and Queen Mary University of London, London, United Kingdom, 10 Division of Pharmacology, Faculty of Science, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, Netherlands, 11 Pharmacology and Experimental Therapeutics Unit, Faculty of Medicine, School of Pharmacy, Institute for Drug Research, The Hebrew University of Jerusalem, Jerusalem, Israel, 12 Department of Biochemistry & Cell Biology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, Netherlands, 13 Institute for Risk Assessment Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, Netherlands, 14 Amsterdam UMC, Tytgat Institute for Liver and Intestinal Research, University of Amsterdam, AGEM, Amsterdam, Netherlands Keywords: resolution, inﬂammation, immune ﬁtness, eicosanoids, asthma, chronic inﬂammatory bowel disease Edited by: Fulvio D’Acquisto, University of Roehampton, United Kingdom Reviewed by: Valerio Chiurchiù, Campus Bio-Medico University, Italy Francesco Annunziato, University of Florence, Italy Lorenzo Cosmi, University of Florence, Italy Formerly considered as a passive process, the resolution of acute inﬂammation is now recognized as an active host response, with a cascade of coordinated cellular and molecular events that promotes termination of the inﬂammatory response and initiates tissue repair and healing. In a state of immune ﬁtness, the resolution of inﬂammation is contained in time and space enabling the restoration of tissue homeostasis. There is increasing evidence that poor and/or inappropriate resolution of inﬂammation participates in the pathogenesis of chronic inﬂammatory diseases, extending in time the actions of pro-inﬂammatory mechanisms, and responsible in the long run for excessive tissue damage and pathology. In this review, we will focus on how resolution can be the target for therapy in “Th1/Th17 cell-driven” immune diseases and “Th2 cell-driven” immune diseases, with inﬂammatory bowel diseases (IBD) and asthma, as relevant examples. We describe the main cells and mediators stimulating the resolution of inﬂammation and discuss how pharmacological and dietary interventions but also life style factors, physical and psychological conditions, might inﬂuence the resolution phase. A better understanding of the impact of endogenous and exogenous factors on the resolution of inﬂammation might open a whole area in the development of personalized therapies in non-resolving chronic inﬂammatory diseases. Formerly considered as a passive process, the resolution of acute inﬂammation is now recognized as an active host response, with a cascade of coordinated cellular and molecular events that promotes termination of the inﬂammatory response and initiates tissue repair and healing. In a state of immune ﬁtness, the resolution of inﬂammation is contained in time and space enabling the restoration of tissue homeostasis. There is increasing evidence that poor and/or inappropriate resolution of inﬂammation participates in the pathogenesis of chronic inﬂammatory diseases, extending in time the actions of pro-inﬂammatory mechanisms, and responsible in the long run for excessive tissue damage and pathology. In this review, we will focus on how resolution can be the target for therapy in “Th1/Th17 cell-driven” immune diseases and “Th2 cell-driven” immune diseases, with inﬂammatory bowel diseases (IBD) and asthma, as relevant examples. We describe the main cells and mediators stimulating the resolution of inﬂammation and discuss how pharmacological and dietary interventions but also life style factors, physical and psychological conditions, might inﬂuence the resolution phase. A better understanding of the impact of endogenous and exogenous factors on the resolution of inﬂammation might open a whole area in the development of personalized therapies in non-resolving chronic inﬂammatory diseases. *Correspondence: Cindy Barnig cindy.barnig@chru-strasbourg.fr Specialty section: This article was submitted to Inﬂammation, a section of the journal Frontiers in Immunology Received: 06 November 2018 Accepted: 08 July 2019 Published: 23 July 2019 INTRODUCTION The acute inﬂammatory response is a highly coordinated sequence of events characterized by an onset phase coordinated by several families of chemokines, cytokines, and pro-inﬂammatory mediators that is followed in health by an active resolution phase brought about by the engagement of speciﬁc cellular mechanisms under the control of several pro-resolving mediators to promote resolution of the tissue inﬂammation as well as healing and repair. A failure in pro-resolving pathways can extend in time the actions of pro-inﬂammatory mechanisms resulting in prolonged or chronic inﬂammation with recurrent exacerbations. FIGURE 1 \| Dynamics of the inﬂammatory response in chronic inﬂammation. The acute inﬂammatory response is a highly coordinated sequence of events characterized by an onset phase coordinated by several families of chemokines, cytokines, and pro-inﬂammatory mediators that is followed in health by an active resolution phase brought about by the engagement of speciﬁc cellular mechanisms under the control of several pro-resolving mediators to promote resolution of the tissue inﬂammation as well as healing and repair. A failure in pro-resolving pathways can extend in time the actions of pro-inﬂammatory mechanisms resulting in prolonged or chronic inﬂammation with recurrent exacerbations. FIGURE 1 \| Dynamics of the inﬂammatory response in chronic inﬂammation. The acute inﬂammatory response is a highly coordinated sequence of events Poor and/or inappropriate resolution of inﬂammation has indeed emerged as a critical process in the pathogenesis of numerous chronic inﬂammatory and auto-immune diseases including inﬂammatory bowel diseases (IBD) (such as Crohn’s disease and ulcerative colitis) (4). Persistent airway inﬂammation in chronic lung diseases, such as asthma, may also be due to defects in pro-resolving molecular pathways (5, 6). (Figure 1). A post-resolution phase of inﬂammation that links innate and adaptive immune systems has also been described (9, 10). For eﬀective resolution of inﬂamed tissues to occur and to restore tissue homeostasis, speciﬁc cellular mechanisms that are under the control of pro-resolving mediators are enlisted to promote termination of the inﬂammatory response and initiate tissue repair and healing (Figure 2). Better understanding of how the environment can impact on the resolution of inﬂammation will lead to an improved understanding of why the chronic inﬂammatory diseases persist. The possibility to promote resolution of the inﬂammatory response as a therapeutic approach has only become apparent in the twenty-ﬁrst century (7, 8). Macrophages One of the key events in determining the initiation of the resolution phase is the recruitment of non-phlogistic monocytes and their diﬀerentiation into macrophages at sites of inﬂammation. Indeed, central to the successful resolution of inﬂammation, is the process of local leukocyte clearance by apoptosis and subsequent phagocytosis of the apoptotic cells by surrounding monocyte-derived phagocytes (Figure 2). Engulfment of apoptotic cells signals to the phagocytosing macrophages that the inﬂammatory response is ending, and alters macrophage mediator production from a predominantly pro- inﬂammatory (M1) to an anti-inﬂammatory and pro-resolving phenotype (M2), that further enhances phagocytosis of apoptotic Citation: Barnig C, Bezema T, Calder PC, Charloux A, Frossard N, Garssen J, Haworth O, Dilevskaya K, Levi-Schaffer F, Lonsdorfer E, Wauben M, Kraneveld AD and te Velde AA (2019) Activation of Resolution Pathways to Prevent and Fight Chronic Inﬂammation: Lessons From Asthma and Inﬂammatory Bowel Disease. Front. Immunol. 10:1699. doi: 10.3389/ﬁmmu.2019.01699 July 2019 \| Volume 10 \| Article 1699 Frontiers in Immunology \| www.frontiersin.org 1 Resolution Pathways in Chronic Inﬂammation Barnig et al. INTRODUCTION Better understanding the resolution phase of the inﬂammatory response and how this process might be inﬂuenced by environmental factors might open a whole area of new, aﬀordable, and personalized therapeutic options in chronic inﬂammatory diseases. This article will ﬁrst review the main cellular and molecular mechanisms involved in the resolution of inﬂammation. Finally, we will discuss a series of interventions that can potentially promote resolution with a focus on “Th1/Th17 cell-driven” and “Th2 cell-driven” immune diseases, with IBD and asthma, as relevant examples. INTRODUCTION FIGURE 1 \| Dynamics of the inﬂammatory response in chronic inﬂammation. The acute inﬂammatory response is a highly coordinated sequence of events characterized by an onset phase coordinated by several families of chemokines, cytokines, and pro-inﬂammatory mediators that is followed in health by an active resolution phase brought about by the engagement of speciﬁc cellular mechanisms under the control of several pro-resolving mediators to promote resolution of the tissue inﬂammation as well as healing and repair. A failure in pro-resolving pathways can extend in time the actions of pro-inﬂammatory mechanisms resulting in prolonged or chronic inﬂammation with recurrent exacerbations. Inﬂammation is part of the normal response of the host to invasion by harmful microorganisms or to tissue injury (1). The acute inﬂammatory response is initiated within minutes of recognition of a danger signal, and begins with an onset phase coordinated by several families of chemokines, cytokines, eicosanoids, proteases, vasoactive amines, neuropeptides and neurotransmitters, and other pro-inﬂammatory mediators produced by resident immune and structural cells in the injured/infected tissue, which is followed by a rapid inﬂux of granulocytes from blood to the tissue inﬂammatory site (2). Self-amplifying networks of pro-inﬂammatory pathways perpetuate leukocyte recruitment and activation. In a state of immune ﬁtness, the inﬂammatory response is contained in time and space, and is programmed to resolve, i.e., return from the infected or injured state to a “healthy” state corresponding to that of pre-inﬂamed tissue. Formerly considered as a passive process, the natural resolution of acute inﬂammation is now known as an active host response, with highly coordinated cellular and molecular events with release of anti-inﬂammatory cytokines, loss of receptors for pro- inﬂammatory signals, and production of a wide range of pro- resolving mediators including recently uncovered specialized pro-resolving lipid mediators (SPMs) that enable restoration of tissue homeostasis (3). A failure in pro-resolving pathways may extend in time the actions of pro-inﬂammatory mechanisms resulting in prolonged or chronic inﬂammation with recurrent exacerbations, responsible in the long run for excessive tissue damage and pathology (Figure 1). FIGURE 1 \| Dynamics of the inﬂammatory response in chronic inﬂammation. THE MAIN DETERMINANTS OF THE RESOLUTION PHASE Overall there are two distinct phases in an inﬂammatory reaction: the initiation of inﬂammation and the resolution phase July 2019 \| Volume 10 \| Article 1699 Frontiers in Immunology \| www.frontiersin.org 2 Barnig et al. Resolution Pathways in Chronic Inﬂammation FIGURE 2 \| Key cellular actors of resolution. For effective resolution of inﬂamed tissues to occur and to restore tissue homeostasis, speciﬁc cellular mechanisms that are under the control of pro-resolving mediators are enlisted. They promote termination of the inﬂammatory response and initiate tissue repair and healing. Pro-inﬂammatory mediators: red circles, pro-resolving mediators: blue circles. Anx, annexin; DCs, Dendritic cells; Eos, eosinophils; Gal, Galectin; IBD, inﬂammatory bowel disease; IL, interleukin; ILC2, Type 2 innate lymphoid cells; ILC3, Type 3 innate lymphoid cells; MDSCs, Myeloid-derived suppressor cells; MiRs, MicroRNAs; NK, Natural killer; PMN, polymorphonuclear cells; TGF-beta, Transforming growth factor beta; Th1, Type 1 T helper cells; Th2, Type 2 T helper cells; Treg, regulatory T cells; SPMs, specialized pro-resolution lipid mediators; VIP, vasoactive intestinal peptide. FIGURE 2 \| Key cellular actors of resolution. For effective resolution of inﬂamed tissues to occur and to restore tissue homeostasis, speciﬁc cellular mechanisms that are under the control of pro-resolving mediators are enlisted. They promote termination of the inﬂammatory response and initiate tissue repair and healing. Pro-inﬂammatory mediators: red circles, pro-resolving mediators: blue circles. Anx, annexin; DCs, Dendritic cells; Eos, eosinophils; Gal, Galectin; IBD, inﬂammatory bowel disease; IL, interleukin; ILC2, Type 2 innate lymphoid cells; ILC3, Type 3 innate lymphoid cells; MDSCs, Myeloid-derived suppressor cells; MiRs, MicroRNAs; NK, Natural killer; PMN, polymorphonuclear cells; TGF-beta, Transforming growth factor beta; Th1, Type 1 T helper cells; Th2, Type 2 T helper cells; Treg, regulatory T cells; SPMs, specialized pro-resolution lipid mediators; VIP, vasoactive intestinal peptide. CD25), signaling of surface molecules, cytolysis, and metabolic control (15, 16). cells and promotes the return to tissue homeostasis (11, 12). This shifting balance between pro-inﬂammatory M1 and wound- healing M2 macrophages over time is essential for proper resolution of inﬂammation (13). Treg cells are important players for maintaining homeostatic balance in the intestine [reviewed in (17)]. Acute Treg cell deﬁciency results in an exacerbated inﬂammatory immune response toward commensal intestinal bacteria leading to a chronic inﬂammatory state as found in IBD (18). Frontiers in Immunology \| www.frontiersin.org Innate Lymphoid Cells y Innate lymphoid cells (ILCs) are a large family of cells with various immunological functions (22). They can be classiﬁed into diﬀerent subgroups based on their cytokine production and their expression of key transcription factors, similar to T cell subsets. In various mouse models of asthma and IBD, studies suggest a role for ILCs in the induction of inﬂammation [for recent reviews see (23, 24)]. Recent evidence suggests a more complex role for these cells, with dual roles in the induction of inﬂammatory diseases but also the control of chronic inﬂammation. Type 2 ILC (ILC2) cells demonstrate a ﬂexibility and plasticity dependent on the local microenvironment and can potentially act both as eﬀectors and suppressors [reviewed in (25, 26)]. ILC2 cells, by producing IL-5 and IL-13, promote the development of type 2 allergic inﬂammation, independent of Th2 cells (9, 27). In contrast, the production of IL-9 by ILC2s was recently reported to mediate resolution of inﬂammation in a model of chronic arthritis, another chronic non-resolving disease (28). Also, a potential role for ILC2 has been suggested in tissue repair after acute lung injury in a mouse model of H1N1 inﬂuenza virus infection through the production of amphiregulin (29). During the inﬂammatory response, diverse mediators are synthesized in a strict temporal and spatial manner to act on speciﬁc receptor targets and to actively prevent the overshooting of acute inﬂammatory mechanisms, and ultimately restore tissue homeostasis. Functionally, these anti- inﬂammatory and pro-resolving mediators counter-regulate key events of inﬂammation. Diﬀerent from solely anti- inﬂammatory actions, pro-resolving mediators actions typically target speciﬁc pro-resolution mechanisms: limitation and/or cessation of neutrophil recruitment; promotion of non-phlogistic monocyte recruitment; induction of neutrophil apoptosis and their subsequent eﬀerocytosis by macrophages, enhancement of eﬀerocytosis, reprogramming of macrophages from classically activated to alternatively activated cells; return of non-apoptotic cells to the blood or egress via the lymphatic vasculature; stimulation of tissue repair and cellular repopulation of the tissue, leading to “adapted homeostasis” [recently reviewed in (43)]. Pro-resolving mediators are diverse in nature, and include SPMs (lipoxins, resolvins, protectins, and maresins), proteins and peptides [annexin A1 (AnxA1), galectins, adrenocorticotropic hormone (ACTH), and IL-10], gaseous mediators including hydrogen sulﬁde (H2S) and carbon monoxide (CO), nucleotides (e.g., adenosine), as well as neuromodulators released under the control of the vagus nerve such as acetylcholine and neuropeptides released from non-adrenergic non-cholinergic neurons (44). Regulatory T Cells Regulatory T (Treg) cells can also play roles in the resolution process, by promoting repair and regeneration of various organ systems and may link innate and adaptive immune systems [for a recent review see (14)]. Treg cells, like T helper (Th) cells, derive from the progenitor CD4+ naive T cell. The population of Treg cells consists of thymus-derived Treg cells called natural Treg (nTregs) cells, and Treg cells induced in the periphery or induced Treg (iTregs) cells. Treg cells suppress the activation and function of inﬂammatory leukocytes, speciﬁcally macrophages, through the production of anti-inﬂammatory cytokines (IL-10 and TGF-β) and by scavenging IL-2 (high expression of IL-2R Similarly, in mouse models of allergic asthma, resolution of allergic airway inﬂammation was dependent on CD4+CD25+Foxp3+ expressing Treg cells (19). Accumulation of Treg cells in local draining lymph nodes of the lung correlated with spontaneous resolution of chronic asthma in another murine model (20). Moreover, in the lung, Treg cells have also been described to directly stimulate lung tissue repair, as a consequence of the production of amphiregulin, an autocrine growth factor (21). July 2019 \| Volume 10 \| Article 1699 Frontiers in Immunology \| www.frontiersin.org 3 Resolution Pathways in Chronic Inﬂammation Barnig et al. Innate Lymphoid Cells As diverse as their nature is their origin, where mediators of resolution can be produced locally, acting in paracrine and autocrine manners, or produced at distant sites, followed by their systemic release and extravasation to sites of inﬂammation (43). Below the main pro-resolving mediators will be described. The same holds true for type 3 ILC (ILC3) cells, the most abundant ILC subtype in the human intestine at steady state (30). ILC3 cells are the main contributors to intestinal IL-22 production, which is a tightly regulated mediator for immune homeostasis in the intestinal tract (31). NK cells are also members of the ILC family with potential roles of SPM-induced resolution of eosinophilic inﬂammation in Th2 asthma (32, 33). Frontiers in Immunology \| www.frontiersin.org Myeloid-Derived Suppressor Cells Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of cells, consisting of myeloid progenitor cells, and immature macrophages, granulocytes and dendritic cells. These cells are not present in the normal healthy steady state, and appear in pathological situations related to chronic inﬂammatory situations and stress. Their main function is the suppression of T cell function (34–36). Specialized Pro-resolving Lipid Mediators (SPMs) Recently, a new array of lipid molecules that function in the resolution of inﬂammation were elucidated and collectively named SPMs (3, 45, 46). These mediators, such as lipoxins (Lx), resolvins (Rv), protectins (PD), and maresins (Mar), are produced during the inﬂammatory response and derive from polyunsaturated fatty acids (PUFAs). Whereas, Lx derive from the omega-6 PUFA arachidonic acid, the omega-3 PUFAs eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) give rise to Rv, PD, and Mar (Figure 3). More recently SPMs produced from both the omega-6 and omega- 3 docosapentaenoic acids have been described (47). The SPMs are produced via biosynthetic circuits engaged during cell– cell interactions including diﬀerent innate immune cells, for example macrophages or neutrophils, and structural cells at sites of inﬂammation. SPMs can also been produced through interactions of platelets with leukocytes (48). Recently, a recommendation was published to classify these cells into two diﬀerent subsets based on their phenotype and function (37). Polymorphonuclear (PMN) and mononuclear (M) MDSCs share several partly overlapping immunosuppressive mechanisms, where inhibition of anti-CD3/CD28-induced T-cell proliferation and IFN-γ production are the general functional tests used for their identiﬁcation. In general, MDSCs use several mechanisms to carry out their immunosuppressive function. As biomarkers, the expression of various transcription factors and apoptotic regulators (pSTAT3, cEBP/b, S100A8/9) and immune-regulatory genes and molecules (ARG1, NOS2, NOX2, and PNT) are associated with MDSCs and/or PMN- MDSC and M-MDSC subsets (37, 38). These molecules have immunosuppressive eﬀects and negatively regulate T cells by impairing IL-2R signaling pathways, trigger apoptosis (39, 40), and induce Treg cell expansion and IL-10 and TGF-ß production (41, 42). Moreover, MDSCs have a relevant role in resolution of inﬂammation by eﬀerocytosis of apoptotic neutrophils (35), a process supported in part by IL-10 (42). These bioactive lipids display potencies in the nanomolar range, and signal through cognate G-protein coupled receptors (GPR) such as the N-formyl peptide receptor 2 (ALX/FPR2), GPR32, and GPR18 with many cell type-speciﬁc actions (49, 50) (Figure 4). Frontiers in Immunology \| www.frontiersin.org FIGURE 3 \| Overview of the pathways for synthesis of resolvins from omega-3 polyunsaturated fatty acids, DHA and EPA. DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid, MaR, maresin; PD, protectin; Rv, resolvin. LXA4 binds to the ALX/FPR2. This receptor displays diverse ligand aﬃnities that extend beyond interactions with LXA4. Indeed, ALX/FPR2 can interact with over 30 ligands with various aﬃnities, and has been identiﬁed as the ﬁrst receptor to engage both bioactive lipids and peptides/proteins, including annexin A1 (50). ALX/FPR2 is widely expressed on human leukocytes, including neutrophils, eosinophils, monocyte-macrophages, T cells, NK cells, and ILC2 cells, as well as on tissue resident cells, such as airway epithelial cells and ﬁbroblasts (33, 51, 52). Its expression is up-regulated by local inﬂammatory-mediators such as IL-13 and IFN-γ (51, 53). intervals, and promote wound healing [for a recent reviews see (6, 16, 64)]. Annexin A1 An important mediator of the resolution of inﬂammation is the glucocorticoid-regulated protein annexin (Anx) A1, also known as lipocortin-1. AnxA1 is highly abundant in myeloid-derived cells such as neutrophils and macrophages, and exerts profound eﬀects on several phases of the resolution of inﬂammation (65). AnxA1 signals through the FPR2, which also binds the SPMs LxA4 and RvD1 (50). Studies in mice indicate that this protein has important modulatory functions in neutrophil traﬃcking by reducing neutrophil inﬁltration and activating neutrophil apoptosis. AnxA1 also promotes monocyte recruitment, clearance of apoptotic neutrophils by macrophages and can switch macrophages toward a pro-resolving M2 phenotype (65). Studies have demonstrated that mast cell-derived AnxA1 is important for the cromones-induced inhibition of allergic mast cell degranulation (16). After initiation of the resolution of inﬂammation, repolarization by resolvin E1 (RvE1) induces a M2 wound healing-type macrophage (54). In addition, diﬀerent Rv and Mar interact with ERV1/ChemR23, GPR32 and GPR18 on macrophages to enhance their eﬀerocytosis, phagocytosis and IL-10 transcription (54–61). Other more recently described targets of these mediators are Treg cells and type 2 ILCs (33, 62). SPMs can prevent naïve CD4+ T cell diﬀerentiation into Th1 and Th17 cells and enhance the generation of Treg cells (63). Myeloid-Derived Suppressor Cells July 2019 \| Volume 10 \| Article 1699 Frontiers in Immunology \| www.frontiersin.org Frontiers in Immunology \| www.frontiersin.org 4 Barnig et al. Resolution Pathways in Chronic Inﬂammation FIGURE 3 \| Overview of the pathways for synthesis of resolvins from omega-3 polyunsaturated fatty acids, DHA and EPA. DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid, MaR, maresin; PD, protectin; Rv, resolvin. IL-10 Evidence for the functional importance of these lipid mediators in the resolution of inﬂammation comes from mouse models of diverse inﬂammatory disorders where SPMs are able to control inﬂammation, limit tissue damage, shorten resolution IL-10 is a cytokine important in controlling excessive inﬂammation. It mediates its major functions through inhibition of cytokine production and down-regulating antigen July 2019 \| Volume 10 \| Article 1699 5 Barnig et al. Resolution Pathways in Chronic Inﬂammation FIGURE 4 \| Specialized pro-resolving lipid mediators signal through G-protein coupled receptors on a variety of cell involved (deranged) immune response leading to cell speciﬁc responses. Akt, protein kinase B; ALX/FPR2, N-formyl peptide receptor 2—LXA4 receptor; AnxA1, annexin A1; BLT1, leukotriene B4 receptor 1; CD, cluster domain; CMKLR1, chemokine like receptor 1 or Chemerin Receptor 23; DVR1, RvD1 receptor or G protein coupled receptor (GRP)32; DVR2, RvD2 receptor or GRP18; ERK, extracellular signal regulated kinases; IL, interleukin; INFγ, interferon γ; Mcl-1, anti-apoptotic protein in mast cells; miR, microRNA; mTOR, mammalian target of rapamycin; NK cell, natural killer cell; NFκB, nuclear factor kappa-light-chain-enhancer of activated B cells; P, phosphorylated; PDK1, phosphoinositide-dependent protein kinase 1; PI3K, phosphatidylinositol 3-kinase; PMN, polymorphonuclear cells; Rv, resolvin; LX, lipoxin; S6K, ribosomal protein S6 kinase; Th17 cell: Thelper 17 lympocyte; Treg cell: regulatory T lymphocyte; TNFα, tumor necrosis factor α; Traf6: TNF receptor associated factor 6. FIGURE 4 \| Specialized pro-resolving lipid mediators signal through G-protein coupled receptors on a variety of cell involved (deranged) immune response leading to cell speciﬁc responses. Akt, protein kinase B; ALX/FPR2, N-formyl peptide receptor 2—LXA4 receptor; AnxA1, annexin A1; BLT1, leukotriene B4 receptor 1; CD, cluster domain; CMKLR1, chemokine like receptor 1 or Chemerin Receptor 23; DVR1, RvD1 receptor or G protein coupled receptor (GRP)32; DVR2, RvD2 receptor or GRP18; ERK, extracellular signal regulated kinases; IL, interleukin; INFγ, interferon γ; Mcl-1, anti-apoptotic protein in mast cells; miR, microRNA; mTOR, mammalian target of rapamycin; NK cell, natural killer cell; NFκB, nuclear factor kappa-light-chain-enhancer of activated B cells; P, phosphorylated; PDK1, phosphoinositide-dependent protein kinase 1; PI3K, phosphatidylinositol 3-kinase; PMN, polymorphonuclear cells; Rv, resolvin; LX, lipoxin; S6K, ribosomal protein S6 kinase; Th17 cell: Thelper 17 lympocyte; Treg cell: regulatory T lymphocyte; TNFα, tumor necrosis factor α; Traf6: TNF receptor associated factor 6. neutrophils and lymphocytes (72, 73). IL-10 DCs that are diﬀerentiated in a Gal-1 rich environment show enhanced regulatory function, reducing the progression of inﬂammation in a mouse model of multiple sclerosis by promotion of IL-10-mediated T- cell tolerance (74). Gal-1 also induces the conversion of macrophages into a pro-resolving M2 phenotype (75). Gal-3 enhances eﬀerocytosis of apoptotic granulocytes by monocyte- derived macrophages (MDMs) (76). Gal-9 promotes apoptosis of extravasated immune cells including neutrophils and Th1 cells, and is protective in diﬀerent experimental animal models of chronic auto-immune diseases (77–79). Finally, Gal-1 and Gal- 9 promote the generation of Treg cells (80, 81) and induce the production of IL-10 by peripheral blood mononuclear cells from healthy donors (82). presentation by macrophages, monocytes, and dendritic cells (DCs) and thereby inhibiting adaptive immune cells such as Th2 and Tregs (66–68). IL-10 can also inhibit eosinophilia, by suppression of IL-5 and GM-CSF and by direct eﬀects on eosinophil apoptosis [(69) #1607; (70) #1622]. Frontiers in Immunology \| www.frontiersin.org ACTH and Melanocortins of the inﬂammatory response upon bacterial peritoneal infections via reduced numbers of group 3 ILCs (102). In macrophages, the nicotine acetylcholine receptor, α7nAChR, mediates anti-inﬂammatory actions and contributes to the regulation of phagocytosis. Especially M2-type macrophages express this receptor that has a protective and pro-survival role (103) and M2-type macrophages are important producers of protectin conjugates in tissue regeneration (PCTR)1 during resolution (104). Melanocortins, including adrenocorticotrophic hormone (ACTH) and the α, β and γ-melanocyte-stimulating hormone (MSH) are derived from a larger precursor molecule known as the pro-opiomelanocortin (POMC) protein. They exert their numerous biological eﬀects by activating 7 transmembrane GPCR (83). ACTH does not only induce cortisol production, as previously assumed, but also exerts anti-inﬂammatory actions by targeting melanocortin receptors present on immune cells (84). The protective actions of melanocortins include inhibition of leukocyte transmigration and reduction of pro- inﬂammatory cytokine production (85, 86). Melanocortins also promote clearance of apoptotic cells (86) and cutaneous wound healing (87). Frontiers in Immunology \| www.frontiersin.org Neuropeptides and Neurotransmitters It is important to consider that not only immunological mediators, but also factors produced by the nervous system, like neuropeptides and neurotransmitters, contribute to the resolution of inﬂammation. During an inﬂammatory response several anti-inﬂammatory neuropeptides with an immunomodulatory role are produced. One example is vasoactive intestinal peptide (VIP) displaying anti-inﬂammatory functions in various models of chronic inﬂammatory disease. VIP impairs the development and inﬁltration of self-reactive Th1 cells into target organs, as well as the release of inﬂammatory cytokines and chemokines and the subsequent recruitment and activation of macrophages and neutrophils (94). In addition, VIP stimulates the production of IL-10 and IL-1RA, both important mediators of resolution, and induces the generation of tolerogenic DCs regulating the Th/Treg cells balance (95–99). Very recently, VIP was shown to modulate the diﬀerentiation of human macrophages toward the M2 phenotype, which is important in the resolution of inﬂammation (100). More recently, IL-4 has been reported to induce macrophage proliferation and activation with reduced pulmonary injury after infection with a lung-migrating helminth (123). Other Mediators Participating to the Resolution of Inﬂammation Anti-inﬂammatory Cytokines TGF-β is a potent inhibitor of classical pro-inﬂammatory macrophage activation (105). TGF-β is also a mediator in critical processes in wound healing, stimulating angiogenesis, ﬁbroblast proliferation, collagen synthesis and deposition and remodeling of extracellular matrix (106, 107). Additionally, TGF-β regulates immune responses through the development and diﬀerentiation of Th17 cells and FoxP3+ Treg cells (108, 109). TGF-β inhibits the diﬀerentiation of T helper subsets as it inhibits the expression of Tbet and GATA3, thereby blocking the diﬀerentiation of Th1 and Th2 cells respectively (110, 111). Gaseous Mediators Carbon monoxide (CO) and hydrogen sulﬁde (H2S) are the best characterized gaseous substances that, in addition to their important roles in physiological and pathophysiological processes, have conﬁrmed pro-resolving actions during inﬂammatory processes [reviewed in (88)]. H2S promotes neutrophil apoptosis and stimulates macrophage phagocytosis (89, 90). CO can inhibit leukocyte migration and reduce pro-inﬂammatory cytokine production (91). CO has shown therapeutic potential in animal models of acute lung injury (92). IL-22 primarily targets non-hematopoietic cells and plays a role in host defense at barrier surfaces where it promotes tissue regeneration (112). IL-22 is produced by Th17 and Th22 cells, ILCs and NKT cells (113). It has diﬀerent roles in the gastrointestinal tract including tissue regeneration, maintenance of the intestinal barrier and intestinal defense against pathogens (113–116). IL-22 levels are enhanced in the lungs of patients with asthma (117). However, in inducible lung- speciﬁc IL-22 transgenic mice, a signiﬁcant decrease in allergic airway hyperresponsiveness and allergic inﬂammation occurred indicating an immune modulating eﬀect of IL-22 (118). Galectins Galectins are ß-galactoside-binding lectins produced by, and acting upon, cells of both the innate and adaptive immune systems, modulating multiple processes within the host. Some members of this family of lectins are proposed to play pro- resolving functions, namely Galectin (Gal)-1 and 9. Gal-1 is found in resolving exudates in a murine model of peritonitis induced by zymosan (71), where it stops recruitment of July 2019 \| Volume 10 \| Article 1699 Frontiers in Immunology \| www.frontiersin.org 6 Resolution Pathways in Chronic Inﬂammation Barnig et al. Adenosine Adenosine, is a purine nucleoside generated by the dephosphorylation of adenine nucleotides. In addition to being a potent endogenous physiologic and pharmacologic regulator of many functions, adenosine has pro-resolving mechanisms including inhibition of neutrophil and T cell functions, eﬀerocytosis and macrophage reprogramming [reviewed in (93)]. IL-1RA (receptor antagonist) is a natural inhibitor of the pro- inﬂammatory cytokine IL-1 as it functions as an IL-1 receptor competitor (119). It is produced by CD163+ wound healing M2 macrophages (120). In IBD, polymorphism in the IL-1RA gene have been demonstrated and an imbalance of IL-1 and IL1RA has been suggested to induce mucosal inﬂammation associated with IBD (121, 122). Extracellular Vesicles The paracrine manner of the cellular communication in resolution may be achieved not only by secretion of immune mediators but also through extracellular vesicles. Extracellular vesicles are small membrane vesicles (exosomes, microvesicles, and apoptotic bodies) secreted by all cell types including immune cells in a controlled manner. Extracellular vesicles have recently been reported both as immune activators and immune suppressors as they contain for example MHC class I and II and T cell co-stimulatory molecules (128, 129). However, the most described function of extracellular vesicles is triggering of the immune system, and very recently involvement of extracellular vesicles in inﬂammation resolution, tissue repair and regeneration was reported (130, 131). MicroRNAs ( These results indicate that MiRs actively contribute to resolution of inﬂammation. These results indicate that MiRs actively contribute to resolution of inﬂammation. MicroRNAs ( MicroRNAs (MiRs) are small non-coding RNA molecules that can bind to complementary sequences of mRNA molecules thereby regulating/inhibiting post-translational gene expression. MiRs are contributors to the resolution of inﬂammation by targeting pro-inﬂammatory genes (124). MiRs 21, 146b, 208a, and 219 are increased during the resolution phase of acute resolving peritonitis in mice (125) and RvD1 can regulate expression of these proresolving MiRs (126). MiR-146b down- regulates NF-κB signaling (127), and MiR-219 targets 5- lipoxygenase, with a decreased formation of leukotrienes (19). Another example is the vagal regulation of immune responses, speciﬁcally controlling resolution and the production of SPMs (101). Disruption of the vagal system delays the resolution July 2019 \| Volume 10 \| Article 1699 7 Resolution Pathways in Chronic Inﬂammation Barnig et al. gut microbiota and an excessive immune response (138– 142). The pro-inﬂammatory response is extensively studied, and the suppression of this phase is the main therapeutic strategy in Crohn’s disease, and is still the central research focus, whereas much less is known about the resolution phase. Standard Crohn’s disease therapy involves corticosteroids and immunosuppressants like azathioprine but these therapies are palliative and do not alter the natural history of IBD (143). In the last 20 years biological therapies (antibodies directed against cytokines, like anti-TNFα antibodies) have changed the treatment of more severe IBD. However, only ∼50% of Crohn’s Disease patients achieve clinical remission with the anti- TNFα Humira R⃝or Remicade R⃝. Indeed, the treatment results in a waning of the responsiveness to anti-TNF-α with time and only a minority of patients achieve mucosal healing (143, 144). Alternative therapies such as blocking the migration of eﬀector T cells into the inﬂamed gut by targeting the α4β7 integrin, and recently also the blockade of IL-23 are showing additional success (145, 146) although they are eﬀective only in a minority of patients. These treatments speciﬁcally blocking pro-inﬂammatory mediators cause immuno-suppression, and thereby induce an increased risk of infection. This exempliﬁes why new approaches and new therapies are needed to tackle the problems of chronic intestinal inﬂammation. Therefore, a better understanding of IBD pathophysiology is needed with a focus on the disturbed resolution of inﬂammation. In the line of this, the results of a recent meta-analysis focusing on mucosal healing in IBD as reported from endoscopic studies show that both partial, and full mucosal healing—thus a proper resolution of inﬂammation—predict favorable clinical outcome (147). Frontiers in Immunology \| www.frontiersin.org IMPAIRED RESOLUTION OF CHRONIC INFLAMMATION IN THE INTESTINE AND THE LUNG The pathways involved in the initiation of IBD or asthma diﬀer from each other with respect to cytokine involvement and composition of the resident tissue. IBD is associated with a Th1/Th17 T cell-mediated response induced by interleukin- 12 (IL-12) and IL-23, with concomitant increased production of IL-2, IL-17, IL-18, and IFN-γ (132, 133), whereas asthma and allergic diseases are associated with a typical T helper type 2 (Th2)-mediated response characterized by the production of interleukin-4 (IL-4), IL-5, and IL-13 (134). Therefore, speciﬁc tissue resolution processes exist, guided by the local microenvironment that are impaired in disease [reviewed recently in (135)]. There is increasing evidence that poor and/or inappropriate resolution of inﬂammation participates in the pathogenesis of IBD or asthma, being responsible in the long run for excessive tissue damage and pathology. In this chapter, we give some insights into resolution deﬁciencies in IBD and chronic asthma [for a recent and full review see (6, 136)]. There is more and more evidence that persistent inﬂammation in IBD can occur as a result of inadequate engagement of a series of pro-resolving pathways and many studies have shown that pro-resolving mediators are able to prevent experimental colitis in diﬀerent murine models [reviewed recently in (136, 148)]. MaR1 improves established chronic colitis induced by multiple dextran sulfate sodium (DSS) administrations (149). Systemic treatment of mice with PD1 or RvD5 protects against colitis and intestinal ischemia/reperfusion-induced inﬂammation (150). Other studies report protective roles for SPMs in experimental colitis [summarized in (151)]. Indications that SPM biosynthesis might be dysregulated in patients with IBD come from a study in which RvD5 and PD1 were upregulated in human IBD colon biopsies (150). Interestingly, mucosal expression of LXA4 is elevated exclusively in biopsies from individuals in remission from ulcerative colitis (152). Evidence, whether a defect in SPM signaling exists in IBD remains to be explored. Inﬂammatory Bowel Diseases y IBD aﬄicts around 0.5% of the population in westernized countries (137). It is a chronic relapsing disease that includes Crohn’s disease, a chronic trans-mural inﬂammatory disease of the gastrointestinal tract, mainly aﬀecting the ileum and colon, characterized by leukocyte inﬁltration, granuloma, scarring, and ﬁstulae and ulcerative colitis, a more superﬁcial neutrophilic inﬂammatory lesion of the colon that progresses proximally. The inﬂammation partially, but never completely, resolves leading to tissue remodeling and disruption of the normal epithelial architecture that fails to fully regenerate, resulting in persistent increased epithelial permeability and inﬂammation. AnxA1 stimulates intestinal mucosal wound repair in a murine model of colitis (153) and AnxA1-containing exosomes and microparticles have been shown to accelerate the process of mucosal healing in vivo DSS models of colitis (154). In humans, AnxA1 is released by inﬂamed colonic biopsies from patients having ulcerative colitis (UC) and depends on the severity of inﬂammation (152, 155). In Crohn’s disease, AnxA1 biosynthesis is dysregulated and higher levels correlate with successful intervention with biologicals against TNF-α (156). In IBD multiple factors are identiﬁed that contribute to disease pathogenesis with a focus on host susceptibility genetic factors in combination with a qualitatively and quantitatively abnormal July 2019 \| Volume 10 \| Article 1699 Frontiers in Immunology \| www.frontiersin.org 8 Resolution Pathways in Chronic Inﬂammation Barnig et al. and/or frequent exacerbations despite high doses of inhaled corticosteroids and are often treated with prolonged systemic corticotherapy having many potential side-eﬀects (183). Monoclonal antibodies targeting inﬂammatory pathways that activate immune responses leading to airway inﬂammation have been developed to help broaden the current arsenal of asthma treatment options (184). The ﬁrst anti-body based biological therapy approved for treatment of asthma was omalizumab, targeting IgE, a component of the allergic cascade (185). More recently, monoclonal antibodies have been approved, targeting IL-5 or its receptor (mepolizumab, reslizumab, benralizumab), a key cytokine promoting eosinophil inﬂammation (186). Other monoclonal antibodies targeting a wide variety of intermediaries in the pro-inﬂammatory cascade are currently being tested for their eﬀectiveness in the treatment of asthma (184). These biological therapies can reduce exacerbations and have glucocorticoid-sparing eﬀects, but the clinical responses to these antibody-therapies are variable, with at least 30% of severe asthmatic patients being non-responders (187). In another study in Crohn’s Disease, AnxA1 is involved in intestinal homeostasis after anti-TNF-α treatment and suggested as a potential biomarker of therapeutic eﬃcacy of anti-TNF-α treatment (157). Inﬂammatory Bowel Diseases The production of IL-10 by Tregs is of particular interest in IBD. IL-10 deﬁciency in mice can lead to the development of spontaneous inﬂammatory bowel disease (158) and IL-10 receptor mutations found in patients result in an early-onset enterocolitis (159, 160). Furthermore, a IBD-like colitis can occur in response to recent immune checkpoint inhibitor treatments used in antitumor therapy aiming at blocking Treg cells (161). Tregs accumulate and IL-10 is upregulated in the gut during active IBD (162–166) but a clear demonstration that this pro- resolving mechanism operates in the gut mucosa in IBD is still missing. Several authors report conﬂicting data whether or not it might be possible to use Galectin family member levels as markers for disease activity (167–171). There is also evidence that α-MSH has potent anti- inﬂammatory activity in experimentally induced colitis (172, 173). Oral delivery via Biﬁdobacterium expressing α-melanocyte- stimulating hormone can prevent colitis in an experimental murine model (174). These therapeutic strategies can be combined with allergen- speciﬁc immunotherapies in chronic allergic diseases that are able to improve symptoms but they do not cure allergic disorders (188, 189). As for IBD, there is increasing evidence that chronic and uncontrolled inﬂammation in Th2 immune disorders, might result not just from an excessive uncontrolled pro-inﬂammatory response but also from uncontrolled and insuﬃcient engagement of pro-resolving pathways and thus impaired resolution of exacerbations (5, 33, 190). H2S is able to improve the colonic barrier integrity in a murine model of experimental colitis (175). Administration of inhibitors of H2S synthesis in models of colitis result in an increase in severity of disease (176). In patients with active ulcerative colitis, alterations in the expression of genes involved in the purine metabolic pathway have been demonstrated (177). Like H2S, CO has been shown to exert potent protective eﬀects in the gastro-intestinal tract (178). First, pro-resolving mediators have proved eﬃcient at improving disease and inﬂammatory outcomes in a variety of asthma models. Treatment with SPMs decreases key features of asthma pathobiology, including airway hyperresponsiveness, mucus metaplasia, and Th2 cell bronchial inﬂammation (53, 191–193). AnxA1 deﬁcient mice exhibit spontaneous airway hyperresponsiveness and exacerbated allergen responses (194) and AnxA1 mimetics inhibit eosinophil recruitment (195). Ablation of IL-10 signaling in Th2 cells leads to exacerbated pulmonary inﬂammation (196). In murine models, IL10 knock- out mice develop enhanced allergic airway responses (197, 198) [(199) #1421]. Frontiers in Immunology \| www.frontiersin.org Inﬂammatory Bowel Diseases IL-10 also inhibits pro-inﬂammatory cytokine production by Th2 cells and down-regulates mast cell and eosinophil function (200). Administration of recombinant Gal- 9 or α-MSH diminish allergic airway inﬂammation [(201) #1453; (202)]. Low H2S production in ovalbumin sensitized and challenged mice results in aggravated AHR and increased airway inﬂammation (203). In a rat model of asthma, exogenous administration of H2S reduces airway inﬂammation and airway remodeling (204). VIP can inhibit eosinophil migration (205) and airway remodeling in asthmatic mice (206). Intestinal epithelial cell-derived Gal-9 is involved in the resolution of allergic responses through the induction of tolerogenic DCs and associated Treg cell response (207, 208). In addition, some of these galectins can block IgE binding on mast cells and as such, inhibit allergic inﬂammation (209, 210). The role of adenosine in the resolution of inﬂammation of chronic asthma is not yet elucidated. Several gastro-intestinal neuroendocrine peptides and amines with pro-resolving properties, as members of the chromogranin/secretogranin family, VIP, somatostatin, and ghrelin are aﬀected in experimental colitis and changes of these mediators occur during active IBD in patients [recently reviewed in (179)]. The exact role of neuroendocrine peptides/amines with pro-resolving properties in IBD has to be further elucidated. Pharmacological Interventions Many current therapeutic approaches to manage chronic inﬂammation aim at repressing overactive pro-inﬂammatory responses by reducing pro-inﬂammatory mediator activity (i.e., corticosteroids or biologics). In addition to their potent anti- inﬂammatory properties, steroids can display several pro- resolving properties (7, 65, 231, 232), however, this comes with many potential side-eﬀects, such as osteoporosis, diabetes, systemic hypertension, and impaired immune function. Furthermore, polymorphisms in the IL10 gene resulting in low IL-10 production have been associated with severe asthma (217). T cells from allergic asthmatic patients are partially resistant to IL-10 mediated suppression [(218) #1396]. In humans, galectin-3 production has been reported to be lower in asthma, particularly in neutrophilic asthma (76, 219). Macrophages from sputum samples of asthma patients express reduced levels of Gal-1 and Gal-9 (82). Several human studies have shown a decrease in serum or exhaled-breath H2S levels in both adult and infants (220–222). Moreover, lower H2S levels are correlated with abnormal pulmonary lung function tests and severity of asthma (220, 221). There is relatively limited information available regarding the role of neuropeptides in the resolution of inﬂammation in asthma but Tomaki et al. found that SubP levels in sputum correlate with airway obstruction in asthma (223). Several synthetic pharmaceutical analogs with pro-resolving properties have been proven to be active in animal models [reviewed in (233, 234)]. Toward this end, stable synthetic mimetics to endogenous SPMs are under development, and in matching studies, these mimetics display similar biological actions to the parent mediators in animal models of diverse inﬂammatory disorders with an advantage of resisting local inactivation (including mimetics encapsulated in vesicles) (235– 239). Several of these mimetics are in pre-clinical development programs for diﬀerent chronic inﬂammatory conditions [for reviews see (3, 240)]. In a double-blind placebo-controlled clinical trial, a topical 15-R/S-methyl-LXA4 preparation was tested for the treatment of infantile atopic eczema (241). In this study, the eﬃcacy of the lipoxin mimetic was at least equivalent to gold standard topical steroid therapy for the reduction of eczema severity by quantitative and qualitative measures. HOW TO IMPROVE RESOLUTION IN CHRONIC INFLAMMATORY DISEASES Targeting the inﬂammation phase has been the main focus in medical research for the past decades, resulting in treatment options for immune-mediated diseases that dampen inﬂammation and display immunosuppressive actions (see Figure 1). This comes with a burden for the body, since anti-inﬂammatory immune suppressive therapies, for example corticosteroids or anti-TNF inhibitors, may have increased risks of infection. In addition, the development of expensive targeted anti-inﬂammatory biologicals creates an economic burden on society. Frontiers in Immunology \| www.frontiersin.org Asthma and Allergic Diseases In the industrialized world, millions of individuals suﬀer from inappropriate activation and dysregulation of Th2 cell immune responses responsible for allergic asthma and rhinitis, food allergies and atopic dermatitis (also known as eczema), being part of a process called the atopic march. These disorders are increasingly prevalent and are a major public health problem (180). Th2 cell mediated immune responses are characterized by the release of type 2 signature cytokines (i.e., IL-4, IL-5, IL-9, and IL-13) from cells of both the innate and adaptive immune systems (134, 181). Current therapeutic strategies for chronic Th2 immune disorders are mainly anti-inﬂammatory, and aim at controlling symptoms. In chronic persistent asthma, inhaled corticosteroids are the main anti-inﬂammatory treatment eﬀective in most patients, causing relatively minor adverse eﬀects (182). A subset of asthma patients (∼10%) experience persistent symptoms July 2019 \| Volume 10 \| Article 1699 9 Resolution Pathways in Chronic Inﬂammation Barnig et al. Based on the evidence for the functional importance of pro- resolving mediators in allergic asthma mouse models, defects in the production or the activity of pro-resolving mediators might therefore participate in the chronicity and severity of human asthma. Several studies in distinct populations have reported that SPMs are underproduced in more severe asthma together with a defect in the expression of their related receptors [for a recent review see (6)]. Annexin A1 (AnxA1) levels are also decreased in patients with asthma (211), and in wheezy infants (212). Moreover, plasma and bronchoalveolar AnxA1 levels are correlated with lung function (FEV1 %) (213, 214). Compared with non-asthmatics, asthmatic individuals have reduced levels of IL- 10 in bronchoalveolar lavage ﬂuid (215) and a decreased secretion of IL-10 from alveolar macrophages (216). system (229). There is also evidence that life style factors, physical and psychological conditions can impact on the magnitude of the inﬂammatory response (230). Understanding the mechanism required for adequate resolution of inﬂammation may support the development of new resolution-based strategies able to direct the inﬂammatory processes in a controlled way. Diﬀerent approaches can be considered (Figure 5). Exercise/Physical Activity Exercise/Physical Activity Exercise enhances functional capacity, through increased aerobic capacity and muscle strength, improves quality of life and has the potential to protect from cardiovascular disease, type 2 diabetes mellitus, and certain types of cancer [reviewed in (267)]. The potential mechanisms underlying exercise-mediated protection toward these disorders, include changes in body composition, neuro-hormonal status, as well as eﬀects on resolution pathways. Indeed, acute increases in intramuscular IL-6 following exercise promote resolution processes by increasing the synthesis of anti-inﬂammatory cytokines such as IL-1RA and IL-10, and inhibit pro-inﬂammatory cytokines such as TNF-α (268). Exercise also modulates the production of the PUFA derived y y Exercise enhances functional capacity, through increased aerobic capacity and muscle strength, improves quality of life and has the potential to protect from cardiovascular disease, type 2 diabetes mellitus, and certain types of cancer [reviewed in (267)]. The potential mechanisms underlying exercise-mediated protection toward these disorders, include changes in body composition, neuro-hormonal status, as well as eﬀects on resolution pathways. Indeed, acute increases in intramuscular IL-6 following exercise promote resolution processes by increasing the synthesis of anti-inﬂammatory cytokines such as IL-1RA and IL-10, and inhibit pro-inﬂammatory cytokines such as TNF-α (268). Exercise also modulates the production of the PUFA derived Dietary Interventions Omega-3 PUFAs The main bioactive omega−3 PUFAs, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are poorly synthesized in humans. They are components of seafood, especially oily ﬁsh, of ﬁsh oil, liver oil, krill oil and algal oil supplements, and of a small number of highly concentrated pseudo- pharmaceutical products. p p EPA and DHA have long been known to have beneﬁcial health eﬀects including anti-inﬂammatory, anti-thrombotic, and immuno-regulatory properties (242–244). These n−3 LCPUFAs are substrates for biosynthesis of potent SPMs such as resolvins, protectins, and maresins (Figure 3). DHA is concentrated in neural tissues including brain and retina and in sperm; EPA and DHA are found in membranes of all other cells and tissues and in human milk (245–247). Increased dietary intake of EPA and DHA results in their enrichment in blood and in many cells and tissues. Omega-3 PUFAs can exert signiﬁcant eﬀects on the intestinal environment and modulate the gut microbiota composition (248). EPA and DHA have long been known to have beneﬁcial health eﬀects including anti-inﬂammatory, anti-thrombotic, and immuno-regulatory properties (242–244). These n−3 LCPUFAs are substrates for biosynthesis of potent SPMs such as resolvins, protectins, and maresins (Figure 3). DHA is concentrated in neural tissues including brain and retina and in sperm; EPA and DHA are found in membranes of all other cells and tissues and in human milk (245–247). Increased dietary intake of EPA and DHA results in their enrichment in blood and in many cells and tissues. Omega-3 PUFAs can exert signiﬁcant eﬀects on the intestinal environment and modulate the gut microbiota composition (248). Immune responses are very complex and only recently the ﬁrst initiative to deﬁne the naturally occurring variation and the boundaries of a healthy immune response to complex stimuli was published (224). Interestingly, there is considerable variation in the ability of tissue inﬂammation to resolve within a healthy population (225). Diﬀerent endogenous factors such as age, genetics, sex, ethnicity, might inﬂuence the nature and extent of the acute inﬂammatory response including the resolution process (226). Studies have highlighted how epigenetic reprogramming can lead to chronic inﬂammation and impede resolution resulting in inﬂammatory diseases (227, 228). Gut microﬂora plays a critical role in the stimulation and maturation of a balanced immune The airway mucosa is also enriched with DHA in healthy individuals (249). Interestingly, airway mucosal levels of n−3 July 2019 \| Volume 10 \| Article 1699 10 Barnig et al. Dietary Interventions Omega-3 PUFAs Resolution Pathways in Chronic Inﬂammation FIGURE 5 \| New resolution-based strategies able to direct the inﬂammatory processes in a controlled way. FIGURE 5 \| New resolution-based strategies able to direct the inﬂammatory processes in a controlled way. mouse models for food allergy and in infants suﬀering from cow’s milk allergy (209). In vitro studies showed that the epithelial release of gal 9 by this speciﬁc combination of pre- and probiotic induces tolerogenic DCs that in turn upregulated Treg cells (209, 261). In addition, the combination of Biﬁdobacterium longum with inulin-oligofructose resulted in resolution of inﬂammation in patients suﬀering from active colitis (262, 263). Several preclinical studies have demonstrated that treatment with speciﬁc bacterial strains induces an IL-10 response associated with a faster resolution of inﬂammation in allergy and Crohn’s Disease models (264–266). Overall, there are some indications that dietary intervention with pre- and probiotics promotes the induction of resolution of inﬂammation. However, the exact mechanisms of resolution induced by pre- and probiotics remains to be examined. PUFAs are lower in patients with asthma than in people without asthma (249). Population surveys report that diets rich in n-3 fatty acids are associated with lower asthma prevalence (250). It is noteworthy that SPMs are present at signiﬁcant levels in placenta and human milk (251–253), which suggests an important role for SPMs in health maintenance during a particularly vulnerable period of infant development. In a recent randomized placebo-controlled study from a Danish birth cohort, supplementation with a high dose of n-3-LCPUFAs (a dose corresponding to a 10–20-times increase of the normal intake) during the third trimester of pregnancy was associated with a signiﬁcantly lower risk of asthma symptoms and fewer respiratory infections in children at 3 years (254). This eﬀect was most prominent among children of women who had low pre-intervention EPA and DHA blood levels (255). Recent human studies have shown that increased intake of EPA and DHA results in higher concentrations of selected SPMs in the bloodstream (256–258). High doses of n-3 PUFAs reduce pain and other symptoms in patients with rheumatoid arthritis (259, 260). Many of the mechanisms of action of EPA and DHA suggest that they reduce the pro-inﬂammatory response (242, 243). However, the discovery of SPMs derived from EPA and DHA and the potency of those SPMs in animal models (see earlier) hints that their main action might be promotion of resolution. Frontiers in Immunology \| www.frontiersin.org CONCLUSION SPMs described earlier. Indeed, maximal physical exertion was found to result in a rapid post-exercise increase in the urinary excretion of arachidonic acid (AA) derived lipoxin A4 (LXA4) in healthy subjects (269). Similarly, EPA derived resolvin E1 (RvE1) transiently increases early in human serum following exercise and DHA derived resolvin D1 (RvD1) and protectins increased later during recovery (270). There are a number of immune-mediated chronic diseases that might, at least in part, be controlled or prevented in an immune ﬁt person, including IBD, allergy, and asthma developed in this review, as well as rheumatoid arthritis, chronic obstructive pulmonary disease (COPD), Parkinson’s disease, Alzheimer’s disease, multiple sclerosis, diabetes or myalgic encephalomyelitis. The focus of research on resolution of the immune response as a possible therapeutic approach has only been apparent in the twenty-ﬁrst century and there is now increasing evidence that poor and/or inappropriate resolution of inﬂammation participates in the pathogenesis of IBD or asthma, being responsible in the long run for excessive tissue damage and pathology. This might now open a whole area in the development of personalized therapeutic options for chronic immune diseases driven in part by maladaptive, non-resolving inﬂammation. Interestingly, levels of LXA4 are found to increase immediately after exercise in exhaled air condensate of asthmatic children with exercise induced bronchoconstriction (EIB) (271). The authors hypothesized that airway LXA4 increases to compensate bronchoconstriction and to suppress acute inﬂammation, and that spontaneous bronchodilation after EIB may be due to LXA4. In murine studies in relation to asthma, physical exercise reduced asthma associated bronchial inﬂammation (IL- 4, IL-5 expression and eosinophil inﬁltrate) which was associated with an increase of IL-10 (272, 273). Exercise in animal models of colitis also reduced levels of TNF-α, and decreased markers of oxidative stress and histological damage to the colon in parallel to increased levels of the resolution-promoting and anti-inﬂammatory cytokine IL-10 (274). Moreover, since there is a strong link between a compromised immune system and the brain, individuals can experience a reduced quality of life and lack of well-being (280, 281). Many chronic inﬂammatory diseases are associated with depression, anxiety, and reduced cognitive function (282) and it is becoming apparent that many brain diseases (psychiatric and neurological) are associated with activation of the immune system (281, 283). FUNDING FL-S received funding from Aimwell Charitable Trust (UK), Israel Science Foundation 472/15, Rosetrees Trust (UK), and Emalie Gutterman Memorial Endowed Fund for COPD related research (USA) and is aﬃliated with the Adolph and Klara Brettler Center at the Hebrew University. Stress Management Emotional, cognitive, and psychosocial factors are now widely recognized as signiﬁcant determinants of health outcomes including impacts on the immune system (275). In this sense, a broad variety of mind-body therapies that are able to decrease stress, including meditation-based stress reduction programs (MBSR) and yoga have been increasingly proposed over the past years, as substantial adjuncts to conventional medical treatment in chronic inﬂammatory diseases and cancer patients (275). Convergent evidence suggests that these mind-body therapies may have eﬀects on immune functions including eﬀects on the hypothalamic-pituitary- adrenocortical (HPA) axis function (276) and on NK cell functions and IL-10 levels within patients suﬀering from chronic inﬂammatory disorders (277–279). More precise impacts of the mental state on resolution parameters remain to be examined. Pre- and Probiotics The prebiotic galacto- and fructo-oligosaccharides, so-called non-digestible oligosaccharides, in combination with probiotic bacteria induced the resolution-inducing lectin galectin 9 in July 2019 \| Volume 10 \| Article 1699 Frontiers in Immunology \| www.frontiersin.org 11 Resolution Pathways in Chronic Inﬂammation Barnig et al. CONCLUSION Therefore, deviant immune ﬁtness because of overreaction and poor or defective resolution of the immune system has an enormous impact and is a central issue in these chronic immune diseases. AUTHOR CONTRIBUTIONS All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. REFERENCES Pro-resolving mediators in regulating and conferring macrophage function. Front Immunol. (2017) 8:1400. doi: 10.3389/ﬁmmu.2017.01400 34. Gabrilovich DI, Nagaraj S. Myeloid-derived suppressor cells as regulators of the immune system. Nat Rev Immunol. 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https://openalex.org/W1998949529	https://europepmc.org/articles/pmc2483355?pdf=render	English	null	Up-Regulation of Mitochondrial Activity and Acquirement of Brown Adipose Tissue-Like Property in the White Adipose Tissue of Fsp27 Deficient Mice	PloS one	2,008	cc-by	16,219	Abstract Fsp27, a member of the Cide family proteins, was shown to localize to lipid droplet and promote lipid storage in adipocytes. We aimed to understand the biological role of Fsp27 in regulating adipose tissue differentiation, insulin sensitivity and energy balance. Fsp272/2 mice and Fsp27/lep double deficient mice were generated and we examined the adiposity, whole body metabolism, BAT and WAT morphology, insulin sensitivity, mitochondrial activity, and gene expression changes in these mouse strains. Furthermore, we isolated mouse embryonic fibroblasts (MEFs) from wildtype and Fsp272/2 mice, followed by their differentiation into adipocytes in vitro. We found that Fsp27 is expressed in both brown adipose tissue (BAT) and white adipose tissue (WAT) and its levels were significantly elevated in the WAT and liver of leptin-deficient ob/ob mice. Fsp272/2 mice had increased energy expenditure, lower levels of plasma triglycerides and free fatty acids. Furthermore, Fsp272/2 and Fsp27/lep double-deficient mice are resistant to diet-induced obesity and display increased insulin sensitivity. Moreover, white adipocytes in Fsp272/2 mice have reduced triglycerides accumulation and smaller lipid droplets, while levels of mitochondrial proteins, mitochondrial size and activity are dramatically increased. We further demonstrated that BAT-specific genes and key metabolic controlling factors such as FoxC2, PPAR and PGC1a were all markedly upregulated. In contrast, factors inhibiting BAT differentiation such as Rb, p107 and RIP140 were down-regulated in the WAT of Fsp272/2 mice. Remarkably, Fsp272/2 MEFs differentiated in vitro show many brown adipocyte characteristics in the presence of the thyroid hormone triiodothyronine (T3). Our data thus suggest that Fsp27 acts as a novel regulator in vivo to control WAT identity, mitochondrial activity and insulin sensitivity. Citation: Toh SY, Gong J, Du G, Li JZ, Yang S, et al. (2008) Up-Regulation of Mitochondrial Activity and Acquirement of Brown Adipose Tissue-Like Property in the White Adipose Tissue of Fsp27 Deficient Mice. PLoS ONE 3(8): e2890. doi:10.1371/journal.pone.0002890 Editor: Yihai Cao, Karolinska Institutet, Sweden Received April 14, 2008; Accepted July 8, 2008; Published August 6, 2008 Copyright: 2008 Toh 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: 2008 Toh et al. This is an open-access article distributed under the terms of the Creative Commons Attrib use, distribution, and reproduction in any medium, provided the original author and source are credited. Abstract Funding: This work was supported by grants (704002 to PL) from Ministry of Education of China; (30530350 to PL) from National Natural Science Foundation of China; and National Basic Research program of China (2006CB503900, 2007CB914404) from Ministry of Science and Technology of China. PL is a Cheung Kong Scholar of the Ministry of Education of China. This work was also supported by Program for Changjiang Scholars and Innovative Research Team in University from Ministry of Education in China. Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. * E-mail: li-peng@mail.tsinghua.edu.cn * E-mail: li-peng@mail.tsinghua.edu.cn . These authors contributed equally to this work. fatty acid metabolism, triacylglyceride (TAG) storage and insulin sensitivity [2,3], BAT is known to be functionally more important as a thermogenic tissue [4]. It expresses a unique protein, uncoupling protein 1 (Ucp1), which functions to uncouple oxidative phosphorylation and converting this proton gradient energy into heat to maintain normal body temperature. Besides Ucp1, proteins such as type 2 iodothyronine deiondinase (Dio2) [5] and Cidea [6] have been shown to be highly enriched in BAT. PGC-1a [7,8], a coactivator of multiple transcription factors such as nuclear respiratory factors (NRF-1/2) [9,10] and peroxisome proliferator-activated receptor (PPAR) [11], is also highly expressed in BAT but low in WAT [12]. Furthermore, PGC-1a has been shown to coordinate multiple physiological cues for mitochondrial biogenesis and activity [13]. Up-Regulation of Mitochondrial Activity and Acquirement of Brown Adipose Tissue-Like Property in the White Adipose Tissue of Fsp27 Deficient Mice Shen Yon Toh1,2,3., Jingyi Gong2., Guoli Du2., John Zhong Li2,3, Shuqun Yang2, Jing Ye2,4, Huilan Yao2, Yinxin Zhang2, Bofu Xue3, Qing Li4, Hongyuan Yang5, Zilong Wen6, Peng Li2* 1 Institute of Molecular and Cell Biology, Singapore, Singapore, 2 Department of Biological Sciences and Biotechnology, Protein Science Laboratory of Ministry of Education, Tsinghua University, Beijing, China, 3 Department of Biology, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong, 4 Department of Pathology, Fourth Military Medical University, Xi’an, China, 5 School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, Australia, 6 Department of Biochemistry, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong PLoS ONE \| www.plosone.org Fsp27 is highly expressed in adipose tissue and up- regulated in ob/ob mice g To determine the precise tissue distribution of Fsp27, we generated polyclonal antibody against Fsp27 and evaluated its expression profile by western blot analysis. High levels of Fsp27 protein were detected in WAT and moderate levels in BAT. No Fsp27 protein was detected in other tissues such as liver, kidney, colon and skeletal muscle (SM) (Fig. 1A). Fsp27 was also detected in NIH3T3 L1 adipocytes after 4 days differentiation and its levels remain high during the course of differentiation (Fig. 1B). These data suggest that Fsp27 is rather exclusively expressed at high levels in adipose tissue including BAT and WAT. The specific expression of Fsp27 in adipose tissue prompted us to check whether levels of Fsp27 protein were correlated with the development of obesity by examining the levels of Fsp27 in the WAT of leptin-deficient ob/ob mice. As shown in Fig. 1C, levels of Fsp27 in WAT were dramatically elevated in ob/ob mice compared with that of wild type mice. Furthermore, higher levels of Fsp27 were detected in the liver of ob/ob mice that contains large amount of lipid droplets. Using Fsp27 antibody, we checked the sub- cellular localization of Fsp27 in differentiated 3T3-L1 cells and observed that a fraction of Fsp27 was co-localized with lipid droplet specific marker Perilipin (Fig. 1D), consistent with previous observation that overexpression of GFP-Fsp27 fusion protein is targeted to lipid droplet [31,32]. These data suggest that Fsp27 may positively correlate with the development of obesity and the accumulation of lipid in WAT and hepatocytes. Cide proteins including Cidea, Cideb and Fat specific protein 27 (Fsp27, also known as Cidec in human), share homology with the DNA fragmentation factor DFF40/45 at the N-terminal region [25]. Our previous work demonstrated that Cidea is expressed at high levels in BAT[6], whereas Cideb is expressed at high levels in liver [26]. Mice with deficiency in both Cidea and Cideb have higher energy expenditure, enhanced insulin sensitivity and are resistance to high-fat-diet (HFD)-induced obesity and diabetes [6,26], suggesting a universal role of Cide proteins in the regulation of energy homeostasis. Fsp27 was originally identified in differentiated TA1 adipocytes and its expression is regulated by C/ EBP (or C/EBP like) transcription factor [27]. Fsp27 mRNA is detected at high levels in WAT and moderate levels in BAT [6]. Lower levels of Fsp27 mRNA are present in skeletal muscle (SM) and lung [28]. Introduction Obesity, representing excess amount of body fat, develops as a result of a positive energy balance when energy intake exceeds that of metabolic expenditure. Adipose tissues play crucial roles in the development of obesity, with white adipose tissue (WAT) functioning as an energy storage organ,while brown adipose tissue (BAT) as an energy consumption organ [1]. Morphologically, white adipocytes are characterized by a large unilocular lipid droplet that occupies the majority of the cytoplasmic space, while brown adipocytes contain multiple and relatively smaller lipid droplets. BAT also contains large numbers of mitochondria packed with regularly arranged cristae, a characteristic of high mitochondrial activity. White adipocytes, on the other hand, have fewer mitochondria and their cristae are more compact. Although WAT and BAT both express a set of genes that regulates lipolysis, The exact origin of BAT and WAT and their developmental relationship is not clear. Both BAT and WAT are generally PLoS ONE \| www.plosone.org August 2008 \| Volume 3 \| Issue 8 \| e2890 August 2008 \| Volume 3 \| Issue 8 \| e2890 1 Fsp27 Regulate WAT Identity considered to be derived from a common preadipocyte pool [14]. Whereas recent report by Timmoms et al [15] suggest that brown and white preadipocytes may have different origins. BAT is apparently more closely related to muscle as brown preadipocytes appear to express myogenic specific proteins. Interestingly, differentiated WAT has been shown to acquire certain BAT-like properties and can be converted into an energy consumption organ under special conditions. It has been observed that cold- exposure or administration of b3-agonist could induce the emergence of multilocular brown adipocytes that contain high amount of mitochondria and expresses Ucp1 in WAT depots [16,17]. Furthermore, ablation of b3-adrenoceptor diminishes the formation of BAT in white fat in cold exposed mice [18], suggesting the requirement of b-adrenoceptor signaling for the transdifferentiation of WAT to BAT. A similar increase in the occurrence of BAT in WAT areas was also observed in different strains of inbred mice [19]. Mice harboring increased BAT-like depots show a modest increase in b-adrenoreceptor signaling, which is sufficient to induce Ucp1 in these BAT-like depots. The requirement for PPARc in acquiring BAT-like and energy consuming organ in WAT was also shown in PPARc mutant (P465L) mice in which its WAT is unable to be converted to BAT in cold acclimatized mice [20]. Introduction On the other hand, attainment of fatty acid-oxidizing BAT-like cells in the WAT was also observed in several transgenic and knock-out mouse models. Transgenic knock-in mouse that replaces the C/EBPa gene with C/EBPb gene (denoted as C/EBP b/b) was shown to lead to an increase in the expression of Gsa subunit, cAMP accumulation, enhanced mitochondrial biogenesis and Ucp1 expression in WAT [21]. WAT in mice with an overexpression of FoxC2 acquired certain BAT-like properties with increased expression levels of PGC1, Ucp1 and cAMP pathway proteins such as b3-adrenoceptor and the protein kinase A alpha regulatory subunit1 [22]. Similarly, in mice lacking negative regulators for BAT differentiation like p107, Rb and RIP140, there is a uniform replacement of WAT with BAT and elevated levels of PGC1a and Ucp1 [23,24]. Although many factors and regulatory pathways have been shown to play important roles in maintaining WAT identity, upstream signals or factors that determine the fate of BAT vs WAT and those that initiate the conversion of WAT to BAT remain unclear. suggesting that Fsp27 expression is positively correlated with the development of obesity. Fsp27 deficiency results in dramatically reduced WAT depot and the acquisition of a brown fat-like morphology in these WAT, typified by the appearance of smaller lipid droplets, increased mitochondrial size and their activity. Furthermore, both Fsp27 deficient and Fsp27/leptin double deficient mice display improved insulin sensitivity and a lean phenotype. Genes such as PGC1a, Ucp1 and Dio2 that are normally expressed in BAT were expressed at high levels in the WAT of Fsp272/2 mice. Furthermore, regulators that maintain BAT identity like FoxC2 which was known to promote BAT differentiation was upregulated, whereas factors that inhibit BAT differentiation such as Rb, p107 and RIP140 were down-regulated in WAT of Fsp272/2 mice. Finally, differentiated mouse embryonic fibroblasts (MEFs) isolated from Fsp272/2 mice showed an increased propensity to acquire brown adipocyte characteristics, which is consistent with the in vivo observations in WAT of the mutant mice. Introduction On the other hand, attainment of fatty acid-oxidizing BAT-like cells in the WAT was also observed in several transgenic and knock-out mouse models. Transgenic knock-in mouse that replaces the C/EBPa gene with C/EBPb gene (denoted as C/EBP b/b) was shown to lead to an increase in the expression of Gsa subunit, cAMP accumulation, enhanced mitochondrial biogenesis and Ucp1 expression in WAT [21]. WAT in mice with an overexpression of FoxC2 acquired certain BAT-like properties with increased expression levels of PGC1, Ucp1 and cAMP pathway proteins such as b3-adrenoceptor and the protein kinase A alpha regulatory subunit1 [22]. Similarly, in mice lacking negative regulators for BAT differentiation like p107, Rb and RIP140, there is a uniform replacement of WAT with BAT and elevated levels of PGC1a and Ucp1 [23,24]. Although many factors and regulatory pathways have been shown to play important roles in maintaining WAT identity, upstream signals or factors that determine the fate of BAT vs WAT and those that initiate the conversion of WAT to BAT remain unclear. considered to be derived from a common preadipocyte pool [14]. Whereas recent report by Timmoms et al [15] suggest that brown and white preadipocytes may have different origins. BAT is apparently more closely related to muscle as brown preadipocytes appear to express myogenic specific proteins. Interestingly, differentiated WAT has been shown to acquire certain BAT-like properties and can be converted into an energy consumption organ under special conditions. It has been observed that cold- exposure or administration of b3-agonist could induce the emergence of multilocular brown adipocytes that contain high amount of mitochondria and expresses Ucp1 in WAT depots [16,17]. Furthermore, ablation of b3-adrenoceptor diminishes the formation of BAT in white fat in cold exposed mice [18], suggesting the requirement of b-adrenoceptor signaling for the transdifferentiation of WAT to BAT. A similar increase in the occurrence of BAT in WAT areas was also observed in different strains of inbred mice [19]. Mice harboring increased BAT-like depots show a modest increase in b-adrenoreceptor signaling, which is sufficient to induce Ucp1 in these BAT-like depots. The requirement for PPARc in acquiring BAT-like and energy consuming organ in WAT was also shown in PPARc mutant (P465L) mice in which its WAT is unable to be converted to BAT in cold acclimatized mice [20]. Fsp27 is highly expressed in adipose tissue and up- regulated in ob/ob mice In human, it is also detected in heart and many other tissues [29]. Furthermore, Fsp27 protein is detected in the lipid droplet enriched fraction [30] and over-expressed Fsp27 protein was shown to be targeted to lipid droplets and promotes TAG storage [31]. Recently, Fsp27 was also found to be a direct mediator of PPARc-dependent hepatic steatosis [28]. However, its physiological role in regulating adipocyte function and the development of obesity is largely unknown. Here, we observed that Fsp27 protein was detected in both BAT and WAT, was dramatically up-regulated in the WAT and liver of ob/ob mice To elucidate the in vivo function of Fsp27 in adipocyte differentiation and the development of obesity, we isolated Fsp27 genomic DNA and generated Fsp27-null mice by homologous recombination. The translation initiation site ATG in exon 2 of the Fsp27 gene was replaced with a Neo gene cassette that is transcribed by a PGK promoter from the opposite direction (Fig. S1A). This strategy effectively deleted the first 31 amino acids of Fsp27. We confirmed the gene disruption by PCR and western blot analyses. Genetic deletion generates a PCR product of a 3 Kb instead of a 5 Kb wild-type band (Fig. 1E). Western blot analysis also showed that no Fsp27 protein was detected in either WAT or BAT of Fsp272/2mice, suggesting that Fsp27 gene was successfully disrupted (Fig. 1F). To investigate the role of Fsp27 in adipocyte differentiation, we first examined the morphology of fat pads. White fat pads from various locations such as gonadal fat and subcutaneous fat from August 2008 \| Volume 3 \| Issue 8 \| e2890 PLoS ONE \| www.plosone.org August 2008 \| Volume 3 \| Issue 8 \| e2890 2 Fsp27 Regulate WAT Identity Figure 1. Fsp27 expression in mice tissue and the morphology of WAT and BAT in Fsp272/2 mice. A. Tissue distributions of Fsp27 protein by western blot analysis. SI, small intestine; BAT, brown adipose tissue; GWAT, gonadal white adipose tissue; SM, skeletal muscle. b-tubulin was used as a loading control. B. Fsp27 is expressed in differentiated 3T3-L1 adipocytes. FABP (fatty acid binding protein) is used as an adipocyte differentiation marker. Day 0 represents undifferentiated 3T3-L1 fibroblasts. . C. Elevated Fsp27 protein levels in the WAT and liver of leptin deficient (ob/ob) mice. WT, wild type. D. Indirect immunofluorescence showing co-localization of Fsp27 with perilipin (Plin) on lipid droplet surface in Day 8 post differentiated 3T3-L1 cells. Fsp27 is highly expressed in adipose tissue and up- regulated in ob/ob mice E & F. Genotype analyses by PCR (E) and western blot (F) assays using tail genomic DNA and gonadal white adipose tissue (GWAT) lysate, respectively. +/+, +/2, 2/2 represent wild-type, Fsp27 heterozygous and homozygous mice, respectively. G & H. Morphology of WAT and BAT of wild type and Fsp272/2 mice. HE, hemotoxylin and eosin staining. EM, electron microscope image. 3 months old mice were used for the experiments. LD, lipid droplets; N, nuclei. Scale bar = 25 mm and 1 mm for HE and EM, respectively. . doi:10.1371/journal.pone.0002890.g001 Figure 1. Fsp27 expression in mice tissue and the morphology of WAT and BAT in Fsp272/2 mice. A. Tissue distributions of Fsp27 protein by western blot analysis. SI, small intestine; BAT, brown adipose tissue; GWAT, gonadal white adipose tissue; SM, skeletal muscle. b-tubulin was used as a loading control. B. Fsp27 is expressed in differentiated 3T3-L1 adipocytes. FABP (fatty acid binding protein) is used as an adipocyte differentiation marker. Day 0 represents undifferentiated 3T3-L1 fibroblasts. . C. Elevated Fsp27 protein levels in the WAT and liver of leptin deficient (ob/ob) mice. WT, wild type. D. Indirect immunofluorescence showing co-localization of Fsp27 with perilipin (Plin) on lipid droplet surface in Day 8 post differentiated 3T3-L1 cells. E & F. Genotype analyses by PCR (E) and western blot (F) assays using tail genomic DNA and gonadal white adipose tissue (GWAT) lysate, respectively. +/+, +/2, 2/2 represent wild-type, Fsp27 heterozygous and homozygous mice, respectively. G & H. Morphology of WAT and BAT of wild type and Fsp272/2 mice. HE, hemotoxylin and eosin staining. EM, electron microscope image. 3 months old mice were used for the experiments. LD, lipid droplets; N, nuclei. Scale bar = 25 mm and 1 mm for HE and EM, respectively. . doi:10.1371/journal.pone.0002890.g001 Fsp272/2 mice are all significantly reduced in sizes compared with those from wild type mice. Furthermore, WAT of Fsp272/2 mice isolated from the gonadal region had a dark-red color and abundant blood vessel circulation (data not shown). Staining by hemotoxylin and eosin (HE) revealed that white adipocytes from gonadal region of Fsp272/2 mice had small and multiple lipid droplets, whereas white adipocytes of wild type mice had large and unilocular lipid droplet (Fig. 1G). Electron microscope (EM) analysis using semi-thin sections of the WAT showed that white adipocytes of Fsp272/2 mice had defined minuscule lipid droplets distinguishable from each other, and occupied the cytosol like in brown adipocytes (Fig. Lean phenotype in Fsp272/2 and leptin/Fsp27 double deficient mice Furthermore, TAG levels in the WAT of ob/ob/Fsp272/2 mice were much lower (27.763.9 vs 586.96100.7 mmol/mg protein, P,0.01, Fig. 2E) compared with that of ob/ob mice. In addition to a reduced amount of TAG in WAT, the amount of TAG in the liver and skeletal muscle of Fsp272/2 mice was also significantly reduced (Fig. S1B). Therefore, the reduced TAG levels in WAT were not due to alternative lipid storage in other tissues such as liver and SM. In accordance with the presence of lower amount of WAT, Fsp27-null mice had significantly lower levels of serum leptin under either a ND (2.72061.732 pg/ml vs 1.41961.023 pg/ml, Table 1, P,0.05) or a HFD feeding condition (13.15768.824 pg/ml vs 4.5265.588 ng/ml, P,0.001). Levels of serum leptin under fasting condition were also significantly reduced in Fsp272/2 mice (Fig. 2F, Table 1). Consequently, food intake, which is regulated by plasma levels of leptin, was 10% higher in Fsp27-null mice compared to that of wildtype mice (Fig. 2G, 4.260.036 g/d vs 3.760.026 g/d, P,0.01). We also measured the levels of plasma TAG and NEFA and found that when animals were fed with a HFD, levels of plasma TAG were Lean phenotype in Fsp272/2 and leptin/Fsp27 double deficient mice The drastic reduction of lipid droplet size in WAT prompted us to investigate if the atrophy of WAT was due to its loss of lipid content by measuring the total amount of lipid in gonadal WAT. We found that total lipid in gonadal white fat (GWAT) pad was reduced by almost 6 fold (from 0.899560.1436 g in wild type to 0.151260.0079 g in Fsp272/2 mice, P,0.001, Fig. 2A), which strongly suggests that the reduction in fat pad size observed in WAT of Fsp272/2 mice is a result of decreased lipid content in the adipocytes. The total amount of protein and DNA content within this fat pad was similar between wild-type and Fsp27 null mice (data not shown). We then investigated the potential role of Fsp27 in regulating overall body weight and adiposity. Under normal diet (ND) conditions, the body weight of wild type and Fsp272/2 mice were similar (Fig. 2B), while the average body weight of ob/ ob mice was dramatically higher than that of wild type or Fsp272/ y g yp p 2 mice. However, the body weight of leptin/Fsp27 double deficient (ob/ob/Fsp272/2) mice was significantly lower than that of ob/ob mice (Fig. 2B, P,0.001), suggesting that Fsp27-deficiency could counteract with weight gain in ob/ob mice. When fed with a ND, the adiposity index of Fsp272/2 mice (0.061160.0021) was approximately 45% lower than that of wild type mice (0.110160.0008, P,0.0001). We also observed no difference in overall size with a slightly increased weight of some individual organs such as liver and heart in Fsp272/2 mice compared to control animals (data not shown). When mice were fed with a high fat diet (HFD), there was a 54% reduction in adiposity index in Fsp27 null mice (0.077260.0042) compared to that of wild type mice (0.169560.0052, P,0.0001, Fig. 2C). While the adiposity index of ob/ob mice is approximately 30%, the adiposity index for ob/ob/Fsp272/2 mice (10%) was markedly lower (P,0.001), representing a 70% reduction in total body fat (Fig. 2D). Consistent with the decreased adiposity index, the weight of fat pads from different anatomical locations in Fsp272/2 as well as WAT and BAT in ob/ob/Fsp272/2 mice was significantly reduced (Table 1 & 2). Levels of TAG in the WAT of Fsp272/2 mice (25.362.6 mmol/mg protein) were much lower than that of wild type mice (213.2662.5 mmol/mg protein), representing a 90% reduction (P,0.01, Fig. 2E). Fsp27 is highly expressed in adipose tissue and up- regulated in ob/ob mice The rate of oxygen consumption measured by indirect calorimetry was approximately 20% higher in Fsp27-null mice than in wildtype mice under both ND and HFD conditions (Fig. 2H), suggesting of an increased whole-body energy expenditure in Fsp272/2 mice. As Fsp27 has been suggested to play a role in TAG storage and lipolysis [31], we measured the rate of lipolysis in WAT in the absence or presence of b-agonist (isoproterenol) and observed that WAT of Fsp27 null mice had higher rate of basal lipolysis. The rate of b-agonist induced lipolysis was similar between wild type and Fsp27 null mice (Fig. 2I, Fig. S1C). We then measured the body temperature at ambient conditions under both feeding and fasting conditions and observed that Fsp272/2 mice had slightly higher body temperature compared with that of wild type mice (Fig. S2). Upon exposure to cold, the core body temperature of wild type and Fsp272/2 mice remain similar at the first 30 min after cold exposure (Fig. 2J). However, the core body temperature decreased rapidly in Fsp272/2 mice after prolonged cold exposure (Fig. 2J). Our data therefore clearly suggest that Fsp27-deficiency in both wild type and leptin deficient mice could lead to a lean phenotype and less fat accumulation in the WAT. liver was observed between wild type and Fsp27 mutant mice (data not shown). These data suggest that WAT of Fsp272/2 mice acquires a morphology similar to that of BAT with smaller and multiple lipid droplets and abundant blood vessel circulation. Lean phenotype in Fsp272/2 and leptin/Fsp27 double deficient mice Fsp27 is highly expressed in adipose tissue and up- regulated in ob/ob mice 1G). We also noted that the morphological features were homogeneous throughout the entire WAT. As Fsp27 is also expressed in BAT, we next compared the morphology of BAT of wild type and Fsp272/2 mice. The BAT dissected from Fsp272/2 mice appear to be pale and slightly enlarged compared with the wild type BAT. Histological examination of BAT from wild type and Fsp272/2 mice indicates that both contain multilocular lipid droplets. However, the sizes of lipid droplet in BAT of Fsp272/2 mice were significantly larger than that of wildtype mice. EM analysis confirmed such an observation (Fig. 1H). No gross morphological difference for SM, heart or Fsp272/2 mice are all significantly reduced in sizes compared with those from wild type mice. Furthermore, WAT of Fsp272/2 mice isolated from the gonadal region had a dark-red color and abundant blood vessel circulation (data not shown). Staining by hemotoxylin and eosin (HE) revealed that white adipocytes from gonadal region of Fsp272/2 mice had small and multiple lipid droplets, whereas white adipocytes of wild type mice had large and unilocular lipid droplet (Fig. 1G). Electron microscope (EM) analysis using semi-thin sections of the WAT showed that white adipocytes of Fsp272/2 mice had defined minuscule lipid droplets distinguishable from each other, and occupied the cytosol like in August 2008 \| Volume 3 \| Issue 8 \| e2890 3 PLoS ONE \| www.plosone.org Fsp27 Regulate WAT Identity significantly lower in Fsp27-null mice under fasting conditions (Table 1, P,0.001). Levels of plasma non-esterified fatty acids (NEFA) were also significantly lower in Fsp27-null mice under both HFD and ND feeding or fasting conditions (Table 1). Surpris- ingly, we observed lower levels of plasma ketone body under both ND and HFD fed conditions and under ND fasting condition for Fsp272/2 mice compared with that of wild type mice (Table 1). Levels of free cholesterol, LDL and HDL were similar between wild type and Fsp27 null mice (Table 1). Levels of plasma NEFA and TAG in ob/ob/Fsp272/2 mice were also lower than that of ob/ ob mice (Table 2). Levels of plasma glucose under both fed and fasting conditions were similar between wild type and Fsp272/2 mice. However, levels of plasma insulin under ND and fasting condition were varied and generally higher in Fsp272/2 mice than that of wild type (Table 1). Improved insulin sensitivity in Fsp272/2 and leptin/Fsp27 double deficient mice Adiposity index of 10 months old wild-type (+/ +) and Fsp27 mutant mice (2/2) fed with a normal diet (ND) or high fat diet (HFD). Fsp27 mutant mice have significantly less adipose tissue. (ND: +/+ n = 13, 2/2 n = 14 , HFD: +/+ n = 12, 2/2 n = 15) (*p,0.001). D. Adiposity index of 3 months old ob/ob and ob/ob/Fsp272/2 mice. ob/ob: n = 8, ob/ ob/ Fsp272/2: n = 8) (p,0.001). E. TAG content in gonadal white fat (GWAT) of wild type (WT), Fsp27 mutant (Fsp272/2), ob/ob, and ob/ob/ Fsp272/2 mice fed with normal diet. 3 mice were used for each genotype. p,0.01. F. Fasting serum leptin level in mice under normal diet (ND) or high-fat diet (HFD) feeding conditions. ND: +/+ n = 11, 2/2 n = 8; HFD: +/+ n = 14, 2/2 n = 11. p,0.05, *p,0.001. G. Increased food intake in Fsp27 mutant and ob/ob/Fsp272/2 mice. Wild type: n = 15, Fsp272/2: n = 17; ob/ob: n = 6, ob/ob/Fsp272/2: n = 6. p,0.01. H. Whole body O2 consumption rate of wild-type or Fsp27 mutant mice fed with a ND or HFD. ND: +/+ n = 14, 2/2 n = 13, HFD: +/+ n = 16, 2/2 n = 18. p,0.01, p,0.001. I. Lipolysis rate in GWAT of 3 months old wild type (+/+) and Fsp27 mutant (2/2) mice. Basal refers to no isoproterenol (Iso,1 mM) treatment.. N = 4 for each genotype. p,0.05. J. Core Body temperature of wild-type (+/+) or Fsp27 mutant (2/2) mice after animals were exposed to cold ( 4uC). 21 refers to core body temperature 1 hr before transfer of mice to 4uC. Fsp27 mutant mice had lower core body temperature when exposed to cold. (+/+ n = 25, 2/2 n = 20, p,0.05, p,0.01, p,0.001). d i 10 1371/j l 0002890 002 Fsp27 Regulate WAT Identity Figure 2. Lean phenotype of Fsp27 deficient and leptin/Fsp27 double deficient mice. A. Total lipid content of gonadal white fat pad from 3- month old wild-type (+/+) and Fsp27 mutant (2/2) mice, showing a decrease in lipid content in the WAT of Fsp272/2 mice. 5 pairs of mice were used for analysis. p,0.001. B. Body weight of wild type (WT), Fsp27 deficient (Fsp272/2), leptin deficient (ob/ob) and leptin/Fsp27 double deficient (ob/ ob/Fsp272/2) mice. Improved insulin sensitivity in Fsp272/2 and leptin/Fsp27 double deficient mice Improved insulin sensitivity in Fsp272/2 and leptin/Fsp27 double deficient mice The lean phenotype and lower plasma TAG and NEFA in Fsp272/2 and ob/ob/Fsp272/2 mice raised the possibility that their insulin sensitivity might be increased. We then measured the rates of glucose disposal and insulin sensitivity in Fsp272/2 and ob/ob/Fsp272/2 mice by glucose tolerance test (GTT) and insulin tolerance test (ITT). Fsp272/2 mice had significantly lower levels of blood glucose after administration of an exogenous load of glucose compared with that of wildtype mice, suggesting an enhanced glucose disposal (Fig. 3A). Whereas ob/ob mice had much higher levels of plasma glucose in the GTT test, a typical phenotype reflecting insulin resistance in these mice. Importantly, we also observed significantly reduced plasma glucose levels for ob/ob/Fsp272/2 mice (Fig. 3A) compared with that of ob/ob mice. These data suggest that mice with Fsp27 deficiency in both wild type and leptin deficient background all have improved glucose disposal rate. When administered with excessive amounts of insulin in an ITT test, Fsp272/2 mice showed reduced levels of blood glucose compared with that of wildtype mice (Fig. 3B, P,0.001). In addition, ob/ob/Fsp272/2 mice had significantly lower levels of blood glucose compared with that of ob/ob mice in an ITT test (Fig. 3B). Improved glucose disposal and lower blood glucose levels in ITT experiments suggest that Fsp272/2 and ob/ ob/Fsp272/2 mice have enhanced insulin sensitivity. To gain mechanistic insight into the enhanced insulin sensitivity, we measured levels of IRS1, AKT2 and GLUT4 (principal mediators of insulin signaling pathway in adipocytes) and assessed To gain mechanistic insight into the enhanced insulin sensitivity, we measured levels of IRS1, AKT2 and GLUT4 (principal mediators of insulin signaling pathway in adipocytes) and assessed PLoS ONE \| www.plosone.org August 2008 \| Volume 3 \| Issue 8 \| e2890 4 Fsp27 Regulate WAT Identity Figure 2. Lean phenotype of Fsp27 deficient and leptin/Fsp27 double deficient mice. A. Total lipid content of gonadal white fat pad from 3- month old wild-type (+/+) and Fsp27 mutant (2/2) mice, showing a decrease in lipid content in the WAT of Fsp272/2 mice. 5 pairs of mice were used for analysis. p,0.001. B. Body weight of wild type (WT), Fsp27 deficient (Fsp272/2), leptin deficient (ob/ob) and leptin/Fsp27 double deficient (ob/ ob/Fsp272/2) mice. (wildtype: n = 12; Fsp2/2: n = 12, ob/ob, n = 10; ob/ob/ Fsp272/2, n = 11, P,0.001)C. Improved insulin sensitivity in Fsp272/2 and leptin/Fsp27 double deficient mice Blood Chemistry and Fat Pad Weight of 10 months old wildtype (+/+) and Fsp272/2 (2/2) mice Normal Diet High Fat Diet +/+ 2/2 +/+ 2/2 N Mean6SEM N Mean6SEM P N Mean6SEM N Mean6SEM P NEFA (mEq/l) (Fed) 8 1.01460.243 8 0.63560.150 0.0021 9 1.30560.192 9 1.08660.147 0.0314* NEFA (mEq/l) (Fast) 8 1.35860.148 8 1.18960.120 0.0250* 9 1.56160.277 9 1.12060.283 0.0012 TG (mmol/l) (Fed) 8 0.56060.162 8 0.46360.056 0.0525 9 0.61560.136 9 0.24760.032 ,0.0001* TG (mmol/l) (Fast) 8 1.56360.308 8 1.04560.192 ,0.0001* 9 2.33560.277 9 1.31460.254 ,0.0001* Ketone Bodies (mmol/L) (Fed) 14 0.07060.025 12 0.03560.016 0.0003* 8 0.03660.01 14 0.06660.026 0.0010* Ketone Bodies (mmol/L) (Fast) 12 1.48760.107 14 0.53460.310 ,0.0001*** 16 0.73060.44 18 0.51560.295 0.1 Insulin (ng/ml) (Fed) 12 0.94660.550 11 1.07660.594 0.591 7 1.52260.657 12 2.06761.07 0.242 Insulin (ng/ml) (Fast) #9 #0.3576#0.084 #7 #0.3776#0.166 0.7586 13 1.76860.464 8 1.76760.836 0.999 Leptin (pg/ml) (Fed) 12 2.72061.732 12 1.41961.023 0.0350* 12 21.15768.824 13 13.5265.588 0.0190* Leptin (pg/ml) (Fast) 11 1.37560.955 7 0.53160.498 0.0480* 14 13.19864.091 11 3.32961.328 ,0.0001* Glucose (mmol/l) (Fed) 16 11.7062.635 17 11.75364.479 0.9675 18 11.1063.378 19 10.60561.504 0.5651 Glucose (mmol/l) (Fast) 15 6.88761.132 17 7.72961.768 0.1244 18 10.71761.827 19 11.96861.995 0.0548 Inguinal Fat 15 0.02360.009 14 0.01360.002 0.008 13 0.03960.011 15 0.01760.004 ,0.0001* Gonadal Fat 15 0.02460.006 14 0.00460.001 ,0.0001* 13 0.02860.007 15 0.00660.002 ,0.0001* Retroperitoneal Fat 15 0.01460.004 14 0.00860.002 0.0001* 13 0.02360.01 15 0.01760.007 0.07 Mesenteric Fat 15 0.01460.003 14 0.00960.002 ,0.0001* 13 0.01660.005 15 0.00860.002 ,0.0001* Subcuteneous Fat 15 0.01860.007 14 0.01260.004 0.009** 13 0.02360.009 15 0.01560.005 0.012* All values are means6s.e.m. Statistical analysis was done with two-tailed unpaired student t-test. N represents number of mice used. # Data obtained from 3 months old mice. P,0.05, P,0.01, P,0.001 doi:10.1371/journal.pone.0002890.t001 Table 2. Blood Chemistry and Tissue Weight of ob/ob and ob/ob/Fsp272/2 mice Table 2. Improved insulin sensitivity in Fsp272/2 and leptin/Fsp27 double deficient mice (wildtype: n = 12; Fsp2/2: n = 12, ob/ob, n = 10; ob/ob/ Fsp272/2, n = 11, P,0.001)C. Adiposity index of 10 months old wild-type (+/ +) and Fsp27 mutant mice (2/2) fed with a normal diet (ND) or high fat diet (HFD). Fsp27 mutant mice have significantly less adipose tissue. (ND: +/+ n = 13, 2/2 n = 14 , HFD: +/+ n = 12, 2/2 n = 15) (p,0.001). D. Adiposity index of 3 months old ob/ob and ob/ob/Fsp272/2 mice. ob/ob: n = 8, ob/ ob/ Fsp272/2: n = 8) (p,0.001). E. TAG content in gonadal white fat (GWAT) of wild type (WT), Fsp27 mutant (Fsp272/2), ob/ob, and ob/ob/ Fsp272/2 mice fed with normal diet. 3 mice were used for each genotype. p,0.01. F. Fasting serum leptin level in mice under normal diet (ND) or high-fat diet (HFD) feeding conditions. ND: +/+ n = 11, 2/2 n = 8; HFD: +/+ n = 14, 2/2 n = 11. p,0.05, p,0.001. G. Increased food intake in Fsp27 mutant and ob/ob/Fsp272/2 mice. Wild type: n = 15, Fsp272/2: n = 17; ob/ob: n = 6, ob/ob/Fsp272/2: n = 6. p,0.01. H. Whole body O2 consumption rate of wild-type or Fsp27 mutant mice fed with a ND or HFD. ND: +/+ n = 14, 2/2 n = 13, HFD: +/+ n = 16, 2/2 n = 18. p,0.01, p,0.001. I. Lipolysis rate in GWAT of 3 months old wild type (+/+) and Fsp27 mutant (2/2) mice. Basal refers to no isoproterenol (Iso,1 mM) treatment.. N = 4 for each genotype. p,0.05. J. Core Body temperature of wild-type (+/+) or Fsp27 mutant (2/2) mice after animals were exposed to cold ( 4uC). 21 refers to core body temperature 1 hr before transfer of mice to 4uC. Fsp27 mutant mice had lower core body temperature when exposed to cold. (+/+ n = 25, 2/2 n = 20, p,0.05, p,0.01, *p,0.001). doi:10.1371/journal.pone.0002890.g002 August 2008 \| Volume 3 \| Issue 8 \| e2890 PLoS ONE \| www.plosone.org 5 Fsp27 Regulate WAT Identity ble 1. All values are means6s.e.m. Statistical analysis was done with two-tailed unpaired student t-test. N represents number of mice used # Data obtained from 3 months old mice. values are means 6 s.e.m. Statistical analysis was done with two-tailed unpaired student t-test. N represents number of mice used. 0 05 Improved insulin sensitivity in Fsp272/2 and leptin/Fsp27 double deficient mice Blood Chemistry and Tissue Weight of ob/ob and ob/ob/Fsp272/2 mice ob/ob ob/ob/Fsp272/2 N Mean6SEM N Mean6SEM P NEFA (mEq/l) (Fed) 8 3.90660.383 8 3.61760.212 0.2946 NEFA (mEq/l) (Fast) 8 4.58260.686 8 3.87360.382 0.01148 TG (mg/l) (Fed) 8 0.318560.0142 8 0.368260.0216 ,0.0001* TG (mg/l) (Fast) 8 0.358460.0222 8 0.425660.0232 0.0028 Cholesterol (mmol/l) (Fed) 8 5.80560.014 8 6.51860.013 0.016* Cholesterol (mmol/l) (Fast) 8 5.51760.020 8 6.14760.007 0.04748* Insulin (ng/ml) (Fed) 8 7.98660.162 8 6.29060.209 ,0.0001* Insulin (ng/ml) (Fast) 8 7.42660.320 8 6.01160.133 ,0.0001* Glucose (mmol/l) (Fed) 12 17.24 6 1.129 12 16.766 1.210 0.3226 Glucose (mmol/l) (Fast) 12 11.90 6 1.369 12 11.376 0.884 0.2692 Gonadal Fat Pad 10 0.061 6 0.007 10 0.004 6 0.002 ,0.0001* BAT 10 0.061 6 0.007 10 0.004 6 0.002 ,0.0001* Liver 10 0.102 6 0.009 10 0.009 6 0.002 ,0.0001*** All values are means 6 s.e.m. Statistical analysis was done with two-tailed unpaired student t-test. N represents number of mice used. P,0.05, P,0.01, ** August 2008 \| Volume 3 \| Issue 8 \| e2890 Fsp27 Regulate WAT Identity Figure 3. Increased insulin sensitivity in Fsp272/ and ob/ob/Fsp272/2 mice. GTT (A) and ITT (B) experiments using 10-week-old wild type (WT), Fsp27 mutant (Fsp272/2), leptin deficient (ob/ob) and leptin/Fsp27 double deficient (ob/ob/Fsp272/2) mice. 6 mice from each group were used for the experiment. p,0.05; p,0.01; *p,0.001. Western blot analysis for levels of IRS-1 and insulin stimulated IRS-1 tyrosine phosphorylation (p- IRS) in the WAT of wild type (WT) and Fsp27 mutant (Fsp272/2) mice (C) and in the WAT of leptin deficient (ob/ob) and leptin/Fsp27 double deficient (ob/ob/Fsp272/2) mice (D). . E & F. Western blot analysis of total AKT2, phosphor-AKT2 (pAKT2) and GLUT4 protein levels in WAT of wild type (WT), Fsp27 mutant (Fsp272/2), leptin deficient (ob/ob) and leptin/Fsp27 double deficient (ob/ob/Fsp272/2) mice. b-actin was used as loading control. doi:10.1371/journal.pone.0002890.g003 Figure 3. Increased insulin sensitivity in Fsp272/ and ob/ob/Fsp272/2 mice. GTT (A) and ITT (B) experiments using 10-week-old wild type (WT), Fsp27 mutant (Fsp272/2), leptin deficient (ob/ob) and leptin/Fsp27 double deficient (ob/ob/Fsp272/2) mice. 6 mice from each group were used for the experiment. p,0.05; p,0.01; p,0.001. Western blot analysis for levels of IRS-1 and insulin stimulated IRS-1 tyrosine phosphorylation (p- IRS) in the WAT of wild type (WT) and Fsp27 mutant (Fsp272/2) mice (C) and in the WAT of leptin deficient (ob/ob) and leptin/Fsp27 double deficient (ob/ob/Fsp272/2) mice (D). . E & F. Improved insulin sensitivity in Fsp272/2 and leptin/Fsp27 double deficient mice Western blot analysis of total AKT2, phosphor-AKT2 (pAKT2) and GLUT4 protein levels in WAT of wild type (WT), Fsp27 mutant (Fsp272/2), leptin deficient (ob/ob) and leptin/Fsp27 double deficient (ob/ob/Fsp272/2) mice. b-actin was used as loading control. doi:10.1371/journal.pone.0002890.g003 the phosphorylation levels of IRS1 and AKT2 in the absence or presence of insulin. Levels of IRS1 were slightly increased in the WAT of Fsp272/2 mice (P,0.01, Fig. 3C, Fig. S3A). However, levels of tyrosine phosphorylation on IRS1 were drastically increased in the WAT of Fsp27 mutant mice (approximately 8 fold higher, P,0.001, Fig. 3C, Supplementary 3A). In addition, levels of IRS1 tyrosine phosphorylation in the WAT of ob/ob/ Fsp272/2 mice were also significantly increased compared with that in ob/ob mice (Fig. 3D, P,0.001, Fig. S3A). Levels of AKT2 were also significantly increased in Fsp272/2 and ob/ob/Fsp272/2 mice (Fig. 3 E&F, P,0.01 & P,0.001, Fig. S3B). Furthermore, levels of phosphorylated-AKT2 in the presence of insulin were signifi- cantly increased in the WAT of Fsp272/2 and ob/ob/Fsp272/2 mice. No difference in IRS2 phosphorylation was observed in Fsp27-null mice (data not shown). Levels of IRS1 and AKT2, and their phosphorylated products in BAT, liver and skeletal muscle are similar between wild type and Fsp272/2 mice (Fig. S4), suggesting no change in insulin sensitivity in these tissues. Surprisingly, we observed significantly increased levels of GLUT4 in the WAT of Fsp272/2 and ob/ob/Fsp272/2 mice (Fig. 3 E&F, P,0.001, Fig. S3C). These data suggest that insulin sensitivity in the WAT of Fsp272/2 and ob/ob/Fsp272/2 mice was improved possibly due to the increased protein levels of IRS1, AKT2 and GLUT4, as well as IRS1 tyrosine phosphorylation. Increases in mitochondrial size, activity and proteins in WAT of Fsp272/2 mice To investigate the underlying mechanism for reduced lipid accumulation, increased whole body metabolism in WAT and lean PLoS ONE \| www.plosone.org August 2008 \| Volume 3 \| Issue 8 \| e2890 7 Fsp27 Regulate WAT Identity Fsp27 Regulate WAT Identity Fsp27 Regulate WAT Identity wavy cristae that transverse the width of the mitochondria, a typical characteristic of metabolically active mitochondria (Fig. 4A). While brown adipocytes of wild type mice have larger mitochondria in their cytosol with dense and regular cristea aligned along the mitochondrial matrix, mitochondria from Fsp272/2 brown adipocytes were irregular in size and had significantly fewer cristea in its matrix, suggesting their lower phenotypes in Fsp27 null mice, we examined the mitochondrial morphology of Fsp272/2 white adipocytes by EM analysis. The mitochondria in wildtype white adipocytes were smaller and more compact. However, the mitochondrial volume appears to be dramatically increased in gonadal white adipocytes of Fsp27-null mice compared with that of wild type mice (Fig. 4A). Mitochon- dria in Fsp272/2 white adipocytes were full of straight or slightly mice compared with that of wild type mice (Fig. 4A). Mitochon- dria in Fsp272/2 white adipocytes were full of straight or slightly Fsp272/2 brown adipocytes were irregular in size and had significantly fewer cristea in its matrix, suggesting their lower Figure 4. Increased mitochondria activity in the WAT of Fsp27 mutant mice. A. Electron microscope image of mitochondria in gonadal WAT (GWAT) (Scale Bar = 0.2 mm) and BAT (Scale Bar = 0.5 um) of wild-type and Fsp272/2 mice showing enlarged mitochondria in Fsp272/2 mice. N: nucleus, L: lipid droplet, M: mitochondria. B. Cytochrome oxidase IV (Cox) activity in GWAT, BAT, muscle and liver (n = 4). COXIV activity in Fsp272/2 mice is higher in GWAT (p,0.05), but lower in BAT (*p,0.01) as compared with that of wild type mice. No difference in COX activity is detected in muscle or liver. C. Mitochondrial oxygen consumption rate is higher in WAT of Fsp272/2 mice. (p,0.05) (n = 3). D. Western blot analysis of various mitochondrial proteins in the WAT of wild-type and Fsp27 mutant mice, showing higher level in Fsp27 mutant WAT. (n = 4), CytoC, cytochrome C. b- tubulin was used as loading control. D. Relative mRNA levels of COXIV, CPT1 and CPT2 in the WAT of wild type (+/+) and Fsp27 mutant mice (2/2). Increases in mitochondrial size, activity and proteins in WAT of Fsp272/2 mice 5B & C). These data suggest that Fsp27 acts upstream to control insulin signaling pathway and modulates programs that maintain WAT identity such as FoxC2, Rb, p107 and RIP140. Furthermore, Fsp27 regulates the expression of PPAR and PGC1 that in turn regulate their downstream targets such as Ucp1, Dio2 and Cidea expression. WAT of Fsp272/2 mice. On the other hand, Rb, p107 and RIP140, factors that were negatively associated with WAT to BAT transformation were substantially decreased in WAT of Fsp272/2 mice (Fig. 5A). Furthermore, mRNA levels for GLUT4 and AKT2 were significantly increased (Fig. 5A), consistent with increased insulin sensitivity in Fsp27 null mice. In accordance with their increased mRNA levels, protein levels for Cidea, PGC1a, Ucp1 and Fabp were significantly increased in WAT of Fsp272/2 mice (Fig. 5B &C). We then checked the protein levels of Cidea, PGC1a, Ucp1 and FABP in the WAT of ob/ob and ob/ob/Fsp272/ 2 mice and observed that their levels were all significantly up- regulated in ob/ob/Fsp272/2 mice (Fig. 5B & C). These data suggest that Fsp27 acts upstream to control insulin signaling pathway and modulates programs that maintain WAT identity such as FoxC2, Rb, p107 and RIP140. Furthermore, Fsp27 regulates the expression of PPAR and PGC1 that in turn regulate their downstream targets such as Ucp1, Dio2 and Cidea expression. activity in BAT (Fig. 4A). No change in mitochondrial morphology was observed in muscle cells and hepatocytes of wild type and Fsp272/2 mice (data not shown). activity in BAT (Fig. 4A). No change in mitochondrial morphology was observed in muscle cells and hepatocytes of wild type and Fsp272/2 mice (data not shown). To investigate whether the increased mitochondrial size is correlated with enhanced activity, we measured the activity of mitochondrial cytochrome C oxidase (COX), which is the terminal electron acceptor that transfers electron from cytochrome C to molecular oxygen. We observed that COX activity in the mitochondria of Fsp272/2 WAT (12.2960.7381 U/min/mg) is approximately 5 fold higher than that of wild type WAT (3.39460.9319 U/min/mg, P#0.01, Fig. 4B). In contrast, COX activity in BAT of Fsp272/2 mice (114.769.402 U/min/mg) was slightly lower than that of wild type mice (15968.622 U/min/mg). Consistent with their decreased mitochondrial activity, levels of mitochondrial proteins such as Cpt1, Cpt2 and BAT specific proteins such as Ucp1 and PGC1 were decreased in the BAT of Fsp272/2 mice (Fig. S5). Differentiated Fsp272/2 MEFs display characteristics of brown adipocyte p y To check whether the conversion of WAT to BAT in Fsp272/2 mice is cell autonomous or requires exogenous signals, we isolated mouse embryonic fibroblasts (MEFs) from wild type and Fsp272/2 mice and induced them to differentiate into adipocytes in vitro. The morphologies of differentiated MEFs from wildtype and Fsp272/2 mice are very different. Wildtype adipocytes have fewer but larger lipid droplets while differentiated Fsp272/2 MEFs possesses more but smaller lipid droplets (Fig. 6A). The small lipid droplets in Fsp272/2 adipocytes tend to cluster together, fully resembling the morphology seen in WAT from Fsp272/2 mice. Furthermore, levels of TAG from differentiated Fsp272/2 MEFs were significantly lower than that of differentiated wild type MEFs (Fig. 6B). The lower amount of TAG and smaller lipid droplets in Fsp272/2 adipocytes was not due to their defect in differentiation as the levels of adipocyte-specific markers such as Fabp and Perilipin-A were in fact slightly higher in Fsp272/2 adipocytes (data not shown). Furthermore, the lower amount of TAG in differentiated Fsp272/2 MEFs was not due to a decrease in FFA uptake as the rate of fatty acid uptake was similar between differentiated wildtype and Fsp272/2 MEFs (data not shown). Consistent with data observed with WAT of Fsp272/2 mice, rates of basal lipolysis were all significantly increased in differentiated Fsp272/2 MEFs compared with that of wild type cells (Fig. 6C, P,0.05). These data suggest that Fsp27 deficiency in differenti- ated MEFs leads to lower TAG accumulation, increased basal lipolysis and smaller lipid droplets. y To assess the molecular basis of increased mitochondria activity in Fsp272/2 mice, we measured the expression levels of several genes, including cytochrome c, COXIV, CPT1 and CPT2, that are involved in mitochondrial oxidative phosphorylation and fatty acid oxidation. We observed that their protein levels were markedly increased in Fsp272/2 WAT. Hsp60, a chaperon protein localized in the mitochondrial matrix, was also signifi- cantly increased (Fig. 4D), consistent with the increased mitochondrial biogenesis in Fsp272/2 white adipocytes. We further measured their mRNA levels by semi-quantitative PCR and observed that the mRNA levels of COXIV, CPT1 and CPT2 were significantly increased in Fsp272/2 mice, consistent with the increase in their protein levels (Fig. 4E). These data suggest that WAT of Fsp272/2 mice have increased mitochondrial activity due to increases mitochondrial size and higher levels of mitochondrial gene expression. Up-regulation of crucial transcriptional factors and BAT- specific proteins in Fsp272/2 WAT The morphological resemblance of WAT to that of BAT, and the increased mitochondrial activity in WAT of Fsp272/2 mice, suggest that Fsp272/2 WAT might have acquired BAT-like properties. To determine the extent of shifting of the physiology and gene expression profiles of WAT to BAT in Fsp272/2 mice, we checked the expression levels of BAT-specific genes and several transcriptional regulators by real-time PCR analysis. We observed that the mRNA levels of the brown fat specific gene Ucp1 were significantly elevated in the WAT of Fsp272/2 mice compared with that of wild type mice (approximately 20 folds, P,0.05, Fig. 5A). The mRNA levels of Cidea and Dio2, a protein that catalyzes the conversion of T4 (thyroxine) into the active substance T3 (3, 5, 39-tri-iodothyronine) in BAT, were also significantly increased in the WAT of Fsp272/2 mice. In addition, mRNA levels of PGC1a, PPARa and PPARc were all increased in the WAT of Fsp272/2 mice. Consistently, mRNA levels for FABP (aP2), a downstream target of PPARc, were also increased (Fig. 5A). Importantly, FoxC2, a crucial regulator of cAMP pathway that promotes BAT differentiation, was increased in the We then checked the expression levels of PGC1a, Ucp1, Cidea and mitochondrial proteins in differentiated Fsp272/2 MEFs in the presence or absence of T3. In the absence of T3, protein levels for PGC1a were similar for differentiated wild type and Fsp272/2 MEFs (Fig. 6D), which is in drastic contrast to the phenotypic changes seen between the wildtype and mutant adipocytes derive from the mice. In addition, protein levels for mitochondrial proteins for oxidative phosphorylation such as cytochrome-C and COXIV were also similar in differentiated wild type and Fsp272/2 MEFs. Furthermore, no up-regulation of Ucp1 and Cidea was observed in differentiated Fsp272/2 MEFs compared to wild type (Fig. 6D). These data suggest that in vitro molecular induction of Ucp1 expression and upregulation of mitochondrial proteins for oxidative phosphorylation in Fsp272/2 adipocytes requires exogenous signals or factors, and are likely influenced by endocrine regulation. We then tested several factors known to promote BAT differentiation such as b-agonist, retinoid acid and We then checked the expression levels of PGC1a, Ucp1, Cidea and mitochondrial proteins in differentiated Fsp272/2 MEFs in the presence or absence of T3. In the absence of T3, protein levels for PGC1a were similar for differentiated wild type and Fsp272/2 MEFs (Fig. Increases in mitochondrial size, activity and proteins in WAT of Fsp272/2 mice No difference in COX activity was observed in muscle and liver between wild type and Fsp272/2 mice (Fig. 4B). To further confirm that mitochondrial activity is increased in mutant WAT, we measured the rate of mitochondrial oxygen consumption from WAT of wildtype and Fsp272/2 mice using a Clark-type electrode. Mitochondrial oxygen consumption rate of Fsp272/2 WAT was 2 fold higher (44.5263.186 mol/mg) than that of wild- type mice (22.3964.037 mol/ng, P,0.05, Fig. 4C). These data all indicate that Fsp272/2 white adipocytes showed increased mito- chondrial size and enhanced mitochondrial activity. Differentiated Fsp272/2 MEFs display characteristics of brown adipocyte Increases in mitochondrial size, activity and proteins in WAT of Fsp272/2 mice doi:10.1371/journal.pone.0002890.g004 PLoS ONE \| www plosone org 8 August 2008 \| Volume 3 \| Issue 8 \| e2890 Figure 4. Increased mitochondria activity in the WAT of Fsp27 mutant mice. A. Electron microscope image of mitochondria in gonadal WAT (GWAT) (Scale Bar = 0.2 mm) and BAT (Scale Bar = 0.5 um) of wild-type and Fsp272/2 mice showing enlarged mitochondria in Fsp272/2 mice. N: nucleus, L: lipid droplet, M: mitochondria. B. Cytochrome oxidase IV (Cox) activity in GWAT, BAT, muscle and liver (n = 4). COXIV activity in Fsp272/2 Figure 4. Increased mitochondria activity in the WAT of Fsp27 mutant mice. A. Electron microscope image of mitochondria in gonadal WAT (GWAT) (Scale Bar = 0.2 mm) and BAT (Scale Bar = 0.5 um) of wild-type and Fsp272/2 mice showing enlarged mitochondria in Fsp272/2 mice. N: nucleus, L: lipid droplet, M: mitochondria. B. Cytochrome oxidase IV (Cox) activity in GWAT, BAT, muscle and liver (n = 4). COXIV activity in Fsp272/2 mice is higher in GWAT (p,0.05), but lower in BAT (p,0.01) as compared with that of wild type mice. No difference in COX activity is detected in muscle or liver. C. Mitochondrial oxygen consumption rate is higher in WAT of Fsp272/2 mice. (p,0.05) (n = 3). D. Western blot analysis of various mitochondrial proteins in the WAT of wild-type and Fsp27 mutant mice, showing higher level in Fsp27 mutant WAT. (n = 4), CytoC, cytochrome C. b- tubulin was used as loading control. D. Relative mRNA levels of COXIV, CPT1 and CPT2 in the WAT of wild type (+/+) and Fsp27 mutant mice (2/2). doi:10.1371/journal.pone.0002890.g004 August 2008 \| Volume 3 \| Issue 8 \| e2890 PLoS ONE \| www.plosone.org 8 Fsp27 Regulate WAT Identity WAT of Fsp272/2 mice. On the other hand, Rb, p107 and RIP140, factors that were negatively associated with WAT to BAT transformation were substantially decreased in WAT of Fsp272/2 mice (Fig. 5A). Furthermore, mRNA levels for GLUT4 and AKT2 were significantly increased (Fig. 5A), consistent with increased insulin sensitivity in Fsp27 null mice. In accordance with their increased mRNA levels, protein levels for Cidea, PGC1a, Ucp1 and Fabp were significantly increased in WAT of Fsp272/2 mice (Fig. 5B &C). We then checked the protein levels of Cidea, PGC1a, Ucp1 and FABP in the WAT of ob/ob and ob/ob/Fsp272/ 2 mice and observed that their levels were all significantly up- regulated in ob/ob/Fsp272/2 mice (Fig. Up-regulation of crucial transcriptional factors and BAT- specific proteins in Fsp272/2 WAT 6D), which is in drastic contrast to the phenotypic changes seen between the wildtype and mutant adipocytes derive from the mice. In addition, protein levels for mitochondrial proteins for oxidative phosphorylation such as cytochrome-C and COXIV were also similar in differentiated wild type and Fsp272/2 MEFs. Furthermore, no up-regulation of Ucp1 and Cidea was observed in differentiated Fsp272/2 MEFs compared to wild type (Fig. 6D). These data suggest that in vitro molecular induction of Ucp1 expression and upregulation of mitochondrial proteins for oxidative phosphorylation in Fsp272/2 adipocytes requires exogenous signals or factors, and are likely influenced by endocrine regulation. We then tested several factors known to promote BAT differentiation such as b-agonist, retinoid acid and PLoS ONE \| www.plosone.org August 2008 \| Volume 3 \| Issue 8 \| e2890 9 Fsp27 Regulate WAT Identity T3 during MEF differentiation We observed that in the presence mRNA levels for GLUT4 were also increased in differentiated Figure 5. Altered expression levels of crucial metabolic regulators and BAT-specific factors in Fsp27 mutant mice. A. Real-time PCR analysis of regulatory factors and BAT specific gene expression in the WAT of wild type (+/+) and Fsp272/2 mice (2/2). Significantly higher levels of BAT markers expression in Fsp27 mutant mice WAT were detected (p,0.05; p,0.01). B. Western blot analysis showing increased protein levels of Cidea, PGC1a, Ucp1 and FABP in the WAT of wildtype, ob/ob, Fsp272/2 and ob/ob/Fsp272/2of Fsp27 mutant mice. b-tubulin was used as loading control. C. Densitometric reading of relative levels of the indicated proteins in western blot analysis (as in B) were quantitated in these mouse strains. Similar experiments were done five times and the intensity of individual band in each western blot was quantified by TOTAL-LAB software (Nonlinear Dynamics, UK) and used for statistical analysis. The relative protein level in wildtype and ob/ob mice was designated as 1.0. p,0.01. doi:10.1371/journal.pone.0002890.g005 Figure 5. Altered expression levels of crucial metabolic regulators and BAT-specific factors in Fsp27 mutant mice. A. Real-time PCR analysis of regulatory factors and BAT specific gene expression in the WAT of wild type (+/+) and Fsp272/2 mice (2/2). Significantly higher levels of BAT markers expression in Fsp27 mutant mice WAT were detected (p,0.05; p,0.01). B. Western blot analysis showing increased protein levels of Cidea, PGC1a, Ucp1 and FABP in the WAT of wildtype, ob/ob, Fsp272/2 and ob/ob/Fsp272/2of Fsp27 mutant mice. b-tubulin was used as loading control. C. Up-regulation of crucial transcriptional factors and BAT- specific proteins in Fsp272/2 WAT Densitometric reading of relative levels of the indicated proteins in western blot analysis (as in B) were quantitated in these mouse strains. Similar experiments were done five times and the intensity of individual band in each western blot was quantified by TOTAL-LAB software (Nonlinear Dynamics, UK) and used for statistical analysis. The relative protein level in wildtype and ob/ob mice was designated as 1.0. p,0.01. doi:10.1371/journal.pone.0002890.g005 mRNA levels for GLUT4 were also increased in differentiated Fsp272/2 MEFs. The rate of fatty acid oxidation, an indicator of mitochondrial activity, is increased in differentiated Fsp272/2 adipocytes in the presence of T3 at various time points tested (Fig. 6F). These data suggest that Fsp272/2 MEFs attain certain characteristics of brown adipocyte-like cells in vitro and that the thyroid hormone T3 plays a critical role in this phenotypic changes. T3 during MEF differentiation. We observed that in the presence of T3 in the differentiation medium, basal lipolysis rate (Fig. 6C) and protein levels for PGC1a were significantly increased in differentiated Fsp272/2 MEFs compared with that of wild type cells (Fig. 6D, Fig. S6, P,0.05). Ucp1 protein was also induced in the presence of T3 in differentiated wild type MEFs but its levels were increased by approximately two fold in differentiated Fsp272/2 MEFs (P,0.05). Levels of Cidea were also significantly increased by more than 3 fold in differentiated Fsp272/2 MEFs (Fig. 6D, Fig. S6, P,0.05) in the presence of T3, although no difference in mRNA levels for Ucp1 or Cidea was observed between wild type and Fsp272/2 MEFs. Furthermore, levels of mitochondrial proteins such as COXIV, cytochrome C and Hsp60 were also higher. Consistent with the increase in PGC1a protein levels, the mRNA levels for PGC1a were also significantly increased (approximately 4 fold) and FoxC2 (2 fold) in differen- tiated Fsp272/2 MEFs in the presence of T3 (Fig. 6E). Consistent with our in vivo data, levels of Rb and RIP140 mRNAs were significantly decreased in differentiated Fsp272/2 MEFs. The T3 during MEF differentiation. We observed that in the presence of T3 in the differentiation medium, basal lipolysis rate (Fig. 6C) and protein levels for PGC1a were significantly increased in differentiated Fsp272/2 MEFs compared with that of wild type cells (Fig. 6D, Fig. S6, P,0.05). Ucp1 protein was also induced in the presence of T3 in differentiated wild type MEFs but its levels were increased by approximately two fold in differentiated Fsp272/2 MEFs (P,0.05). Up-regulation of crucial transcriptional factors and BAT- specific proteins in Fsp272/2 WAT Levels of Cidea were also significantly increased by more than 3 fold in differentiated Fsp272/2 MEFs (Fig. 6D, Fig. S6, P,0.05) in the presence of T3, although no difference in mRNA levels for Ucp1 or Cidea was observed between wild type and Fsp272/2 MEFs. Furthermore, levels of mitochondrial proteins such as COXIV, cytochrome C and Hsp60 were also higher. Consistent with the increase in PGC1a protein levels, the mRNA levels for PGC1a were also significantly increased (approximately 4 fold) and FoxC2 (2 fold) in differen- tiated Fsp272/2 MEFs in the presence of T3 (Fig. 6E). Consistent with our in vivo data, levels of Rb and RIP140 mRNAs were significantly decreased in differentiated Fsp272/2 MEFs. The Discussion Fields shown were visualized under fluorescence microscope at appropriate wavelengths for GFP (green) and Hoechst (blue). B. Lower TAG accumulation in differentiated Fsp272/2 MEFs (2/2) than in wildtype (+/+) cells. (n = 4) (p,0.01; p,0.001). C. Increased basal lipolysis rate in differentiated Fsp272/2 MEFs in the absence or presence of T3 (10 nM) in the differentiation medium. (n = 3) (p,0.05; *p,0.01). D. Western blot analysis showing levels of various proteins in differentiated MEF cells in the absence or presence of T3 (10 nM). b-tubulin was used as a loading control. E. Relative mRNA levels of various genes in differentiated wildtype (+/+) and Fsp272/2(2/2)MEFs. mRNA levels for Rb were measured using MEFs that were differentiated for 3 days. (n = 4) (p,0.05). F. Higher levels of fatty acid b-oxidation rate in differentiated Fsp272/2 (2/2) MEFs as compared to wildtype (+/+) MEFs in the presence of T3 (10nM). (n = 4) (p,0.01). Results of relative amount of 3H2O released into the medium after four (4h) or six (6h) hours chase of pre-labelled MEFs with cold palmitate. Wildtype oxidation rate at 4h is set to 100. G. Regulatory and metabolic pathways that are altered in the WAT of Fsp272/2 mutant mice, leading to anti-obesity and insulin sensitive phenotype. doi:10.1371/journal.pone.0002890.g006 Figure 6. Differentiated Fsp272/2 MEFs display many brown adipocyte characteristics. A. Smaller lipid droplets in differentiated Fsp27 mutant mouse embryonic fibroblasts (MEFs). Lipid droplets were stained with BODIPY493/503, and nuclei were stained with Hochest 366243. Fields shown were visualized under fluorescence microscope at appropriate wavelengths for GFP (green) and Hoechst (blue). B. Lower TAG accumulation in differentiated Fsp272/2 MEFs (2/2) than in wildtype (+/+) cells. (n = 4) (p,0.01; **p,0.001). C. Increased basal lipolysis rate in differentiated Fsp272/2 MEFs in the absence or presence of T3 (10 nM) in the differentiation medium. (n = 3) (p,0.05; *p,0.01). D. Western blot analysis showing levels of various proteins in differentiated MEF cells in the absence or presence of T3 (10 nM). b-tubulin was used as a loading control. E. Relative mRNA levels of various genes in differentiated wildtype (+/+) and Fsp272/2(2/2)MEFs. mRNA levels for Rb were measured using MEFs that were differentiated for 3 days. (n = 4) (p,0.05). F. Higher levels of fatty acid b-oxidation rate in differentiated Fsp272/2 (2/2) MEFs as compared to wildtype (+/+) MEFs in the presence of T3 (10nM). (n = 4) (p,0.01). Discussion Through genetic deletion of Fsp27, we have found that Fsp27 deficiency causes reduced white fat pads, decreased adiposity, increased whole body metabolism, enhanced insulin sensitivity and a lean phenotype not only in a wild type background but also in leptin deficient obese mice. The anti-obesity effect of Fsp27 deficiency in wild type and ob/ob background is likely due to increased energy expenditure in WAT of Fsp272/2 mice as it has markedly increased mitochondrial activity, which could in turn August 2008 \| Volume 3 \| Issue 8 \| e2890 PLoS ONE \| www.plosone.org August 2008 \| Volume 3 \| Issue 8 \| e2890 10 Fsp27 Regulate WAT Identity Figure 6. Differentiated Fsp272/2 MEFs display many brown adipocyte characteristics. A. Smaller lipid droplets in differentiated Fsp27 mutant mouse embryonic fibroblasts (MEFs). Lipid droplets were stained with BODIPY493/503, and nuclei were stained with Hochest 366243. Fields shown were visualized under fluorescence microscope at appropriate wavelengths for GFP (green) and Hoechst (blue). B. Lower TAG accumulation in differentiated Fsp272/2 MEFs (2/2) than in wildtype (+/+) cells. (n = 4) (p,0.01; **p,0.001). C. Increased basal lipolysis rate in differentiated Fsp272/2 MEFs in the absence or presence of T3 (10 nM) in the differentiation medium. (n = 3) (p,0.05; *p,0.01). D. Western blot analysis showing levels of various proteins in differentiated MEF cells in the absence or presence of T3 (10 nM). b-tubulin was used as a loading control. E. Relative mRNA levels of various genes in differentiated wildtype (+/+) and Fsp272/2(2/2)MEFs. mRNA levels for Rb were measured using MEFs that were differentiated for 3 days. (n = 4) (p,0.05). F. Higher levels of fatty acid b-oxidation rate in differentiated Fsp272/2 (2/2) MEFs as compared to wildtype (+/+) MEFs in the presence of T3 (10nM). (n = 4) (**p,0.01). Results of relative amount of 3H2O released into the medium after four (4h) or six (6h) hours chase of pre-labelled MEFs with cold palmitate. Wildtype oxidation rate at 4h is set to 100. G. Regulatory and metabolic pathways that are altered in the WAT of Fsp272/2 mutant mice, leading to anti-obesity and insulin sensitive phenotype. doi:10 1371/journal pone 0002890 g006 Figure 6. Differentiated Fsp272/2 MEFs display many brown adipocyte characteristics. A. Smaller Figure 6. Differentiated Fsp272/2 MEFs display many brown adipocyte characteristics. A. Smaller lipid droplets in differentiated Fsp27 mutant mouse embryonic fibroblasts (MEFs). Lipid droplets were stained with BODIPY493/503, and nuclei were stained with Hochest 366243. Discussion Furthermore, the increase in Dio2 expression could also boost the local concentration of T3, and serve as a source for systemic T3 in WAT of Fsp272/2 mice, resulting in the increased PGC1a and Ucp1 expression. These alterations all point to a possibility that the WAT tissue of Fsp272/ 2 mice has acquired certain BAT-like properties, and hence was transformed from a typical energy storage tissue into an energy consuming tissue. Although the exact mechanism by which Fsp272/2 WAT is transformed into a BAT-like tissue is not clear, we observed enhanced expression of genes regulating cAMP signaling pathway such as FoxC2 in WAT of Fsp272/2 mice. Enhanced FoxC2 expression could contribute to the increased expression of PGC1a and Ucp1. We also observed dramatically reduced expression of Rb and its homolog p107 that were known to negatively regulate PGC1a expression and BAT differentiation. Furthermore, we observed reduced expression of a co-repressor of many nuclear receptors, RIP140, in the WAT of Fsp272/2 mice. Therefore, the attainment of BAT–like property in the WAT of Fsp272/2 mice is likely to be a combinatory effect of activation of pathways promoting the conversion and downregulation of pathways that inhibit the conversion of BAT to WAT. Fsp27 appears to act somewhere upstream to control signaling networks such as cAMP pathway, Rb, p107 and RIP140 expression and insulin signaling that maintains WAT identity (Fig. 6G). The phenotype in Fsp272/2 WAT is reminiscent of that of FoxC2 overexpression transgenic mice [22] and p107 and Rb knockout mice [23], where PGC1a and Ucp1 are upregulated and that WAT of these genetically modified mice all showed brown adipocyte-like metabolic activity. However, these Fsp272/2 WAT may not fully function as a brown adipocyte as the relative levels of Ucp1 is only approximately 5–10% of that of wild type BAT (data not shown). The level of PGC1a and Cidea in Fsp272/2 WAT were also not as high as in wild type BAT. Similarly, Paradoxically, we observed larger lipid droplets, slightly reduced mitochondrial protein expression and mitochondrial activity, irregular structure of mitochondrial cristae and reduced levels of mitochondrial proteins in Fsp272/2 brown adipocytes. All these changes are somewhat opposite to the changes observed in the Fsp272/2 WAT and are apparently associated with BAT dysfunction. The precise role of Fsp27 in regulating BAT function is not clear. Discussion However, under the condition that animals are already insulin resistant (such as in the ob/ob background), increased insulin sensitivity in WAT of Fsp27 deficiency in leptin and Fsp27 double deficient mice caused drastically reduced glucose levels after glucose and insulin administration. phosphorylation of IRS1 in the WAT. We only notice a marginal difference in glucose levels after glucose and insulin administration under normal diet condition (Fig. 3A & B), which is in contrast to the drastic difference in the phosphorylation of IRS1 and AKT2 (Fig. 3C to 3F) in the WAT of Fsp272/ mice. Glucose uptake and insulin sensitivity are contributed by multiple organs such as adipose tissue, liver and muscle, depending on the status of energy homeostasis within the body. As we observed no difference in the levels of AKT phosphorylation in the skeletal muscle, liver or BAT, it is possible that under normal diet feeding condition when animals are insulin sensitive, the impact of increased insulin sensitivity in WAT on whole body glucose level control would be marginal. However, under the condition that animals are already insulin resistant (such as in the ob/ob background), increased insulin sensitivity in WAT of Fsp27 deficiency in leptin and Fsp27 double deficient mice caused drastically reduced glucose levels after glucose and insulin administration. g y p g y g Remarkably, the BAT-like phenotype in the WAT of Fsp272/2 mice can be mostly reproduced in MEFs differentiated in vitro. Our observations suggest that the acquirement of BAT-like property in WAT may require two steps, the first of which is T3-independent and cell autonomous, and the subsequent step is T3-dependent. In the first step, the changes mainly include smaller lipid droplets, increased lipolysis, and reduced TAG storage in the basic differentiation medium that contains pioglitazone and insulin. In the second step, which requires T3 in the differentiation medium, fatty acid b-oxidation was increased, accompanied by the increased protein levels of brown fat specific genes (PGC1a, Cidea, Ucp1) and mitochondrial proteins such as Cox IV. In this study, we have also revealed the importance of T3 in increasing mitochondrial activity and altering transcriptional program in the WAT of Fsp272/2 mice. Fsp27 could functionally antagonize the T3 action, which is a pleiotropic hormone regulating the expression of many genes involved in adipocyte differentiation, lipogenesis and lipolysis, and controls the induction and maintenance of the WAT identity in vivo. Discussion Based on these data, the phenotype of reduced lipid droplet size, increased lipolysis and reduced TAG storage appears to be the primary defects in the WAT of Fsp272/2 mice, while increased mitochondrial activation and up-regulation of BAT specific markers are secondary effects. The mechanism by which Fsp27 controls lipid droplet function and mitochondrial activity is not clear. As certain portion of endogenous Fsp27 proteins are localized to the lipid droplet [31,32] (Fig. 1D), it may function to control lipid droplet formation or inhibit lipolysis. Increased mitochondrial oxidative activity and Ucp1 expression in the WAT of Fsp272/2 mice may be a result of increased release of fatty acids to the cytosolic fraction. Attainment of a BAT-like cell type with increased mitochondrial activity in Fsp272/2 white adipocytes may reflect a feedback control in regulating TG storage, resulting from a defect in lipid droplet formation, lower TG storage and a lean phenotype. Although Fsp272/2 and Perilipin (Plin)2/2 mice have a similar lean phenotype, the molecular mechanism by which Fsp27 and Perilipin control energy storage and lipid droplet formation appears to be different [31,33,34]. Plin2/2 mice had a defect in lipid storage due to increase lipolysis. As a result, lipids were channelled into the circulation and other tissues. No increase in mitochondrial activity and upregulation of mitochondrial gene expression was observed in Plin2/2 mice. Fsp27, on the other hand, appears to control lipid storage and insulin sensitivity by regulating the expression levels of FoxC2, Rb/p107, RIP140, PPAR, PGC1, AKT2, GluT4 and mitochondrial proteins in addition to regulating lipid droplet size. We observed that unlike wild type white adipocytes that contain a large unilocular lipid droplet, Fsp272/2 white adipocytes have small but numerous lipid droplets in their cytosol, with nuclei localize at the center of the cell. Furthermore, Fsp272/2 white adipocytes contain significantly larger mitochondria, dramatically higher mitochondrial activity and markedly elevated levels of many mitochondrial proteins for fatty acid oxidation, electron transport chain and TCA cycles. BAT enriched proteins such as Ucp1, Cidea and Dio2 were all significantly up-regulated in Fsp272/2 white adipocytes. Expression of PPAR family proteins (PPARa and c) and PGC1a, crucial factors for controlling adipocytes differentiation, lipolysis, fatty acid transport and oxidation, glycolysis and uncoupling are also upregulated in WAT of Fsp272/2 mice. Discussion Results of relative amount of 3H2O released into the medium after four (4h) or six (6h) hours chase of pre-labelled MEFs with cold palmitate. Wildtype oxidation rate at 4h is set to 100. G. Regulatory and metabolic pathways that are altered in the WAT of Fsp272/2 mutant mice, leading to anti-obesity and insulin sensitive phenotype. doi:10.1371/journal.pone.0002890.g006 lead to the increased consumption of TAG and FFA in WAT and enhanced overall whole body energy expenditure. Furthermore, we could rule out the contribution of other tissues such as muscle and liver to the decreased overall adiposity as we observed no difference in morphology and mitochondrial activity in these tissues between wild type and Fsp272/2 mice. Besides its role in regulating adiposity, Fsp27 also plays an important role in modulating insulin sensitivity. Both Fsp272/2 and ob/ob/Fsp272/2 mice have increased glucose uptake and improved insulin sensitivity. The mechanism by which Fsp272/2 mice had increased insulin sensitivity is likely due to an increased expression of GLUT4 and AKT2 and increased tyrosine lead to the increased consumption of TAG and FFA in WAT and enhanced overall whole body energy expenditure. Furthermore, we could rule out the contribution of other tissues such as muscle and liver to the decreased overall adiposity as we observed no difference in morphology and mitochondrial activity in these tissues between wild type and Fsp272/2 mice. August 2008 \| Volume 3 \| Issue 8 \| e2890 August 2008 \| Volume 3 \| Issue 8 \| e2890 PLoS ONE \| www.plosone.org 11 Fsp27 Regulate WAT Identity mitochondrial cristea of Fsp272/2 white adipocytes were not as tightly packed and regularly arranged as that of BAT. phosphorylation of IRS1 in the WAT. We only notice a marginal difference in glucose levels after glucose and insulin administration under normal diet condition (Fig. 3A & B), which is in contrast to the drastic difference in the phosphorylation of IRS1 and AKT2 (Fig. 3C to 3F) in the WAT of Fsp272/ mice. Glucose uptake and insulin sensitivity are contributed by multiple organs such as adipose tissue, liver and muscle, depending on the status of energy homeostasis within the body. As we observed no difference in the levels of AKT phosphorylation in the skeletal muscle, liver or BAT, it is possible that under normal diet feeding condition when animals are insulin sensitive, the impact of increased insulin sensitivity in WAT on whole body glucose level control would be marginal. PLoS ONE \| www.plosone.org Discussion The reduced activity in the BAT of Fsp272/2 mice may be related to a decrease of sympathetic innervation and activity surrounding the BAT. Further analysis will be needed to address Fsp27’s molecular role in BAT. Furthermore, increased WAT activity coupled with reduced BAT activity was also observed in several genetically modified lean mouse models such as transgenic mice over-expressing A-ZIP, a nuclear form of SREBP1, Ucp1 and FoxC2 in WAT [22,35–37] as well as PLoS ONE \| www.plosone.org August 2008 \| Volume 3 \| Issue 8 \| e2890 12 Fsp27 Regulate WAT Identity Caveolin-1 knockout mice[38]. It is possible that increased WAT activity could in turn inhibit BAT activity. Caveolin-1 knockout mice[38]. It is possible that increased WAT activity could in turn inhibit BAT activity. Caveolin-1 knockout mice[38]. It is possible that increased WAT activity could in turn inhibit BAT activity. 1 mg of total RNA with oligo-(dT) 20 primers using Superscript III RT kit (Invitrogen, USA) according to the manufacturer’s protocol. Real-time PCRs were performed with ABI SYBR GREEN PCR Master Mix in the MX3000P real-time PCR system (Stratagene, USA) according to the manufacturer’s instruction. Results were normalized to b-actin level in each sample. Primer sequences for genotype and real-time PCR analyses are available upon request. 1 mg of total RNA with oligo-(dT) 20 primers using Superscript III RT kit (Invitrogen, USA) according to the manufacturer’s protocol. Real-time PCRs were performed with ABI SYBR GREEN PCR Master Mix in the MX3000P real-time PCR system (Stratagene, USA) according to the manufacturer’s instruction. Results were normalized to b-actin level in each sample. Primer sequences for genotype and real-time PCR analyses are available upon request. y y We observed that Fsp272/2 mice had slightly increased body temperatures under both feeding and fasting conditions in particular for female mice under high fat diet condition, indicating a possible increase in thermogenic activity. We found that the body temperature within the first hour of cold exposure was similar between wild type and Fsp272/2 mice. Intriguingly, the body temperature in Fsp272/2 mice became lower than the wild type mice at one hour of cold exposure and decreased even further thereafter. Therefore, Fsp272/2 mice appear to have slightly increased thermogenic activity in ambient condition, but they become hypothermic with prolonged cold exposure. Discussion The slight increase in thermogenic activity in ambient temperature in Fsp272/2 mice may be due to increased Ucp1 expression and enhanced mitochondrial activity in their WAT. However, we have evaluated Ucp1 protein levels in WAT of Fsp272/2 mice and observed that the amount of Ucp1 in Fsp272/2 WAT is approximately only 5% of that in wild type BAT (data not shown). Furthermore, mitochondrial cristea structure of Fsp272/2 white adipocytes was not arranged as regularly as wild type brown adipocytes. In addition, significantly lower amount of TG was stored in Fsp272/2 white adipocytes, resulting in limited fuel for WAT to produce heat when exposed to cold. Therefore, although the white adipocytes of Fsp272/2 mice had increased Ucp1 expression, their capacity in generating heat and to increase body temperature when animals were exposed to cold with prolonged time remains unclear. Importantly, maintenance of body temper- ature under prolonged cold treatment would require coordination between BAT (non-shivering) and muscle (shivering). As Fsp272/2 mice are leaner and had a problem storing lipid, hence hypothermia seen in Fsp272/2 mice may be due to the reduced lipid storage, which translates into lesser fuel for muscle activity. We speculate that the BAT-like property in the WAT of Fsp272/2 mice could contributes to the increased energy expenditure and lean phenotype, but could not serve to maintain body temperature when animals are exposed to prolong cold treatment. Taken together, our study has clearly demonstrated that Fsp27 acts as a novel regulator to control gene expression of crucial metabolic regulators, mitochondrial activity and white adipocyte identity. Therefore, Fsp27 could serve as a potential therapeutic target for the control of obesity and diabetes. Glucose and insulin tolerance tests (GTT and ITT) We carried out glucose and insulin tolerance tests (GTT and ITT) by intraperitoneal injection of glucose (1.0 g per kg of body weight) and recombinant human insulin (0.5 unit per kg body weight) into fasting mice (16 hour or 4 hour for GTT and ITT, respectively). We took tail blood samples before (0 min) and 15, 30, 60, 90, and 120 min after injection, to measure glucose levels. Glucose levels were measured by a blood glucose meter (Roche). Mitochondria O2 consumption from tissue Procedures for the isolation of Fsp27 genomic clones, generation of Fsp27-deficient mice, and routine maintenance of mouse strain were essentially the same as previously described [6]. Animals were fed with either normal chow diet (5053, PicoLab Rodent Diet 20) or high-fat diet (D12331, 58% of kilocalories from fat; Research Diet). Mice experiments were carried out in the In Vivo Modeling Unit, Institute of Molecular and Cell Biology and animal facility in Department of Biological Sciences and Biotechnology, Tsinghua University. Mice handling procedures were in accordance with the Responsible Care and Use of Laboratory Animals (RCULA) guideline set by the National University of Singapore and Tsinghua University. Mitochondria were isolated essentially as described [40]. Isolated mitochondria was resuspended in MRB buffer (120 mM KCl, 20 mM sucrose, 3 mM HPEPS-KOH, pH 7.2, 3 mM KH2PO4, 2 mM MgCl2 and 2 mM EGTA) containing 0.5% (w/v) BSA. 200 mg of mitochondrial protein was added to a chamber equipped with a Clark-type O2 electrode (Hansatech, USA) that contained 1 ml of MRB buffer maintained at 37uC. Mitochondrial activity measurement was initiated by the addition of 5 mM succinate and 1 mM ADP. Rate of oxygen consumption was monitored for 5-8 minutes before termination of experiment with 1mM KCN. Blood chemistry NEFA, TG, HDL, LDL and Cholesterol were quantified using NEFA-C, L-Type Triglyceride H, HDL-Cholesterol E, L-Type LDL-C and Cholesterol E kits according to manufacturer’s instructions (Wako Pure Chem, Japan). Insulin levels (CRYSTAL CHEM., USA) were measured according to manufacturer’s instruction. Adiposity index, electron microscopy, histological analysis, whole body O2 consumption, food intake measurement and core body temperature Procedures for adiposity index analysis, electron microscopy, H&E staining and whole body O2 consumption were essentially the same as described by Li et al [26]. For histological analysis, WAT was fixed, dehydrated, embedded and transversely sectioned into 5 mm piece for further staining. For whole body O2 consumption assessment, mice were adapted for 8 hours in the metabolic chamber before reading were taken periodically at 15 min intervals for the next 10 h. Mice were given free access to food and water during measurement. For food intake measure- ment, two or three mice were house in a cage. The amount of normal diet consumed was carefully monitored at 10:00 a.m. local time very morning for a period of one month. Measurement of core body temperature of the mice is essentially the same as describe by Zhuo et al [6] Cytochrome oxidase activity Genotyping, RNA extraction, semi-quantitative real-time PCR PLoS ONE \| www.plosone.org Lipolysis assay in WAT and MEF cells WAT isolated from three month old mice were cut into small pieces and 8 days post differentiated MEF were used for lipolysis assay [45]. Tissue pieces or cells were incubated in DMEM (without phenol red) containing 1% fatty acid free BSA with or without 1 mM isoproterenol as indicated. 100 ml of medium was withdrawn at the indicated time points and used for the assay. Glycerol level was determined using Free Glycerol determination kit according to the manufacturer’s instruction (Sigma, USA). Fatty acid b-oxidation Total lipid was isolated from mouse tissue or cells as previously described [41]. Dried lipids were reconstituted in chloroform/ methanol 2:1 and loaded onto a TLC plate (Sigma, USA). Lipids were resolved in hexane/diethyl ether/acetic acid 70:30:1 v/v. TLC plates were sprayed with 10% CuSO4 in 10% phosphoric acid and developed by drying in a 150uC oven. The procedure for measurement of fatty acid oxidation in MEF was essentially the same as described [44]. MEF cells were seeded in 12-well plates. After 8 days of differentiation, the cells were washed three times with HBSS (Hanks’ balanced salt solution with calcium and magnesium, 10 mM HEPES) buffer and prelabeled with [9, 10(n)-3H] palmitatic acid (1.0 mCi/well) with 22 mM unlabeled palmitic acid and 0.5 mg/ml fatty acid-free BSA in HBSS. After incubation at 37uC for 2 h, cells were washed with HBSS three times and incubated in 0.5 ml HBSS buffer. At each time point, the reaction medium was transferred to glass tube and the cells were then washed 3 times with 0.5 ml HBSS which was collected in the same tube. The resultant medium was extracted with 8 ml Chloroform/Methanol (2:1), and the aqueous phase was collected and used for radioactivity measurement in a liquid scintillation counter. Immunofluoresence assay All statistical analyses were calculated using Graphpad Prism v4.0. Data consolidated were expressed as mean6sem and p value was calculated using non-parametric student t-test. Immunofluorescence study was carried out on cells grown on cover-slips. Cells were fixed in 3% paraformaldehyde/PBS solution for 30 min at room temperature, followed by permeabi- lization with 0.1% saponin/PBS for 15 mins. The cells were then incubated with purified Fsp27 antibody and perilipin antibody (Research Diag. Inc.). In MEF cells, intracellular lipids were visualize with 20 mg/ml Bodipy 493/503. Fluoresencing cells were visualize with Zeiss LSM510Meta cofocal and Zeiss Axiovert 200M microscope. Supporting Information Figure S1 Generation of Fsp27 knockout mice. A. Fsp27 partial genomic structure, gene targeting construct, and expected homologous recombinant allele. Transcriptional disruption is achieved with a copy of the Neo gene being inserted in place of exon 2 and part of exon 3 in the opposite direction to transcriptional orientation. B. Total triacylglycerol content (TAG) in skeletal muscle (SM) and liver of 3 months old wildtype (+/+) and Fsp272/2 (2/2) mice (n = 3). C. Lipolysis rate of WAT from wildtype (+/+) and Fsp272/2 (2/2) mice treated with (Iso) or without (Basal) 1uM isoproterenol (n = 4) for the indicated time. Genotyping, RNA extraction, semi-quantitative real-time PCR Cytochrome oxidase activity (Cox) activity was measured as described [21] with the following modifications. Reduced cytochrome c was prepared by incubating 5 mM DTT with 21.8 mM cytochrome C on ice for 15 min. To test Cox activity, tissue was homogenized in Buffer C (30 mM potassium phosphate, Genomic DNA was isolated from mouse tails as previously described [39]. Total RNA was isolated using the TRIzol reagent (Invitrogen, USA). The first-strand cDNAs were synthesized from PLoS ONE \| www.plosone.org PLoS ONE \| www.plosone.org August 2008 \| Volume 3 \| Issue 8 \| e2890 August 2008 \| Volume 3 \| Issue 8 \| e2890 13 Fsp27 Regulate WAT Identity 8 mg/ml Biotin, 1 mM DEX , 0.5 mM IBMX, 5 mg/ml insulin and 1.0 mM pioglitazone (TAYANG Pharmaceutical Industry Co. LTD, China), with or without 0.5 mM 3, 5, 39-tri-iodothyronine (T3). Two days later, the medium was changed to DMEM /FBS, 8 mg/ml D-Pantothenic acid, 8 mg/ml Biotin, 5 mg/ml insulin, 1 mM pioglitazone, with or without 0.5 mM T3. Cells were collected after 8 days of differentiation and used for further experiments. 8 mg/ml Biotin, 1 mM DEX , 0.5 mM IBMX, 5 mg/ml insulin and 1.0 mM pioglitazone (TAYANG Pharmaceutical Industry Co. LTD, China), with or without 0.5 mM 3, 5, 39-tri-iodothyronine (T3). Two days later, the medium was changed to DMEM /FBS, 8 mg/ml D-Pantothenic acid, 8 mg/ml Biotin, 5 mg/ml insulin, 1 mM pioglitazone, with or without 0.5 mM T3. Cells were collected after 8 days of differentiation and used for further experiments. pH 7.4, with a protease inhibitor cocktail [Roche, USA]) and centrifuged at 600g for 2 minutes to remove tissue clumps and fat cake. 50 mg of total protein from the infranatant was added to Buffer C containing 0.01 mmol of reduced cytochrome C and measured immediately with a spectrophotometer. Cytochrome oxidase activity was monitored as the change of OD 550/565 ratio per unit time. Western blot analysis Tissue or cultured cells for western blot analysis of total lysate were lysed in RIPA buffer (20 mM HEPES-KOH pH 7.5, 150 mM NaCl, 1 mM EDTA, 10% Glycerol, 0.5% sodium deoxycholate, 1% NP40, 0.1% SDS and protease inhibitor) and analysed as previously described [6]. Western blot analysis for insulin stimulated phosphorylation of IRS1, AKT and GLUT4 was performed as described [26]. Fsp27 and Cidea was detected using polyclonal antibody raised against mouse Fsp27 and mouse Cidea by injecting His-Tagged truncated Fsp27 (aa 1–190) and Cidea (aa 1–195) into rabbit respectively as previously described [42]. Antibodies against anti-tubulin (Sigma), anti-rat FABP (Alpha Diagnostic.), anti-cytochrome c (Pharmingen), anti-CoxIV (Molecular Probes), anti-Cpt1 (Alpha Diag.), anti-Cpt2 (Alpha Diag.), anti-UCP3 (Research Diag. Inc.), Hsp60 (Chemicon), anti- Pgc1a (Chemicon), Ucp1 (Research Diag. 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Puigserver P, Wu Z, Park CW, Graves R, Wright M, et al. (1998) A cold- inducible coactivator of nuclear receptors linked to adaptive thermogenesis. Cell 92: 829–839. 24. Hansen JB, Kristiansen K (2006) Regulatory circuits controlling white versus brown adipocyte differentiation. Biochem J 398: 153–168. 8. Wu Z, Puigserver P, Andersson U, Zhang C, Adelmant G, et al. (1999) Mechanisms controlling mitochondrial biogenesis and respiration through the thermogenic coactivator PGC-1. Cell 98: 115–124. 25. Acknowledgments We thank members in our laboratory in Tsinghua University for technical assistance and helpful discussion and Dr. B.L. Tang (National University of Singapore, Singapore) and Dr. SC Lin (Xiamen University, China) for their critical comments on the manuscript. PL is a Cheung Kong Scholar of the Ministry of Education of China. This work was also supported by Program for Changjiang Scholars and Innovative Research Team in University from Ministry of Education in China. Found at: doi:10.1371/journal.pone.0002890.s005 (1.03 MB TIF) Found at: doi:10.1371/journal.pone.0002890.s005 (1.03 MB TIF) Found at: doi:10.1371/journal.pone.0002890.s005 (1.03 MB TIF) Figure S6 Densitometric reading of relative protein level in western blot analysis performed for the indicated protein of wildtype (Fsp27+/+) or Fsp27 null (Fsp272/2) mice in Day 8 post-differentiated MEF cells with (+T3) or without (2T3) triiodothyronine (n = 3). Figure S6 Densitometric reading of relative protein level in western blot analysis performed for the indicated protein of wildtype (Fsp27+/+) or Fsp27 null (Fsp272/2) mice in Day 8 post-differentiated MEF cells with (+T3) or without (2T3) triiodothyronine (n = 3). Found at: doi:10.1371/journal.pone.0002890.s003 (0.32 MB TIF) Found at: doi:10.1371/journal.pone.0002890.s006 (0.44 MB TIF) Found at: doi:10.1371/journal.pone.0002890.s006 (0.44 MB TIF) Figure S4 No difference of levels of AKT and phosphor-AKT in Fsp272/2and ob/ob/Fsp272/2 mice. 3 months old mice that were fasted for 4 hours were intraperitoneally injected with 40mg/ kg body weight of insulin. AKT proteins were immunoprecipitated with antibody against AKT and subsequently immunoblotted with antibodies again AKT or phosphor-AKT (pAKT). A, C& E. Western blot analysis for levels of total AKT and pAKT stimulated with and without insulin in BAT, skeleton muscle (SM) and liver of wild type (WT) and Fsp27 mutant (Fsp272/2) mice. B, D& F. Western blot analysis for levels of total AKT and insulin stimulated pAKT in BAT, skeletal muscle (SM) and liver of ob/ob and ob/ ob/Fsp272/2 mice. Actin was used as the loading control. Found at: doi:10.1371/journal.pone.0002890.s004 (1.80 MB TIF) Author Contributions Conceived and designed the experiments: PL. Performed the experiments: SYT JG GD JZL SY JY. Analyzed the data: SYT JG GD JZL HY ZW PL. Contributed reagents/materials/analysis tools: SY HY YZ BX QL HY ZW. Wrote the paper: PL. Figure S5 Western Blot analysis of total BAT tissue lysate from wildtype (Fsp27+/+) or Fsp27 null (Fsp272/2) mice. b-tubulin was used as the loading control. Each panel is a representative of 4 individual experiments. Tubb represents tubulin. Cell culture 3T3-L1 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, USA) containing 10% fetal bovine serum (FBS, Invitrogen, USA). Differentiation of 3T3-L1 cells were initiated at 2 days post-confluent cells by the addition of 5 mg/ml of insulin, 1 mM dexamethasone (DEX), and 0.5 mM isobutylmethylxanthine (IBMX) for 2 days and subsequently replaced by DMEM/FBS supplemented only with 5 mg/ml insulin for a further 2 days before replacing it with normal culture medium. Mouse embryonic fibroblasts (MEF) were isolated from 13.5 day wild-type and Fsp27 mutant mouse embryos as described [43]. Passage 2 of MEF was used for differentiation. For the induction of MEF to differentiate into adipocytes, MEF cells were grown for 2 days post confluence, and then differentiated in DMEM/FBS supplemented with 8 mg/ml D-Pantothenic acid, Found at: doi:10.1371/journal.pone.0002890.s001 (0.77 MB TIF) Found at: doi:10.1371/journal.pone.0002890.s001 (0.77 MB TIF) Found at: doi:10.1371/journal.pone.0002890.s001 (0.77 MB TIF) Figure S2 Body temperature of animals under ambient conditions. Retal temperature was taken from 6 months old wildtype (+/+) and Fsp272/2 (2/2) mice fed ad libitum with normal diet (ND) or high fat diet (HFD) or fasted for 18 hours (ND: Male: +/+: n = 16, 2/2: n = 17; Female: +/+: n = 16, 2/2: n = 16. HFD: Male: +/+: n = 18, 2/2: n = 19; Female: +/+: n = 15, 2/2: n = 22.). Found at: doi:10.1371/journal.pone.0002890.s002 (0.59 MB TIF) PLoS ONE \| www.plosone.org August 2008 \| Volume 3 \| Issue 8 \| e2890 14 Fsp27 Regulate WAT Identity Figure S3 Densitometric reading of relative protein level in western blot analysis performed for, A. IRS1 or phosphor-IRS1 (pIRS1), B. AKT2 or phosphor-AKT2 (pAKT2) and C. GLUT4 in WAT of 3 months old wildtype (WT), Fsp272/2, leptin deficient (ob/ob) and leptin/Fsp27 double deficient (ob/ob/ Fsp272/2) mice (n = 3). F d t d i 10 1371/j l 0002890 003 (0 32 MB TIF) References (2007) Fat-specific protein 27, a novel lipid droplet protein that enhances triglyceride storage. J Biol Chem 282: 34213–34218. 16. Bouillaud F, Ricquier D, Mory G, Thibault J (1984) Increased level of mRNA for the uncoupling protein in brown adipose tissue of rats during thermogenesis induced by cold exposure or norepinephrine infusion. J Biol Chem 259: 11583–11586. 32. Keller P, Petrie JT, De Rose P, Gerin I, Wright WS, et al. (2008) Fat-specific protein 27 regulates storage of triacylglycerol. J Biol Chem 283: 14355–14365. 17. Yoshida T, Umekawa T, Kumamoto K, Sakane N, Kogure A, et al. (1998) beta 3-Adrenergic agonist induces a functionally active uncoupling protein in fat and slow-twitch muscle fibers. Am J Physiol 274: E469–475. 33. Martinez-Botas J, Anderson JB, Tessier D, Lapillonne A, Chang BH, et al. (2000) Absence of perilipin results in leanness and reverses obesity in Lepr(db/ db) mice. Nat Genet 26: 474–479. 18. Jimenez M, Barbatelli G, Allevi R, Cinti S, Seydoux J, et al. (2003) Beta 3- adrenoceptor knockout in C57BL/6J mice depresses the occurrence of brown adipocytes in white fat. Eur J Biochem 270: 699–705. 34. Castro-Chavez F, Yechoor VK, Saha PK, Martinez-Botas J, Wooten EC, et al. (2003) Coordinated upregulation of oxidative pathways and downregulation of PLoS ONE \| www.plosone.org 15 August 2008 \| Volume 3 \| Issue 8 \| e2890 August 2008 \| Volume 3 \| Issue 8 \| e2890 Fsp27 Regulate WAT Identity Fsp27 Regulate WAT Identity Fsp27 Regulate WAT Identity 40. Hofmann WE, Liu X, Bearden CM, Harper ME, Kozak LP (2001) Effects of genetic background on thermoregulation and fatty acid-induced uncoupling of mitochondria in UCP1-deficient mice. J Biol Chem 276: 12460–12465. y g p p 35. Shimomura I, Hammer RE, Richardson JA, Ikemoto S, Bashmakov Y, et al. (1998) Insulin resistance and diabetes mellitus in transgenic mice expressing nuclear SREBP-1c in adipose tissue: model for congenital generalized lipodystrophy. Genes Dev 12: 3182–3194. g g g y mitochondria in UCP1-deficient mice. J Biol Chem 276: 12460–12 41. Folch J, Lees M, Sloane Stanley GH (1957) A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem 226: 497–509. 42. Brock DA, Hatton RD, Giurgiutiu DV, Scott B, Jang W, et al. (2003) CF45-1, a secreted protein which participates in Dictyostelium group size regulation. Eukaryot Cell 2: 788–797. p y p y 36. 42. Brock DA, Hatton RD, Giurgiutiu DV, Scott B, Jang W, et al. (2003) CF45-1, a secreted protein which participates in Dictyostelium group size regulation. Eukaryot Cell 2: 788–797. References Tiraby C, Tavernier G, Lefort C, Larrouy D, Bouillaud F, et al. (2003) Acquirement of brown fat cell features by human white adipocytes. J Biol Chem 278: 33370–33376. 43. Phan J, Peterfy M, Reue K (2004) Lipin expression preceding peroxisome proliferator-activated receptor-gamma is critical for adipogenesis in vivo and in vitro. J Biol Chem 279: 29558–29564. 37. Jaideep Moitra MMM, Michelle Olive, Dmitry Krylov, Oksana Gavrilova, Bernice Marcus-Samuels LF, et al. (1998) Life without white fat: a transgenic mouse. GENES & DEVELOPMENT 12: 3168–3181. 44. Moon A, Rhead WJ (1987) Complementation analysis of fatty acid oxidation disorders. J Clin Invest 79: 59–64. 38. Razani B, Combs TP, Wang XB, Frank PG, Park DS, et al. (2002) Caveolin-1- deficient mice are lean, resistant to diet-induced obesity, and show hypertri- glyceridemia with adipocyte abnormalities. J Biol Chem 277: 8635–8647. 45. Kershaw EE, Hamm JK, Verhagen LA, Peroni O, Katic M, et al. (2006) Adipose triglyceride lipase: function, regulation by insulin, and comparison with adiponutrin. Diabetes 55: 148–157. g y p y J 39. Sambrook JFE, Maniatis T (1989) Molecular Cloning: A Laboratory Manual. NY: Cold Spring Harbor Laboratory Press. 39. Sambrook JFE, Maniatis T (1989) Molecular NY: Cold Spring Harbor Laboratory Press. NY: Cold Spring Harbor Laboratory Press. 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https://openalex.org/W3216690489	https://pureadmin.qub.ac.uk/ws/files/268378731/bmjopen-2020-047018.pdf	English	null	Intervention targeted at physicians’ treatment of musculoskeletal disorders and sickness certification: an interrupted time series analysis	BMJ open	2,021	cc-by	9,066	Published in: BMJ Open Published in: BMJ Open Document Version: Publisher's PDF, also known as Version of record Document Version: Publisher's PDF, also known as Version of record Queen's University Belfast - Research Portal: Link to publication record in Queen's University Belfast Research Portal Queen's University Belfast - Research Portal: Link to publication record in Queen's University Queen's University Belfast - Research Portal: Link to publication record in Queen's University Belfast Research Portal Publisher rights © 2021 The Authors. This is an open access article published under a Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited. Publisher rights © 2021 The Authors. This is an open access article published under a Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited. © 2021 The Authors. This is an open access article published under a Creative Commons Attribution License (https://creativecommons.org/ which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited Intervention targeted at physicians’ treatment of musculoskeletal disorders and sickness certification: an interrupted time series analysis Kausto, J., Rosenström, T. H., Ervasti, J., Pietiläinen, O., Kaila-Kangas, L., Rahkonen, O., Harkko, J., Väänänen, A., Kouvonen, A., & Lallukka, T. (2021). Intervention targeted at physicians’ treatment of musculoskeletal disorders and sickness certification: an interrupted time series analysis. BMJ Open, 11(12), e047018. https://doi.org/10.1136/bmjopen-2020-047018 P bli h d i Intervention targeted at physicians’ treatment of musculoskeletal disorders and sickness certification: an interrupted time series analysis Kausto, J., Rosenström, T. H., Ervasti, J., Pietiläinen, O., Kaila-Kangas, L., Rahkonen, O., Harkko, J., Väänänen, A., Kouvonen, A., & Lallukka, T. (2021). Intervention targeted at physicians’ treatment of musculoskeletal disorders and sickness certification: an interrupted time series analysis. BMJ Open, 11(12), e047018. https://doi.org/10.1136/bmjopen-2020-047018 ABSTRACT To cite: Kausto J, Rosenström TH, Ervasti J, et al. Intervention targeted at physicians’ treatment of musculoskeletal disorders and sickness certification: an interrupted time series analysis. BMJ Open 2021;11:e047018. doi:10.1136/ bmjopen-2020-047018 Strengths and limitations of this study Objective An intervention was carried out at the occupational healthcare services (OHS) of the City of Helsinki beginning in 2016. We investigated the association between the intervention and employee sick leaves using interrupted time series analysis. Design Register-based cohort study with a quasi- experimental study design. Objective An intervention was carried out at the occupational healthcare services (OHS) of the City of Helsinki beginning in 2016. We investigated the association between the intervention and employee sick leaves using interrupted time series analysis. ► ►This study presents novel information on the as- sociation between introducing recommendations for management of pain and prescribing sick leave for musculoskeletal disorders and employee sick leaves. Design Register-based cohort study with a quasi- experimental study design. ► ►Comprehensive individual-level data from employer registers were linked to individual-level data from national registers. Setting Employees of the City of Helsinki. g p y y Participants We analysed individual-level register-based data on all employees who were employed by the city for any length of time between 2013 and 2018 (a total 86 970 employees and 3 014 075 sick leave days). Sick leave days and periods that were OHS-based constituted the intervention time series and the rest of the sick leave days and periods contributed to the comparison time series. Intervention Recommendations provided to physicians on managing pain and prescribing sick leave for low back, shoulder and elbow pain. ► ►Prepublication history and additional supplemental material for this paper are available online. To view these files, please visit the journal online (http://dx.doi.org/10.1136/ bmjopen-2020-047018). ► ►Prepublication history and additional supplemental material for this paper are available online. To view these files, please visit the journal online (http://dx.doi.org/10.1136/ bmjopen-2020-047018). ► ►We used interrupted time series analysis with a comparison time series, which is a strong quasi- experimental design recommended for examining population-level interventions. ► ►Intervention status was not obtainable for the entire follow-up time and information on diagnoses was available only for sick leave periods that lasted over 11 calendar days. ► ►As all employees could be prescribed sick leave both at the occupational healthcare service and else- where, the validity of the comparison group could be debated; however, only the professionals in the intervention group were exposed to the intervention and the time series for the intervention and compar- ison groups were analysed separately. Outcome measures Number of sick leave days per month and sick leave periods per year. 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We would love to hear how access to this research benefits you. – Share your feedback with us: http://go.qub.ac.uk/oa-feedback Open Access This research has been made openly available by Queen's academics and its Open Research team. We would love to hear how access to this research benefits you. – Share your feedback with us: http://go.qub.ac.uk/oa-feedback Download date:24. Oct. 2024 Original research Open access Intervention targeted at physicians’ treatment of musculoskeletal disorders and sickness certification: an interrupted time series analysis Strengths and limitations of this study Results For all sick leave days prescribed at OHS, there was no immediate change in sick leave days, whereas a gradual change showing decreasing number of OHS-based sick leave days was detected. On average, the intervention was estimated to have saved 2.5 sick leave days per year per employee. For other sick leave days, there was an immediate increase in the level of sick leave days after the intervention and a subsequent gradual trend showing decreasing number of sick leave days. 1Finnish Institute of Occupational Health, Helsinki, Finland 2Department of Psychology and Logopedics, University of Helsinki Faculty of Medicine, Helsinki, Finland 3Department of Public Health, University of Helsinki, Helsinki, Finland 4Faculty of Social Sciences, University of Helsinki, Helsinki, Finland 5Administrative Data Research Centre, Queen's University Belfast, Belfast, UK Correspondence to Dr Johanna Kausto; johanna.kausto@ttl.fi © Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY. Published by BMJ. for musculoskeletal pain symptoms, such as back, shoulder or elbow pain. However, scien- tific evidence does not support the benefici- ality of long sick leaves. For example, it has been shown that for low back pain, from a point of view of recovery, staying active is better than bed-rest.2 3 for musculoskeletal pain symptoms, such as back, shoulder or elbow pain. However, scien- tific evidence does not support the benefici- ality of long sick leaves. For example, it has been shown that for low back pain, from a point of view of recovery, staying active is better than bed-rest.2 3 Conclusions The intervention may have reduced employee sick leaves and therefore it is possible that it had led to direct cost savings. However, further evidence for causal inferences is needed. Advice from primary care doctors can have a major impact on whether an individual takes sick leave and for how long.4 A follow-up study found that visiting a medical specialist for a MSD was associated with a delayed full return to work irrespective of the severity of ailment.5 Furthermore, earlier studies have found that sick-listing (sick leave prescribing) practices vary a lot across physicians.6 7 A To cite: Kausto J, Rosenström TH, Ervasti J, et al. Intervention targeted at physicians’ treatment of musculoskeletal disorders and sickness certification: an interrupted time series analysis. BMJ Open 2021;11:e047018. doi:10.1136/ bmjopen-2020-047018 Intervention targeted at physicians’ treatment of musculoskeletal disorders and sickness certification: an interrupted time series analysis Johanna Kausto ‍ ‍ ,1 Tom Henrik Rosenström,2 Jenni Ervasti ‍ ‍ ,1 Olli Pietiläinen,3 Leena Kaila-Kangas,1 Ossi Rahkonen ‍ ‍ ,3 Jaakko Harkko ‍ ‍ ,4 Ari Väänänen,1 Anne Kouvonen ‍ ‍ ,4,5 Tea Lallukka ‍ ‍ 3 To cite: Kausto J, Rosenström TH, Ervasti J, et al. Intervention targeted at physicians’ treatment of musculoskeletal disorders and sickness certification: an interrupted time series analysis. BMJ Open 2021;11:e047018. doi:10.1136/ bmjopen-2020-047018 ► ►Prepublication history and additional supplemental material for this paper are available online. To view these files, please visit the journal online (http://dx.doi.org/10.1136/ bmjopen-2020-047018). Received 17 November 2020 Accepted 09 November 2021 1Finnish Institute of Occupational Health, Helsinki, Finland 2Department of Psychology and Logopedics, University of Helsinki Faculty of Medicine, Helsinki, Finland 3Department of Public Health, University of Helsinki, Helsinki, Finland 4Faculty of Social Sciences, University of Helsinki, Helsinki, Finland 5Administrative Data Research Centre, Queen's University Belfast, Belfast, UK Correspondence to Dr Johanna Kausto; johanna.kausto@ttl.fi © Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY. Published by BMJ. Open access Finnish study showed that differences in the length of prescribed sick leaves can be up to eightfold.8 In line with this, a survey found that Finnish physicians needed more information, guidance and training on sick-listing.9 The study follows the Helsinki Health Study protocol in line with the University of Helsinki’s guidelines and the European Union (EU) and Finnish data legislation. The City of Helsinki and register holders have given permis- sion for data linkage. Against this background, an intervention was carried out at Occupational Health Helsinki, which provides occupational healthcare services (OHS) for the City of Helsinki employees. The intervention was launched in 2016. Introducing and increasing the use of shared guide- lines on the management of pain and work disability and prescribing sick leave for low back, shoulder and elbow pain was the central element of the intervention. To inves- tigate the association of this intervention with employee sick leaves, we carried out prospective controlled time series and binomial regression analyses using register- based data. y Occupational Healthcare in Finland Occupational Healthcare in Finland In the Finnish OHS system, all employees are entitled to statutory preventive OHS provided by the employer. Most employers offer state-subsidised free primary care services for their employees on top of the statutory preventive OHS. OHS providers in Finland provide primary care services to employees in a way that is comparable to primary care in international comparisons in substance, but diﬀers in that the service is targeted only to the working population.13 The statutory tasks of OHS include assessment of the health and safety aspects of work, assess- ment and monitoring of employees’ health and work ability, making initiatives for improvement and moni- toring their implementation, advice and guidance, moni- toring employees with disabilities and referring them to rehabilitation, cooperation with representatives of other healthcare services and social insurance, participation in organising first aid at the workplace, participation in activities that maintain work ability, and monitoring the quality and impact of occupational healthcare activities.14 METHODS Study design and population ll d Data were collected as part of the Helsinki Health Study.10 The study population includes a sample of employees of the City of Helsinki. Women represent over 70% of the employees who work in general local administration, social and healthcare, education and culture, public transport, and technical services. All employees share the same personnel administration, policies and OHS.10 Employees can use the services (including primary care services for other than work-related conditions) of their OHS provider, Occupational Health Helsinki, or they can seek treatment from public or private healthcare. The employees of the City of Helsinki can use self-certification for sick leaves lasting up to 5 days. In the case of self- certification, a medical certificate is not needed, but the employee notifies the supervisor when taken ill. In 2016, self-certification was mostly used among young employees (under 30 years of age).11 Patient and public involvement As this is a register study, no patients were directly involved. INTRODUCTION Musculoskeletal disorders (MSD) are highly prevalent among the working-age population and result in extensive disability costs both for the individual and the society. In Finland in 2020, 26% of the days of sick leave periods lasting at least 11 calendar days were due to MSD.1 Thus, sick leave is frequently granted Musculoskeletal disorders (MSD) are highly prevalent among the working-age population and result in extensive disability costs both for the individual and the society. In Finland in 2020, 26% of the days of sick leave periods lasting at least 11 calendar days were due to MSD.1 Thus, sick leave is frequently granted g A Kausto J, et al. BMJ Open 2021;11:e047018. doi:10.1136/bmjopen-2020-047018 1 Open access Intervention doi:10.1136/bmjopen-2020-047018 Open access in the form of three guideline papers (one for each diagnostic category). Training regarding the implemen- tation of the guidelines was organised at OHS for physi- cians and all other relevant healthcare professionals, such as physiotherapists and nurses. Short educational sessions were offered, and an e-learning course and some coaching sessions were led by a pain specialist. An inten- sive follow-up of the sick leave trends was started at OHS. For instance, recent trends on sick leave were reported in the monthly meetings for the physicians. All new relevant OHS employees were briefed about the guidelines. employees and manual workers), job contract (perma- nent, temporary) valid on 1 January 2016, chronic somatic illnesses (derived from the Register of Special Reimburse- ment for Medication Purchases and included diabetes, heart disease, rheumatoid arthritis, chronic asthma, stage 2 hypertension, Parkinson’s disease, epilepsy, uraemia, bowel disease, multiple sclerosis disease and diseases of the pancreas, and categorised as 0=no or 1=yes) valid on 1 January 2016, and purchases of prescribed psychotropic medication (Anatomical Therapeutic Chemical classifica- tion N06 or N05, categorised as 0=no or 1=yes) between 1 November 2015 and 1 November 2016. p y g As an example, in the case of low back pain, the recom- mendation (online supplemental appendix 1) empha- sised that the condition is very common and that nearly all people experience a disabling low back pain (ie, pain, ache and stiffness in the lower back area) at some point in their lives. The condition is usually benign in nature and heals spontaneously in a few days, and 90% of patients will fully recover in 4–6 weeks. Still, possible serious causes of back pain should be ruled out. In sick-listing, the following points should be considered: (1) in many cases sick leave is not needed; (2) there is no indication that physically strenuous work would slow down recovery; (3) if sick-listing is necessary, a few days up to a week should be sufficient in light duties; (4) if work includes heavy lifting, bending or rotation, 2 weeks of full-time sick leave may be needed; (5) factors that provoke pain should be temporarily modified; (6) and factors that suggest an increased risk of prolonged back pain and work disability include sleeping problems, depression and anxiety, fear avoidance beliefs, multisite pain, and problems at the workplace and dissatisfaction with work. Intervention Occupational Health Helsinki provides occupational healthcare to almost 40 000 employees working annu- ally in the city departments. The employees represent hundreds of different occupations, and a rather large supply of healthcare services supporting employees’ work ability and the functioning of work units are offered. Occupational Health Helsinki staff consists of 150 health- care professionals. y g For the purposes of this study, individual-level register- based data on all employees who were employed by the city for any length of time between 2013 and 2018 (N=86 970) were analysed. Background information on partici- pants was drawn from employer registers. Information on all sick leave periods (start and end dates and diagnostic codes) International Classification of Diseases, 10th revi- sion (ICD-10) during this time was derived from employer registers and the national sickness insurance register of the Social Insurance Institution of Finland (SII). ICD-10 codes for sick leave periods were available from the SII for sick leave periods lasting more than 11 calendar days. Data on chronic somatic and psychiatric illnesses and purchases of prescribed psychotropic medication were obtained from the Register of Special Reimbursement for Medication Purchases (SII). Data from these three registers were linked based on unique individual number of the participants. These numbers are assigned to each citizen of Finland at birth and for migrants when they get a residence permit, and these are common to all admin- istrative registers, enabling extensive record linkages.12 p The aims of the intervention were to increase the quality and effects of occupational healthcare, to increase the use of mutually created and shared guidelines based on available scientific evidence in assessing work disability, to provide a practical instrument for physicians in managing pain and prescribing sick leave in the form of recom- mendations, to help physicians to take into account the complexity of the association between pain and work disability, and to provide up-to-date information to the patient about their pain condition, its treatment and how it may affect work and work ability. The goal was to reduce employee sick leaves and improve work ability. Recommendations on work disability management and prescribing sick leave for low back pain, shoulder pain and elbow pain were launched first in January 2016 and they were the central and most noticeable component of the intervention. Recommendations were launched 2 Kausto J, et al. BMJ Open 2021;11:e047018. Intervention The recommen- dations included a checklist for the physicians to be used at the appointment. Statistical analyses We used an interrupted time series (ITS) design with a comparison time series.15–17 The observational unit in the analyses was a sick leave day or period. The ITS analysis uses time series (a continuous sequence of observations at the population level taken repeatedly at equal intervals over time) for a speciﬁc outcome to establish an underlying trend which is ‘interrupted’ by an intervention at a speciﬁc point of time. A strength of ITS analyses is that they are generally not affected by typical confounding factors (such as age distribution or socioeconomic status) as they model a time trend in a context where the population compo- sition (in terms of the confounding factors) remains rather constant before versus after the intervention. Measured time-varying confounders can be controlled for in the regression models. Unmeasured or unknown time-varying confounders can be controlled by adding a comparison group (not exposed to the interven- tion).15 It has been recommended that the covariate balance between the intervention and comparison time series be explored, although it is not a prerequi- site for the analysis.16 Outcome We calculated the number of sick leave days per month and sick leave periods per year. Intervention status y Our integer-valued (count or proportion) outcome variable was days of sick leave per month (range 0–30, or from 0 to days exposed within the 30-day period, if not with the employer for the full 30-day period), recorded separately for each month. Although each employee was followed up for multiple months, different employees may have different time average rate of sick leave days, introducing statistical depen- dencies to the observations. As our interests pertained to a population-level association, within-employee clustering in the outcome was considered a nuisance variable and its associations were modelled using generalised estimating equations.18 Given the data characteristics above, the outcome was modelled using generalised (binomial) linear regression model19 and the models were estimated with generalised esti- mating equations. Although our data were in the form of monthly counts, an exponentiated binomial regres- sion coefficient can be interpreted as an OR for a sick leave day (vs no sick leave) for a randomly chosen day, adjusting for the other covariates in the model. As a sensitivity analysis, we also tested quasi-binomial Sick leave periods that were OHS-based (if a sick leave was preceded by a likely related appointment at OHS within 11 days, it was approximated that sick-listing had taken place at OHS) constituted the intervention time series, and the rest of the sick leave periods (no appointment at OHS, but appointment and sick-listing had taken place elsewhere, or the employee had used self-certification, in which case there had been no medical appointment at all) contributed to the comparison time series. The avail- able registers contained information on appointments at OHS until 20 April 2017. Descriptive results d l d Our data included information on sick leave of a total of 86 970 employees covering more than 73 months. Due to workforce turnover, the monthly sample size of employees averaged at 41 289, with a minor variance across months (minimum 37 567, maximum 45 520, SD 2054). Overall, 70% of the employees were on sick leave at least once during the follow-up and 33% of the employees were prescribed sick leave at OHS at least once between 1 January 2013 and 21 April 2017. Of the employees who were prescribed sick leave at OHS, 95% were also prescribed sick leave elsewhere (or they used self-certification). Of those who were prescribed sick leave elsewhere (or used self-certification) at least once, 46% were also prescribed sick leave at OHS. Of all employees, 30% were not on sick leave at all. On average, employees were prescribed 0.36 days of sick leave per month at OHS and 0.82 sick leave days per month were prescribed elsewhere or were self-certified. The respective mean numbers of sick leave periods per employee per year were 0.49 and 1.96. where ‍Y‍ is the outcome, ‍g‍ is the logit link function, ‍X‍ is 1 for all months after the intervention launch date (1 January 2016) and 0 before it, and ‍ ( m −m0 ) ‍ is time (months, centred around the time of interven- tion,20 ‍m0‍). Here, ‍βslope‍ is the linear trend estimate before the intervention, whereas ‍βslope + βgradual‍ is the trend after the intervention took place. The intercept before the intervention is ‍β0‍ and after the interven- tion ‍β0 + βimmediate‍. Thus, ‍βimmediate‍ is the immediate trend after the intervention and ‍βgradual‍ is the gradual trend after the intervention that accumulates at the rate ‍βgradual‍ per month after the intervention (for the remaining observation period). Besides this usual ITS setting, we took into account the annual fluctuation of sick leave by modelling an annual trend estimating the coefficients for two additional covariates with values ‍sin ( m2π/12 ) ‍ and ‍cos ( m2π/12 ) ‍, where the factor ‍2π/12‍ scales monthly data points ‍ m‍ to the annual cycles.21 The descriptive statistics for all employees, employees with sick leaves prescribed at OHS and employees with other sick leaves (prescribed elsewhere or self- certificated) are shown in tables 1 and 2. Descriptive results d l d Employees who were prescribed sick leave at OHS at least once by 21 April 2017 were more often permanently employed, older, female, and intermediate-level or lower-level non-manual workers (table 1). Because our outcome data timepoints (months) could include sick leave days from prescribed at OHS and other sources, defining a covariate for interven- tion group membership would have required us to prioritise either OHS or other sources. It is typical in controlled interrupted time series (CITS) design to include a binary covariate that gets a value of 1 when a unit belongs to the intervention group and a value of 0 when in the control group. In this study, partic- ipants had sick leaves both from OHS and non-OHS sources. Instead of formal testing of such covariate, we conducted a separate ITS design and analyses for the intervention and comparison groups. Both crude models and models adjusted for the covariates were fitted. Analyses were run separately for men and women, for short (≤11 calendar days) and longer (>11 calendar days) sick leave periods, and for sick leave periods (≥11 calendar days) in the diagnostic cate- gories of back pain (ICD-10 M54.5), shoulder pain (ICD-10 M75) and elbow pain (ICD-10 M77.1). RESULTS Descriptive results d l d g−1 ( Y ) = β0 + βslope ( m −m0 ) + βimmediateX + βgradualX ( m −m0 ) ‍ Covariates Covariates that were available in the registers and were regarded as potential confounders included age, sex, occupational class (based on occupational title and catego- rised as upper grade non-manual employees, intermediate grade non-manual employees, lower grade non-manual 3 Kausto J, et al. BMJ Open 2021;11:e047018. doi:10.1136/bmjopen-2020-047018 Open access Open access using loess function of ‘stats’ in R package (V.3.5.1), with the default options. The same software was used for all analyses. using loess function of ‘stats’ in R package (V.3.5.1), with the default options. The same software was used for all analyses. generalised linear model with usual maximum like- lihood estimation, which gave similar results with narrower CI. The main test of our hypothesis pertained to the following model equation: Monthly averages of the number of all sick leave days Since it is generally recommended to draw simple plots of ITS analyses before entering into more complex model fitting,15 16 we started by examining the monthly average days of sick leave per employee. Based on a visual inspection of monthly averages, the number of all sick leave days prescribed at OHS declined shortly after the intervention had started in 2016 (figure 1A), and similar temporal changes were not observed for other sick leave periods (figure 1B). After 21 April 2017, our register data could not differentiate between sick leave days that were OHS-based and other sick leave days, but we had a longer stretch of data when counting in all sick leaves. A decline in sick leave days after the intervention was seen on visual inspection of all sick leaves (thick line for local regression smoother), as well as an annual periodic variation (thin dashed line in figure 1C). When removing cyclic annual variation and a linear trend, visual suggestion of an associ- ation between the intervention and sick leaves remained. However, sick leaves appeared to return to preceding rates towards the end of the longest available follow-up data (in 2018) (figure 1D). It should be noted that the panels in p As the sick leave recommendations potentially were associated with both the number of sick leave days and periods, the number of consecutive sick leave periods was computed within the index month plus 5 immediately preceding months. Monthly sample averages of the number of periods were investigated with otherwise similar linear models as in above but using a 6-month moving-average error structure. The ‘arima’ function of R V.3.5.1 (2018-07-02) was used. Local regression lines for the figures were drawn Kausto J, et al. BMJ Open 2021;11:e047018. Monthly averages of the number of all sick leave days doi:10.1136/bmjopen-2020-047018 4 Open access Table 1 Descriptive statistics for all employees, employees with sick leaves prescribed at OHS and employees with sick leave prescribed elsewhere or self-certified (other sick leaves)* Table 1 Descriptive statistics for all employees, employees with sick leaves prescribed at OHS and employees with sick leave prescribed elsewhere or self-certified (other sick leaves)* prescribed elsewhere or self-certified (other sick leaves) Characteristics Sick-listing at OHS (n=25 200) Other sick leaves (n=52 174) All (n=75 962) Age, mean (SD) 44.67 (12.32) 42.16 (13.02) 40.27 (14.08) Men (%) 20 23 27 Occupational class (%) Upper grade non-manual employees 21 24 27 Intermediate grade non-manual employees 27 25 22 Lower grade non-manual employees 37 36 34 Manual workers 16 15 17 Job contract (%) Permanent 82 67 54 Prescribed reimbursed purchases of medication for chronic diseases (%) Yes 21 17 16 Prescribed reimbursed psychotropic medication (%) Yes 12 9 8 Note that many employees were prescribed sick leave both at OHS and elsewhere (or used self-certification). The group ‘All’ contains also employees with no sick leaves. OHS, occupational healthcare services. Note that many employees were prescribed sick leave both at OHS and elsewhere (or used self-certification). The group ‘All’ contains also employees with no sick leaves. OHS, occupational healthcare services. Results on employee-level data: number of all sick leave days We modelled the number of sick leave days using bino- mial regression on monthly sick leave days per employee. Figure 2A,B illustrates the model predictions for the number of all sick leave days. For convenient illustration, we plotted the model estimates overlaid on the above- discussed monthly averages. These models used 2 160 445 observations from 75 962 individuals instead of 52 monthly averages. The exact binomial regression coeffi- cients (with logit link function) as well as the OR for a sick leave day at index month versus the preceding month are presented in table 3. Results on employee-level data: number of all sick leave days We modelled the number of sick leave days using bino- mial regression on monthly sick leave days per employee. Figure 2A,B illustrates the model predictions for the number of all sick leave days. For convenient illustration, we plotted the model estimates overlaid on the above- discussed monthly averages. These models used 2 160 445 observations from 75 962 individuals instead of 52 monthly averages. Monthly averages of the number of all sick leave days The exact binomial regression coeffi- cients (with logit link function) as well as the OR for a sick leave day at index month versus the preceding month are presented in table 3. figures 1 and 2 are not comparable with each other as the y-axes are different. The figures illustrate how the trend in sick leave days changes at the time of the intervention rather than compare the absolute values. Due to relatively small change in trend, similar y-axis would not illustrate the trend. figures 1 and 2 are not comparable with each other as the y-axes are different. The figures illustrate how the trend in sick leave days changes at the time of the intervention rather than compare the absolute values. Due to relatively small change in trend, similar y-axis would not illustrate the trend. Formal modelling (adjusting for seasonal variation) did not reveal an immediate diminishing trend (‍βimmediate ‍=0.01, 95% CI −0.052 to 0.072), but did detect a gradual diminishing trend (‍βgradual‍=−0.007, 95% CI −0.013 to −0.001) in the number of OHS-based sick leave days. The corresponding numbers in the comparison group were similar in direction but not statistically significant (‍βimmediate‍=0.068, 95% CI −0.065 to 0.201; βgradual=−0.009, 95% CI −0.022 to 0.004) (data not shown). These formal findings would support a gradual diminishing trend after the intervention, but we were able to achieve a greater statistical power by accessing employee-level data instead of the above-discussed monthly averages. We observed a clear gradual diminishing trend in the number of all-cause OHS-based sick leave days (model 1; table 3). A similar gradual trend was found in the comparison group (sick-listing elsewhere or self- certification), but this was offset by an increase in the level of the number of sick leave days at the time of 5 Kausto J et al BMJ Open 2021;11:e047018 doi:10 1136/bmjopen-2020-047018 Table 2 Monthly average number of sick leave days per month per employee by intervention status (2013–2017) Sick-listing at OHS Other sick leaves Average Range SD Average Range SD All sick leave days 0.380 0–30 2.59 0.840 0–30 3.40 All short sick leave periods* 0.117 0–25 0.79 0.392 0–24 1.24 Sick leave for low back pain† 0.005 0–30 0.36 0.007 0–30 0.39 Sick leave for shoulder pain† 0.020 0–30 0.71 0.014 0–30 0.59 Sick leave for elbow pain† 0.004 0–30 0.28 0.002 0–30 0.19 ≤11 calendar days. †>11 calendar days. OHS, occupational healthcare services. Results on employee-level data: number of all sick leave periods Results on employee-level data: number of all sick leave periods intervention. Adjusting for the covariates (model 2; table 3) did not affect the estimates significantly. We additionally ran the analyses separately for men and women (not shown in the table). The results were in line with the first model (total sample). The associa- tion between the intervention and sick leaves found in all-cause sick leave was not paralleled in the diagnostic group-specific findings (table 3). As a sensitivity analysis, we investigated the number of all-cause sick leave periods as an outcome. A sick leave period is a consecutive spell of sick leave days. We report the results in units of periods per year per employee, although we computed the periods per index month plus 5 preceding months (figure 2C,D). We analysed the monthly averages with a moving-average regression model. For all sick leave periods prescribed at OHS, we did not detect any immediate change in trend (‍βimmediate‍=0.017, 95% CI −0.011 to 0.046), but a gradual diminishing trend in the number of sick leave periods was found (‍βgradual ‍=−0.009, 95% CI −0.013 to −0.005). The findings on the gradual change of trend were similar in the comparison group (‍βgradual‍=−0.014, 95% CI −0.026 to −0.003), whereas the immediate change in trend indicated that an increase in sick leave periods occurred in the comparison group at the time of the intervention (‍βimmediate‍=0.141, 95% CI 0.067 to 0.214). While a gradual diminishing trend of sick leave days with an OR of 0.986 may appear small, we can illustrate the association between the intervention and sick leave days in a practical way as follows. Assuming employees on average have 1.2 sick leave days per month (cf, figure 1C), their baseline risk rate per day would be 0.04. Small risk ratios closely match the OR. Thus, if the other factors have already been adjusted for, the intercept for a logistic model would be ‍β0 = log ( 0.04 ) ‍. Then the gradual inter- vention estimate of ‍ORgradual = 0.986‍ would imply that the OR of a sick leave day 1 year after the intervention has reduced to ‍exp ( β0 + 12 × log ( ORgradual )) ≈0.034‍ and the risk rate of a sick leave day to approximately 0.033. Monthly averages of the number of all sick leave days 5 Kausto J, et al. BMJ Open 2021;11:e047018. doi:10.1136/bmjopen-2020-047018 Open access Figure 1 Model fits. Monthly averages of the number of all sick leave days. Intervention status could be specified until 21 April 2017 (A and B). In C and D, the intervention status is not differentiated. Vertical dashed lines indicate the timepoint of intervention (A–D). Circles denote observed averages, thick lines their local regression fits (smoothed averages) (A–D) and thin dashed line annual periodic variation (C). Note the differing scales in the figures. OHS, occupational healthcare services; Avg, average; Resid, residual. Figure 1 Model fits. Monthly averages of the number of all sick leave days. Intervention status could be specified until 21 April 2017 (A and B). In C and D, the intervention status is not differentiated. Vertical dashed lines indicate the timepoint of intervention (A–D). Circles denote observed averages, thick lines their local regression fits (smoothed averages) (A–D) and thin dashed line annual periodic variation (C). Note the differing scales in the figures. OHS, occupational healthcare services; Avg, average; Resid, residual. Results on employee-level data: number of all sick leave periods Thus, the gradual intervention estimate on average amounts to ‍ ( 0.04 −0.033 ) × 30 × 12 = 2.52‍ of avoided sick leave days per year per employee. Expressed in another way, the intervention may have saved 2520 sick leave days per year per 1000 employees. DISCUSSION In this register-based study among municipal employees, we analysed sick leave trends before and after an interven- tion targeted at physicians’ management of musculoskel- etal pain-related work disability and prescribing sick leave. Kausto J, et al. BMJ Open 2021;11:e047018. doi:10.1136/bmjopen-2020-047018 6 Open access p Figure 2 Model fits. Monthly averages of the number of all sick leave days (A and B) and periods (C and D). Observed data points denote simple monthly averages and the lines represent GEE model fits to employee-level data. The dashed line represents the full model prediction, including annual variations, whereas the solid line is the ITS part of the model. Note the differing scales in the figures. GEE, generalised estimation equation; ITS, interrupted time series analysis; OHS, occupational healthcare; Avg, average; SA, sickness absence services. Figure 2 Model fits. Monthly averages of the number of all sick leave days (A and B) and periods (C and D). Observed data points denote simple monthly averages and the lines represent GEE model fits to employee-level data. The dashed line represents the full model prediction, including annual variations, whereas the solid line is the ITS part of the model. Note the differing scales in the figures. GEE, generalised estimation equation; ITS, interrupted time series analysis; OHS, occupational healthcare; Avg, average; SA, sickness absence services. The intervention was carried out in an OHS setting. We examined monthly averages of all-cause sick leave days and found no immediate change in trend among OHS- based sick leave days, but did detect a gradual decreasing trend in the number of OHS-based sick leave days by 2.5 days per year per employee. The corresponding findings were similar in direction but not statistically significant for the comparison time series (sick-listing elsewhere or self- certification). In the employee-level data (with a larger statistical power), we found a clear gradual decreasing trend in the number of OHS-based all-cause sick leave days. Similar gradual decreasing trend was found in the comparison time series, but this was offset by an increase in the level of sick leave days and periods at the time of the intervention. A hypothetical organisation with 40 000 employees and an average direct cost of €250 per sick leave day (numbers similar to those of the City of Helsinki)22 is likely to have resulted in substantial annual savings at the time of the intervention. Kausto J, et al. BMJ Open 2021;11:e047018. doi:10.1136/bmjopen-2020-047018 DISCUSSION the intervention was carried out at OHS, the employees increasingly sought treatment elsewhere (or used self- certification increasingly) as an increase in the level of other sick leaves was detected. This trend levelled down, however. As far as we know, there are no previous studies investigating the effects of introducing guidelines on prescribing sick leaves (as compared with the situation where no guidelines exist). A few studies have compared different ways of implementation.23 24 There are many previous studies on interventions attempting to change physicians’ behaviour in some other areas of clinical practice. Findings show that success in changing the behaviour and adherence to different clinical guide- lines vary. Some studies reported success in behaviour change,25 26 whereas other studies reported limited success or found the interventions ineffective.27–29 More- over, a change in physicians’ behaviour does not auto- matically reflect in patient outcomes.28 Guidelines have been found to be a necessary but insufficient step in changing clinical care.30 The importance of active imple- mentation of guidelines and efforts to include the new practice into existing organisational procedures, which both were attempted in this intervention, has been emphasised to achieve sustainable results.31 32 As the context of the intervention is important, the results of The knowledge base on physicians’ sick-listing prac- tices is accumulating. The present study suggests that there was an association between the intervention and the sick-listing practices of physicians working at Occu- pational Health Helsinki. There was a downward trend in the number of OHS-based sick leave days and periods. Nevertheless, the findings may also suggest that soon after 7 Kausto J, et al. BMJ Open 2021;11:e047018. DISCUSSION doi:10.1136/bmjopen-2020-047018 access g p g y Sick-listing at OHS Other sick leaves β coefficient SE OR† 95% CI β coefficient SE OR† 95% CI Sick leave days (all periods, all DC:s) (crude model)‡ (n=2 160 445 employee-months, n=75 962 employees) Immediate change in trend 0.012 0.035 1.012 0.95 to 1.08 0.060* 0.018 1.062 1.03 to 1.10 Gradual change in trend −0.014* 0.003 0.986 0.98 to 0.99 −0.008* 0.002 0.992 0.99 to 1.00 Linear trend before intervention (slope) 0.006* 0.001 1.006 1.00 to 1.01 0.002* 0.001 1.002 1.00 to 1.00 Sick leave days (all periods, all DC:s) (fully adjusted model)‡ (n=2 148 566 employee-months, n=74 701 employees) Immediate change in trend −0.003 0.032 0.997 0.94 to 1.06 0.057 0.018 1.059 1.02 to 1.10 Gradual change in trend −0.010 0.003 0.990 0.98 to 1.00 −0.007* 0.002 0.993 0.99 to 0.99 Linear trend before intervention (slope) 0.007* 0.001 1.007 1.01 to 1.01 0.003* 0.001 1.003 1.00 to 1.01 Sick leave days (short periods, <11 calendar days, all DC:s)‡ (n=2 160 445 employee-months, n=75 962 employees) Immediate change in trend 0.105* 0.024 1.111 1.06 to 1.16 0.196* 0.010 1.217 1.19 to 1.24 Gradual change in trend −0.020* 0.002 0.980 0.98 to 0.98 −0.009* 0.001 0.991 0.99 to 0.99 Linear trend before intervention (slope) 0.001 0.001 1.001 1.00 to 1.00 −0.003* <0.001 0.997 1.00 to 1.00 Sick leave days in low back pain (periods ≥11 calendar days)§ (n=2 160 445 employee-months, n=75 962 employees) Immediate change in trend −0.547 0.340 0.579 0.30 to 1.13 −0.205 0.283 0.815 0.47 to 1.42 Gradual change in trend 0.040 0.035 1.041 0.97 to 1.12 −0.019 0.031 0.981 0.92 to 1.04 Linear trend before intervention (slope) −0.004 0.009 0.996 0.98 to 1.01 −0.004 0.007 0.996 0.98 to 1.01 Sick leave days for shoulder pain (periods ≥11 calendar days)§ (n=2 160 445 employee-months, n=75 962 employees) Immediate change in trend 0.365 0.207 1.441 0.96 to 2.16 −0.426 0.292 0.653 0.34 to 1.16 Gradual change in trend −0.023 0.023 0.977 0.93 to 1.02 0.013 0.025 1.013 0.97 to 1.06 Linear trend before intervention (slope) −0.004 0.008 0.996 0.98 to 1.01 −0.003 0.008 0.997 0.98 to 1.01 Sick leave days for elbow pain (periods ≥11 calendar days)§ (n=2 160 445 employee-months, n=75 962 employees) Immediate change in trend −0.964 0.514 0.381 0.14 to 1.04 −0.398 0.486 0.672 0.26 to 1.74 Gradual change in trend −0.034 0.072 0.967 0.84 to 1.11 −0.079 0.084 0.924 0.78 to 1.09 Linear trend before intervention (slope) 0.003 0.014 1.003 0.98 to 1.03 −0.001 0.018 0.999 0.96 to 1.04 Adjusted for age, sex, occupational class, job contract, prescribed reimbursed purchases of medication for chronic diseases and prescribed reimbursed psychotropic medication. DISCUSSION p<0.05, p<0.01, **p<0.001. †Immediate intervention estimate: OR was estimated for any employee to have any sick leave day after versus before the intervention. Gradual intervention estimate: OR was estimated for any employee to have a sick leave day at index month versus preceding month during the period after the intervention. ‡Follow-up from 1 January 2013 to 21 April 2017. §Follow-up from 1 January 2013 to 31 December 2019 Open access Kausto J, et al. BMJ Open 2021;11:e047018. doi:10.1136/bmjopen-2020-047018 Kausto J, et al. BMJ Open 2021;11:e047018. doi:10.1136/bmjopen-2020-047018 8 Open access an intervention in one environment cannot directly be applied to another context. intervention being effective; however, the causality of the relationship is unclear due to similar findings in the comparison group. The estimated reduction of 2.5 sick leave days per year per employee could leave to substantial savings for a large employer such as the City of Helsinki. This can be considered an economically relevant inter- vention, but further evidence for its causal interpretation is needed. A strength of this study is its design based on register data on all employees. We could link comprehensive data from employer registers to data from national registers using personal identification numbers, resulting in no loss to follow-up. The use of administrative registers in research always entails limitations as they are not typically collected for research purposes. Nevertheless, in general, the quality of registers in Finland, as in other Nordic coun- tries, is good and administrative registers offer unique opportunities and are frequently used in research. Twitter Jenni Ervasti @JenniErvasti1, Jaakko Harkko @JaakkoHarkko and Anne Kouvonen @AKouvonen Twitter Jenni Ervasti @JenniErvasti1, Jaakko Harkko @JaakkoHarkko and Anne Kouvonen @AKouvonen Acknowledgements We would like to thank Eevamaija Tuovinen MD (chief medical officer), Helena Miranda MD, PhD, and Tiina Pohjonen PhD (CEO) of Occupational Health Helsinki for providing information on the intervention and its context. Acknowledgements We would like to thank Eevamaija Tuovinen MD (chief medical officer), Helena Miranda MD, PhD, and Tiina Pohjonen PhD (CEO) of Occupational Health Helsinki for providing information on the intervention and its context. pp q y Applied ITS analysis with a comparison time series has been recommended for analysing population-level inter- ventions.15 33 A limitation was that the intervention status was approximated (an OHS-based sick leave was preceded by a likely relevant appointment at OHS) and the infor- mation was not available for the entire follow-up period. Patient consent for publication Not required. Ethics approval The ethics committees of the Department of Public Health, the University of Helsinki and the health authorities of the City of Helsinki approved the Helsinki Health Study protocol. The ethical approval applies to the current study. Because the intervention was targeted at managing pain and prescribing sick leave from the first day of work disability, a limitation was that we had access to diag- nostic data only for sick leave periods that lasted over 11 calendar days. This, together with the low base rate for specific diagnoses, may explain the result where the asso- ciation between the intervention and sick leaves found in all-cause sick leaves was not replicated in diagnosis-specific findings of sick leaves (lasting over 11 calendar days) due to low back, shoulder and elbow pain. MSDs constitute a major cause of sick leave. The fact that an association between the intervention and sick leaves was seen among all-cause sick leaves (regardless of the length of the sick leave period) may also indicate that recommendations given for these three specific disorders influenced how physicians managed work disability and prescribed sick leave altogether. Unfortunately, we did not have informa- tion on part-time sick leaves, and for this reason we could not take this into account in the analyses. In Finland, partial sickness benefit can be granted only after 11 days of full-time sickness absence. Partial sickness benefit days comprised only 7% of all compensated sickness benefit days in Finland in 2016. Provenance and peer review Not commissioned; externally peer reviewed. Data availability statement All data relevant to the study are included in the article or uploaded as supplementary information. No additional data are available. Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise. ORCID iDs Johanna Kausto http://orcid.org/0000-0002-2898-0018 Jenni Ervasti http://orcid.org/0000-0001-9113-2428 Ossi Rahkonen http://orcid.org/0000-0002-7202-3274 Jaakko Harkko http://orcid.org/0000-0001-8682-1544 Anne Kouvonen http://orcid.org/0000-0001-6997-8312 Tea Lallukka http://orcid.org/0000-0003-3841-3129 The recommendations for physicians constituted chronologically the first and the main element of the intervention. Direct access to physiotherapists without a doctor’s referral was introduced in the following year, and thereafter it was not possible to differentiate the associa- tion between these different elements and sick leaves. Patient consent for publication Not required. Open access This is an open access article distributed in accordance with the Creative Commons Attribution 4.0 Unported (CC BY 4.0) license, which permits others to copy, redistribute, remix, transform and build upon this work for any purpose, provided the original work is properly cited, a link to the licence is given, and indication of whether changes were made. See: https://creativecommons.org/ licenses/by/4.0/. REFERENCES 1 Tilastotietokanta Kelasto (statistics in Finnish). Available: https:// www.kela.fi/kelasto [Accessed 20 Oct 2020]. 2 van Tulder M, Becker A, Bekkering T, et al. Chapter 3. European guidelines for the management of acute nonspecific low back pain in primary care. Eur Spine J 2006;15 Suppl 2:s169–91. 3 Costa-Black KM, Loisel P, Anema JR, et al. Back pain and work. Best Pract Res Clin Rheumatol 2010;24:227–40. Kausto J, et al. BMJ Open 2021;11:e047018. doi:10.1136/bmjopen-2020-047018 DISCUSSION The validity of the comparison group can be debated17 as all employees could be prescribed sick leave both at the OHS and elsewhere (or use self-certification in short sick leaves). However, only OHS professionals were exposed to the intervention and the time series for the intervention and comparison groups were analysed separately. Thus, our analysis should be thought of as being in between ITS and CITS in what comes to scientific value of evidence. Contributors Conceptualisation and methodology: JK, THR, OP, AV, JE, OR, JH, AK and TL. Writing - original draft preparation: JK and THR. Writing - review and editing JK, THR, JE, OP, LK-K, OR, JH, AV, AK and TL. Guarantor and project administrator: JK. All authors have read and agreed to the published version of the manuscript. Funding This work was supported by the Social Insurance Institution of Finland (grant #9/26/2019; JK, THR, JE, AV and LK-K), the Finnish Work Environment Fund (grant #190081; JK, THR, JE, AV and LK-K), the Academy of Finland (grants #1294514 and #319200; OR and TL) and the Economic and Social Research Council (ESRC) (grant #ES/S00744X/1; AK). Competing interests None declared. Patient consent for publication Not required. Patient consent for publication Not required. 1 Tilastotietokanta Kelasto (statistics in Finnish). Available: https:// www.kela.fi/kelasto [Accessed 20 Oct 2020]. Open access Open access 4 Black CM. Sickness absence and musculoskeletal disorders. Rheumatology 2012;51:204–5. 20 Xiao H, Augusto O, Wagenaar BH. Reflection on modern methods: a common error in the segmented regression Parameterization of interrupted time-series analyses. Int J Epidemiol 2020. gy 5 Lötters FJB, Foets M, Burdorf A. Work and health, a blind spot in curative healthcare? A pilot study. 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Eur J Public Health 2012;22:92–6. 24 Becker A, Leonhardt C, Kochen MM, et al. Effects of two guideline implementation strategies on patient outcomes in primary care: a cluster randomized controlled trial. Spine 2008;33:473–80. 9 Hinkka K, Niemelä M, Autti-Rämö I, et al. Physicians' experiences with sickness absence certification in Finland. Scand J Public Health 2019;47:859–66. 25 Bussières AE, Sales AE, Ramsay T, et al. Impact of imaging guidelines on X-ray use among American provider network chiropractors: interrupted time series analysis. Spine J 2014;14:1501–9. 10 Lahelma E, Aittomäki A, Laaksonen M, et al. Cohort profile: the Helsinki health study. Int J Epidemiol 2013;42:722–30. 11 Sumanen Het al. Age-Group differences in sickness absence among the employees of the city of Helsinki 2002−2016. Finnish Medical Journal 2018;73:438–44. 26 Leece P, Shantharam Y, Hassam S, et al. Improving opioid guideline adherence: evaluation of a multifaceted, theory-informed pilot intervention for family physicians. BMJ Open 2020;10:e032167. 12 Gissler M, Haukka J. Finnish health and social welfare registers in epidemiological research. Norsk Epidemiologi 2004;14:113–20. 27 French SD, McKenzie JE, O'Connor DA, et al. Evaluation of a theory- informed implementation intervention for the management of acute low back pain in general medical practice: the implement cluster randomised trial. PLoS One 2013;8:e65471. REFERENCES 1 Tilastotietokanta Kelasto (statistics in Finnish). Available: https:// www.kela.fi/kelasto [Accessed 20 Oct 2020]. 2 van Tulder M, Becker A, Bekkering T, et al. Chapter 3. European guidelines for the management of acute nonspecific low back pain in primary care. Eur Spine J 2006;15 Suppl 2:s169–91. To conclude, we observed a gradual declining trend in the number of sick leave days and periods prescribed at OHS after the intervention. 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The implementation of occupational health guidelines principles for reducing sickness absence due to musculoskeletal disorders. Occup Med 2006;56:237–42. 17 Degli Esposti M, Spreckelsen T, Gasparrini A, et al. Can synthetic controls improve causal inference in interrupted time series evaluations of public health interventions? Int J Epidemiol 2021;49:2010–20. 32 Buyle F, Vogelaers D, Peleman R, et al. Implementation of guidelines for sequential therapy with fluoroquinolones in a Belgian Hospital. Pharm World Sci 2010;32:404–10. 18 Hojsgaard S, Halekoh U, Yan J. The R package Geepack for generalized estimating equations. Journal of Statistical Software 2006;15:1–11. 33 Shadish WR, Cook TD, Campbell DT. Quasi-experimental experiments: Interrupted time-series designs. In: Experimental and quasi-experimental designs for generalized causal inference. Boston, New York: Houghton Mifflin Company, 2002: 623. ; 19 Gelman A, Hill J. Data analysis using regression and Multilevel/ Hierarchical models. Cambridge University Press, 2007. 10 Kausto J, et al. BMJ Open 2021;11:e047018. doi:10.1136/bmjopen-2020-047018
https://openalex.org/W3152955459	https://ri.conicet.gov.ar/bitstream/11336/141159/5/CONICET_Digital_Nro.831d425d-e4a7-4bcf-82f8-f5378a067025_D.pdf	English	null	Environmental Education as a Means for Valuing and Conserving Camelids and Pastoralism in the Argentinean Altiplano of Jujuy	Mountain research and development	2,020	cc-by	9,024	The user has requested enhancement of the downloaded file. See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/351045266 Environmental Education as a Means for Valuing and Conserving Camelids and Pastoralism in the Argentinean Altiplano of Jujuy All content following this page was uploaded by Bibiana Vila on 07 May 2021. The user has requested enhancement of the downloaded file. All content following this page was uploaded by Bibiana Vila on 07 May 2021. Bibiana Vil ´a1,2,3, Yanina Arzamendia1,4, and Ver ´onica Rojo1,5 Over the years, we have proposed different environmental education (EE) strategies targeting local Andean stakeholders and university students, including interventions in primary schools. This paper presents the results of 4 EE interventions focusing on mountain environments, their biodiversity, environmental calendars, and llama caravans. School children participated creatively in this process by writing Keywords: environmental education; Andean pastoralism; mountain biodiversity; local schools. Peer-reviewed: September 2020 Accepted: December 2020 wild vicu~nas share grazing lands. In some cases, depending on human attitudes and animal densities, the wild species are displaced. In other places, there is a certain degree of harmony and potential for the sustainable production of several species (Rojo et al 2012; Arzamendia and Vil´a 2015). Vicu~nas were in danger of extinction due to hunting, because of the high market value of their ﬁne ﬁber. Although conservation efforts have reversed this trend, and vicu~na hunting is prohibited by law, vicu~nas are still subject to poaching. Local communities play a key role in conservation, and situated environmental education (EE) can make a big difference in bringing these communities on board. In some areas of northwest Argentina, projects that promote the sustainable management of wild vicu~nas have employed chakus, a traditional practice of vicu~na capture, shearing, and later release, to obtain the valuable ﬁber and stimulate local economies (Vil´a et al 2010; Arzamendia et al 2010, 2014; Arzamendia and Vil´a 2012). Although this may seem encouraging, pastoralism on the Altiplano of Jujuy faces the same challenges as other pastoral systems around the world (comprising approximately 25% of the land surface worldwide supporting 20 million households; Dong et al 2011). It is vulnerable to climate change, land tenure Bibiana Vil ´a1,2,3, Yanina Arzamendia1,4, and Ver ´onica Rojo1,5 * Corresponding author: bibianavila@gmail.com 1 VICAM (Vicu~nas, Camelidos y Ambiente), Luj´an, 6700 Buenos Aires, Argentina 2 CONICET (Consejo Nacional de Investigaciones Cient´ıficas y Tecnicas), Godoy Cruz 2290, Ciudad Aut´onoma de Buenos Aires C1425FQB, Argentina 3 Department of Social Sciences, National University of Luj´an, Avenida Constituci´on and National Route 5, 6700 Buenos Aires, Argentina 4 INECOA (Instituto de Ecorregiones Andinas)–CONICET–UNJu (National University of Jujuy), Alberdi 47, 4600 San Salvador de Jujuy, Argentina 5 Department of Technology and Department of Basic Sciences, National University of Luj´an, Avenida Constituci´on and National Route 5, 6700 Buenos Aires, Argentina 2020 Vil´a et al. This open access article is licensed under a Creative Commons Attribution 4.0 International License (http://creativecommons.org/ licenses/by/4.0/). Please credit the authors and the full source. 2020 Vil´a et al. This open access article is licensed under a Creative Commons Attribution 4.0 International Lic licenses/by/4.0/). Please credit the authors and the full source. poems, drawing, and playing. The resulting work showed sensitivity, experiential knowledge, and a comprehensive vision of the environment. Most of the artworks were printed and disseminated in the children’s local communities, where they are highly appreciated. We recognize that the usefulness of EE is constrained by social and economic pressures, including extractive activities. However, we also underscore its huge potential to guarantee sustainability during the inevitable process of change in traditional Andean pastoralism. Andean pastoralism, like other pastoral systems around the world, is under stress due to climate change, land tenure regimes, pressures to become sedentary, difficulties in interacting with market-based economies, isolation, and youth emigration. Over the years, we have proposed different environmental education (EE) strategies targeting local Andean stakeholders and university students, including interventions in primary schools. This paper presents the results of 4 EE interventions focusing on mountain environments, their biodiversity, environmental calendars, and llama caravans. School children participated creatively in this process by writing poems, drawing, and playing. The resulting work showed sensitivity, experiential knowledge, and a comprehensive vision of the environment. Most of the artworks were printed and disseminated in the children’s local communities, where they are highly appreciated. We recognize that the usefulness of EE is constrained by social and economic pressures, including extractive activities. However, we also underscore its huge potential to guarantee sustainability during the inevitable process of change in traditional Andean pastoralism. p sedentary, difficulties in interacting with market-based economies, isolation, and youth emigration. Environmental Education as a Means for Valuing and Conserving Camelids and Pastoralism in the Argentinean Altiplano of Jujuy Article in Mountain Research and Development · December 2020 DOI: 10.1659/MRD-JOURNAL-D-20-00009.1 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: VICAM: Vicuñas, Camelidos y ambiente View project MACS: Utilizacion econònica sustentable de camelidos sudamericanos silvestres. View project MountainDevelopment Transformation knowledge MountainDevelopment Transformation knowledge Mountain Research and Development (MRD) An international, peer-reviewed open access journal published by the International Mountain Society (IMS) www.mrd-journal.org Mountain Research and Development (MRD) An international, peer-reviewed open access journal published by the International Mountain Society (IMS) www.mrd-journal.org Environmental Education as a Means for Valuing and Conserving Camelids and Pastoralism in the Argentinean Altiplano of Jujuy Mountain Research and Development Vol 40 No 4 Nov 2020: D39–D49 Introduction Jujuy, the most northwestern province of Argentina, has the largest percentage of mountainous landscape (over 93%) in the country (IGN and SEGEMAR 2019). This includes a wide range of ecosystems. The Puna or Altiplano is a dry, cold, high-elevation (.3500 m above sea level) Andean ecosystem, with a semidesert vegetation community that consists mainly of shrub–steppe, with tussocks and short grasses. Peatlands, locally known as vegas, develop in restricted areas where groundwater is near the surface (Cabrera 1971). The Altiplano ecosystem’s functions and services support one of the most important, long-lived, and culturally distinctive socioecological systems in the world, Andean pastoralism. The uniqueness of the Altiplano ecosystem includes endemic fauna and livestock, with 4 species of South American camelids: wild vicu~nas (Vicugna vicugna) and guanacos (Lama guanicoe), and domestic llamas (Lama glama) and alpacas (Vicugna pacos). Most of the llama breeders in Jujuy are members of indigenous communities and practice subsistence economies. They live in isolated places, with high mobility between different elevational ranges (Wawrzyk and Vil´a 2013; Vil´a et al 2018). In these rangelands, livestock and https://doi.org/10.1659/MRD-JOURNAL-D-20-00009.1 D39 MountainDevelopment knowledge—in collaboration with the teacher, through the development of their curiosity. regimes, pressures on pastoralists to become sedentary, difﬁculties in interacting with market-based economies, isolation, and youth emigration from the area (Dong et al 2011; Coppock et al 2017). Ancestral practices, such as llama caravans, are becoming or are already extinct (Vil´a 2018, 2019). Our research group has developed EE initiatives as a necessary complement to our camelid research program. These initiatives target different stakeholders and include a variety of formats. At the National University of Jujuy, research group staff members teach ‘‘learn by doing’’ courses for undergraduate and graduate students. These involve immersion in the process of sustainable management of vicu~nas and encourage the intercultural exchange of knowledge and experiences among scientists, local communities, and students (Vil´a et al 2020). Dialogue between the scientiﬁc and local communities within an EE framework is a ﬁrst step to addressing this negative scenario. This would involve the coconstruction of interventions such as chakus, design policies (Arzamendia et al 2012), and biocultural conservation events, for example, Ashka llama fairs, workshops about rangeland management, and others. This Andean EE dialogue can promote the social construction of knowledge through the exchange of ideas, feelings, representations, beliefs, concepts, practices, narratives, desires, and experiences (Salas 2013). (UNESCO 1977) We need EE that includes a process of creating new values and knowledge linked to the transformation of reality (Leff 1999) in view of the responsibilities and rights that arise from planetary citizenship. It should also be grounded in the local material and symbolic embodiments of these y responsibilities, such as the holistic concept of Pachamama in the Andean cosmovision. Only from this framework of integrated perspectives can EE help local communities to take charge of their own destiny and reproduce a culture of sustainability (Gutierrez and Prado 1997). As expressed by Leonardo Boff (1996), these new ways of giving meaning to the world envision the emergence of new values, new dreams, and new behaviors. An EE experience engages educators and students in a pedagogical setting that includes elements outside the school: the environment, the pastoral system, llamas, vicu~nas, and so forth. In this scenario, we must build what Freire (2003) described as the possibility for students to produce knowledge—in our case, environmental (UNESCO 1977) A more informal and creative mode of these experiences at schools is what we call ‘‘EE interventions.’’ These are initiated either as a response to requests made by local school members or indigenous communities, or in the context of national (and international) environmentally signiﬁcant dates (ie ‘‘environmental ephemerides’’). EE interventions are short, intensive events with a hands-on approach. They usually take the form of workshops in primary or high schools, where children and teenagers develop a speciﬁc product or output. The deﬁnition includes all the stakeholders in environmental management, so EE must be a cocreation process, respectful of the multicultural nature of Andean socioecosystems. In EE, classic biology dissolves into ethnobiology: the scientiﬁc study of dynamic relationships among peoples, biota, and environments . . . It allows us to examine complex, dynamic interactions between human and natural systems. (Argueta et al 2012: 21) (Argueta et al 2012: 21) The objective of this paper is to describe 4 EE interventions focusing on mountain environments, biodiversity, the environmental calendar, and llama caravans at schools in the Pozuelos Basin (province of Jujuy, Argentina) and to present and analyze some of the resulting artworks, which have become part of the heritage of these communities. One important question is: What kind of EE is appropriate for communities that live embedded in and of nature? We need EE that includes a process of creating new values and knowledge linked to the transformation of reality (Leff 1999) in view of the responsibilities and rights that arise from planetary citizenship. It should also be grounded in the local material and symbolic embodiments of these responsibilities, such as the holistic concept of Pachamama in the Andean cosmovision. Only from this framework of integrated perspectives can EE help local communities to take charge of their own destiny and reproduce a culture of sustainability (Gutierrez and Prado 1997). As expressed by Leonardo Boff (1996), these new ways of giving meaning to the world envision the emergence of new values, new dreams, and new behaviors. An EE experience engages educators and students in a pedagogical setting that includes elements outside the school: the environment, the pastoral system, llamas, vicu~nas, and so forth. In this scenario, we must build what Freire (2003) described as the possibility for students to produce knowledge—in our case, environmental One important question is: What kind of EE is appropriate for communities that live embedded in and of nature? Introduction It also includes the biocultural values of the pastoral world, which are largely ignored by educational institutions. EE activities in local communities focus on technical aspects of wild vicu~na management, including corral and funnel construction, procedures for animal capture and release, animal welfare, and risk management. One product of this interaction is the Handbook on Vicu~na Conservation and Sustainable Use (Baldo et al 2013), which can be downloaded for free from the National Research Council’s website. This has been widely used as a foundation for management techniques by several governmental and nongovernmental organizations. Another focus of joint activities with communities is the exchange of ideas on rangeland management (vegetation, vicu~nas, and livestock) and training for Altiplano school teachers (Vil´a et al 2006, 2009; Vil´a 2014). The purpose of these courses is to familiarize teachers with regional and global environmental issues and provide environmentally focused teaching methodologies that generate signiﬁcant learning opportunities. These can foster a change in attitudes and motivations toward valuing the school’s situation in a mountain ecosystem. As stated by the United Nations Educational, Scientiﬁc and Cultural Organization (UNESCO) in its ofﬁcial deﬁnition: the aim of environmental education is to succeed in making individuals and communities understand the complex nature of the natural and the built environments resulting from the interaction of their biological, physical, social, economic and cultural aspects, and acquire the knowledge, values, attitudes and practical skills to participate in a responsible and effective way in anticipating and solving environmental problems, and the management of the quality of the environment. (UNESCO 1977) https://doi.org/10.1659/MRD-JOURNAL-D-20-00009.1 Mountain Research and Development Study area and methods The ﬁeldwork was carried out in the Pozuelos Basin, in the Laguna de los Pozuelos Natural Monument of the National Parks Administration within the Man and Biosphere Reserve (MAB-UNESCO) and Ramsar site (Figure 1). g Santa Catalina (21856047.4700S; 668307.3200W) is the Santa Catalina (21856047.4700S; 668307.3200W) is the northernmost town in Argentina. It is situated near the border with the Plurinational State of Bolivia. Santa Catalina lies within the valley of the Santa Catalina River at ~3800 m above sea level, in the Rinconada mountain range. The area was inhabited by pre-Hispanic communities, as evidenced by petroglyphs showing camelid iconography discovered in the Mountain Research and Development D40 https://doi.org/10.1659/MRD-JOURNAL-D-20-00009.1 MountainDevelopment FIGURE 1 Map of the Pozuelos basin and the 3 schools: Pozuelos, Rodeo, and Santa Catalina. (Photos by Bibiana Vil´a, Yanina Arzamendia, and Ver´onica Rojo) area. The community of Santa Catalina includes 4 indigenous groups, ofﬁcially recognized as indigenous organizations (Atu Saphi, Aucarpina Chambi, Yurax Rumi, and La Cruz), and a cooperative of local landowners. Santa Catalina is the center of administrative, political, sanitary, commercial, religious, festive, and educational functions for nearby rural areas. The main sources of income in Santa Catalina are from breeding llamas and sheep to produce ﬁber, leather, and meat, governmental subsidies, and state employment. signiﬁcance prompted an intervention. The school authorities and our research team coproposed celebrating key dates (Mountain Day, Biodiversity Day) as part of their school schedule, in line with the national and international agendas. We celebrated the National/International Mountain Day with interventions in the Santa Catalina primary school in 2017 and 2019, with the participation of teachers and 9 and 8 children, respectively. Biodiversity Day activities were carried out in 3 schools from 14 to 16 May 2018. The participants included school children, teachers, and headmistresses of the schools in Pozuelos (9 children and 3 teachers), Rodeo (4 children and 3 teachers), and Santa Catalina (5 children and 1 teacher). area. The community of Santa Catalina includes 4 indigenous groups, ofﬁcially recognized as indigenous organizations (Atu Saphi, Aucarpina Chambi, Yurax Rumi, and La Cruz), and a cooperative of local landowners. Santa Catalina is the center of administrative, political, sanitary, commercial, religious, festive, and educational functions for nearby rural areas. The main sources of income in Santa Catalina are from breeding llamas and sheep to produce ﬁber, leather, and meat, governmental subsidies, and state employment. signiﬁcance prompted an intervention. Study area and methods The school authorities and our research team coproposed celebrating key dates (Mountain Day, Biodiversity Day) as part of their school schedule, in line with the national and international agendas. We celebrated the National/International Mountain Day with interventions in the Santa Catalina primary school in 2017 and 2019, with the participation of teachers and 9 and 8 children, respectively. Biodiversity Day activities were carried out in 3 schools from 14 to 16 May 2018. The participants included school children, teachers, and headmistresses of the schools in Pozuelos (9 children and 3 teachers), Rodeo (4 children and 3 teachers), and Santa Catalina (5 children and 1 teacher). signiﬁcance prompted an intervention. The school Santa Catalina primary (no. 18 G. Rondeau) and secondary school students belong to indigenous groups. Some of them are Quechua speakers and deﬁne themselves as Coyas. The main language used in the school is Spanish, Argentina’s ofﬁcial language, although they also have Quechua lessons. Only 30% of the students’ families are able to meet their basic needs; 40% belong to families of herders and farmers, while the rest are underemployed or unemployed. The school provides 4 daily meals, and several students board in the school through the week. The llama caravan intervention was planned to involve the Santa Catalina school in what was happening in the town, which was waiting for the llama caravans to arrive. The intervention was conducted in November 2017 with 10 seventh grade students and their teachers. All school interventions were launched by asking the children to engage in a triggering activity related to the theme of the day, such as considering the color of animals as a way to approach the issue of biodiversity, or a class trip to climb the neighboring hills to reﬂect upon mountain landscapes, or imagining the everyday life of a llama caravanner. Then, following Freire (2003), we built a pedagogical space of trust in which the children could feel at ease and let their curiosity guide the development of the Two primary schools were added to the initiative for the biodiversity EE intervention, Rodeo and Pozuelos, both located in the Laguna de los Pozuelos MAB Biosphere Reserve. These schools are similar to Santa Catalina’s schools, but they have fewer children in attendance. Study area and methods Teachers consider environmental ephemerides to be important within the educational agenda, and this https://doi.org/10.1659/MRD-JOURNAL-D-20-00009.1 D41 MountainDevelopment FIGURE 2 Santa Catalina school children coming back from the mountain to the town. (Photo by Bibiana Vil´a) FIGURE 2 Santa Catalina school children coming back from the mountain to the town. (Photo by Bibiana Vila) activity. We also proposed the creation of some form of lasting record, a material reminder of this shared experience, to which end participants were invited to engage in creative writing and drawing activities. All the exchanges and the children’s productions were in Spanish. It is important to note that the authors have been working in the area for 10 years. Researchers are relatively familiar in the town, and we have established a very good rapport with the local people. All surveys were conducted following the Code of Ethics for Ethnobiological Research of the Latin American Society of Ethnobiology (SOLAE Ethics Committee et al 2018). All the original work by Santa Catalina community members presented here was translated to English by the authors. Finally, the calendar work was developed as a response to a request made by the indigenous communities of Santa Catalina and its surrounding area, who demanded our collaboration in developing printable material that could be used to record local activities throughout the year. This activity involved 49 participants from Santa Catalina and other communities, including 38 adults—some from the local government (5) and the school staff (3)—and 11 children. The process was published in detail in Spanish (Vila and Arzamendia 2016). Catalina community members presented here was translated to English by the authors. International Mountain Day International Mountain Day is also celebrated as National Mountain Day in Argentina and is therefore part of the educational agenda. To celebrate mountains, we climbed a nearby hill to observe the town of Santa Catalina in its mountainous environment and the springs that feed the river that crosses the town (Figure 2). In all interventions, we took notes and recorded dialogues between participants during the meetings and administered speciﬁc questionnaires. These were used to assess the participants’ experience in the assigned tasks and their perceptions, attitudes, and beliefs regarding the intervention. In the case of the environmental calendar, the speciﬁc questionnaires inquired about environmental management actions undertaken and noteworthy natural processes that occurred in each month of the year. Preliminary responses were discussed with all the participants so that only the most relevant, according to their worldview, remained in the calendar. In the activities involving children, oral exchanges were documented and drawn, and written outputs were compiled. These reﬂected perceptions (ability to see, hear, or become aware of something through the senses), attitudes (learned tendency to evaluate things in a certain way), and beliefs (acceptance that something exists). From the top of the hill, the teacher named the surrounding mountains. Back in the classroom, we discussed mountains and their importance. A crossword with the word ‘‘mountain’’ was proposed, and we wrote it down together on the board (Figure 3A). The students expressed concepts and elements that they associated with these environments, using words such as ‘‘beautiful,’’ ‘‘colorful,’’ ‘‘snow,’’ ‘‘tolas’’ (a local plant), ‘‘lizard,’’ ‘‘vicu~na,’’ and ‘‘cactus.’’ These results demonstrate that physical perceptions of the environment were a signiﬁcant part of the children’s description of ecosystem components (animals and plants), showing a positive attitude and valuation of these components. In terms of nature’s contribution to people, the children discovered intrinsic, Mountain Research and Development https://doi.org/10.1659/MRD-JOURNAL-D-20-00009.1 D42 MountainDevelopment FIGURE 3 (A) Crossword on mountains; (B) children in seventh grade of Santa Catalina school, with their mountain drawings. (Photos by Bibiana Vil´a) FIGURE 3 (A) Crossword on mountains; (B) children in seventh grade of Santa Catalina school, with their mountain drawings. (Photos by Bibiana Vil´a) instrumental and relational values of mountains They ice on top as well as snow and rai instrumental, and relational values of mountains. International Mountain Day They expressed that mountains are good because they have ‘‘good air’’ that is ‘‘nice to breathe,’’ they feed animals, they protect people, and they provide materials, such as rocks, minerals, and healing plants. The children clearly recognized that the instrumental, and relational values of mountains. They expressed that mountains are good because they have ‘‘good air’’ that is ‘‘nice to breathe,’’ they feed animals, they protect people, and they provide materials, such as rocks, minerals, and healing plants. The children clearly recognized that the ice on top, as well as snow and rain in the mountains, are the sources of all rivers in the area. The children told us about their beliefs that mountains are Apus, sacred entities that protect the people. They also expressed that mountains can be harmful when there are landslides or earthquakes, and https://doi.org/10.1659/MRD-JOURNAL-D-20-00009.1 Mountain Research and Development D43 FIGURE 4 Biodiversity collage made by children of 3 schools from the Laguna de los Pozuelos MAB Biosphere Reserve in Jujuy, northwest Argentina. MountainDevelopment MountainDevelopment FIGURE 4 Biodiversity collage made by children of 3 schools from the Laguna de los Pozuelos MAB Biosphere Reserve in Jujuy, northwest Argentina. when volcanoes are ‘‘on’’ (prendidos in Spanish). Two other activities were proposed, drawing the landscape (Figure 3B) and writing poetry, with amazing results (the Spanish originals and further examples are provided in Appendix S1, Supplemental material, https://doi.org/10.1659/MRD-JOURNAL- D-20-00009.1.S1): that lived in their immediate surroundings. As a group, we created a crossword with the letters of ‘‘biodiversity,’’ using local animals and plants to ﬁll in each letter. In the Rodeo school, located on the border of the lagoon, the perceived biodiversity was also remarkable. Children described 9 species of water birds, as well as other species, like shrimp, that are food for the ﬂamingos, and 5 plant species. In agreement with the children, the 3 habitats in the area (lagoon, grassland, and mountains) were drawn on a large sheet. Then, the children made a group collage by drawing animals and plants and pasting them onto their habitat. The purpose of the group collage was to visually capture the local biodiversity. Interestingly, the children included both wild and domestic animals (production and pets) in their drawings; they also added their school, people (mostly women), and a ‘‘Hunting Prohibited’’ sign, showing a clear conservationist attitude. International Mountain Day This reﬂects a holistic perception where biodiversity is part of the daily life of the school, interwoven in the children’s experience and imagination. The ﬁnal image was photographed, edited, and printed on paper by the National Research Council (CONICET) press. Each child received a copy of this poster, and posters were also handed out to different institutions in the area, where they are permanently exhibited (Figure 4). The mountain is very colorful/full of tolas and animals/It is very pretty, beautiful and with memories/On top it has very white snow/and from there the delicious water falls/and many people live [on it],/kind, good [people],/shepherdesses of sheep and llamas,/white and every color. (group creation, 7th grade) I like the mountains/because animals from/the dear Puna live there./Of melted snows,/the river falls like crystal clear water./Mountains of the Coyas/protected by the Pacha/with its llamas and its condors/and a yellow grass/that makes people fall in love. (E.R.C. and U.G.B., 7th grade) (E.R.C. and U.G.B., 7th grade) Celebrating biodiversity The 3 schools where the intervention was conducted are located in the Laguna de los Pozuelos MAB-UNESCO Biosphere Reserve. Biodiversity and the importance of plants and animals are therefore a regular theme in schoolwork. As for Mountain Day, the International Day of Biodiversity is recognized as the National Day of Biodiversity and has a place in the educational agenda. We presented similar initial pedagogical triggers in the 3 schools. Using colors as conversation prompts, we discussed the different living beings and grouped them by color, such as lizards, cacti, frogs, and watermelons for the ‘‘green’’ category and llamas, vicu~nas, armadillos (locally known as quirquinchos), potatoes, and kiwis for ‘‘brown.’’ The children from the Pozuelos school showed a remarkable perception of local biodiversity. They were able to identify 6 wild birds, toads, 7 wild and 2 domesticated mammals, 2 reptiles, and 2 insects We conducted a similar activity in Santa Catalina. Since the children enjoy writing poetry, they wrote the following poem on biodiversity (for Spanish original, see Appendix S2, Supplemental material, https://doi.org/10.1659/MRD-JOURNAL- D-20-00009.1.S1): Biodiversity Biodiversity fascinates me/because it has living beings/that are beautiful and useful/plants and animals./We warm up with the wool/of llamas and sheep/in my town of Santa Catalina./The plants heal me/when my belly hurts/I take mu~na./If I’m cold I build a ﬁre/with the tola and I get warm. Mountain Research and Development https://doi.org/10.1659/MRD-JOURNAL-D-20-00009.1 D44 MountainDevelopment You can knit with the wool of the vicu~na,/that must be taken care of./ The condor ﬂies in the sky to clear the way./The domestic animals,/dogs and cats are my ‘‘friends.’’ You can knit with the wool of the vicu~na,/that must be taken care of./ The condor ﬂies in the sky to clear the way./The domestic animals,/dogs and cats are my ‘‘friends.’’ Llama caravans In the EE llama caravan intervention, the children were asked to imagine an interview with a caravanner, to make up questions, and to think what the possible answers could be. They also took part in a creative writing exercise, where they had to describe their perceptions of what it would be like to be part of a llama caravan during a full day. Most of the children described exhausting, difﬁcult itineraries in their ‘‘imaginary caravan day.’’ They showed a negative attitude in relation to the caravanning activity for their own lives, expressing their concerns regarding this activity (tiring, boring). They also wrote poems about caravanning: (group creation, 7th grade) The poem shows that children have perceived the concept of nature’s contributions in an intuitive or experiential way: plants and animals, both domestic and wild, can be enjoyed and used—for company, medicine, and warmth—but they also need to be cared for and protected. Care and protection were the main attitudes towards nature that emerged from this poem. Puna Caravanner/You, who wake up early,/prepare your food/and load your llamas/You walk your trails/in the sunny hills to the east. (F.C., 7th grade) Environmental calendar The environmental calendar was the result of an intensive 2 day workshop that brought together researchers, The environmental calendar was the result of an intensive 2 day workshop that brought together researchers, representatives from 3 communities (Canchillas-Pe~nas Coloradas, Santa Catalina, Tolamayo), 4 indigenous associations (Athu Saphi, Aucarpina Chambi, Yurax Rumi, and La Cruz), the school community (students of polymodal school no. 7 and primary school no. 18), town authorities, and governmental staff (technicians from the rural extension agency) of the province of Jujuy. The Santa Catalina annual calendar is circular, sliced by month, and composed of 5 rings: climate, nature, festivities, and agronomic and livestock cycles (Figure 5). Each section was illustrated and colored by participants and schoolchildren. For simpliﬁcation, the climate ring was divided into two sections representing the warm and rainy summer season and the dry, cold, and very adverse winter. The calendar expressed a clear environmental perception and attitudes on environmental management using local knowledge, as well as the signiﬁcance of the beliefs in Pachamama and Christianity. Agricultural and animal husbandry activities in the winter period consist primarily of the protection of resources against the inclement weather and the preparation of the land for summer activities. This includes speciﬁc ceremonies to give ‘‘payment’’ to Pachamama (Mother Earth), or Pacha, a deity embodied by Earth and nature, who makes life possible and favors food production and fertility. In return, people are obliged to offer payment to Pacha in a ritual that takes place in midwinter, during August, as well as in other signiﬁcant cultural events. Students drew caravans (an example is provided in Appendix S3, Supplemental material, https://doi.org/10.1659/ MRD-JOURNAL-D-20-00009.1.S1) and decided to make a caravan collage, where each of them created their own llama. They also painted a llama on a small stone, which they pressed against drawing paper, thereby creating a rock painting (Vil´a in press). We developed teaching guidelines for future use in the school, since llama caravans make excellent models for classroom activities. Llama caravans can be used as a topic to explore in different school subjects, for instance, history, geography, biology, and mathematics, as well as music, since there are beautiful tunes dedicated to this traditional Andean practice, such as the gorgeous Camino de Llamas by Argentine folk musician U~na Ramos. Discussion In the Andean Altiplano, the pastoralist livelihood is crucial for the survival of small rural indigenous communities and constitutes the main traditional production activity (Leon- Velarde et al 2000). However, several practices, such as llama caravan travel, are critically threatened. The last recorded caravan arrival at Santa Catalina Fair was in 2018; this event was probably the last of its kind in the region. The Altiplano ecosystem, and consequently its biocultural contribution, is suffering degradation, traditional livelihoods are being threatened, and both the people and the environment are under signiﬁcant pressure from extractive activities, such as mining. This scenario calls for EE and the integration of science within the perspectives of local people, as socioenvironmental conservation and sustainability are based on practical experience and a mixture of knowledges (Gibbs et al 2008). The construction of this calendar represented a signiﬁcant, positive interaction between actors; Box 1 offers testimonies. In the process of building the calendar, the relationships among climate, environment, and the local cosmovision became clear. This was notable at the most climatically hostile time of year, the month of August, when Pachamama ‘‘opens’’ and has to be ritualistically fed (chaya) to foster her fertility. Certain activities occur at ﬁxed times during the year, such as the harvest from January to April, the slaughter of livestock in April, the preparation of the land in July, chaya in August, and sowing during the spring. The elaboration of a calendar provided a rich opportunity for the development of a dialogue between complementary points of view regarding ethnoveterinary and western veterinary medicine treatments. This bridged the gap between scientiﬁc and nonacademic knowledge in the joint search for ways to facilitate local ‘‘good living.’’ Although it is difﬁcult to evaluate the long-term effectiveness of EE interventions, the artwork that constituted the main output provides insight into the participants’ subjective environmental perceptions, attitudes, and beliefs, both during and after the intervention. In their poems about biodiversity, children were able to distinguish nature and nature’s contributions to people, such as wild and domestic animals for food, ﬁber, and pets, and plant resources that are used for ﬁrewood and healing. Discussion In the pioneer ethnographic work on Andean pastoralism in Peru by Flores Ochoa (1974, 1977), the duality between wild and domestic camelids was expressed as salqa–ullya (wild– Mountain Research and Development https://doi.org/10.1659/MRD-JOURNAL-D-20-00009.1 D45 MountainDevelopment FIGURE 5 Participatory calendar produced at Vicu~nas, Camelidos y Ambiente’s environmental calendar workshop in April 2016. The calendar classifies activities and events into 5 categories, 1 in each ring. These rings are (from the center) climate, nature, local festivities, agronomic activities, and livestock management. Each slice represents a month of the year, from January (enero) to December (diciembre). D46 https://doi.org/10.1659/MRD-JOURNAL-D-20-00009.1 Mountain Research and Development MountainDevelopment where the animals can live free under natural selection forces. In contrast, for a domestic llama, it is important to have human assistance, including sanitary care, herding practices, and housing. The human–animal relationship and associated practices are quite different, and this is the main reason that in the local kosmos people believe that vicu~nas are the herds of Pachamama and Coquena, while llamas are the herds of humans. p (M., member of the local municipal government) (M., member of the local municipal government) Seeing the printed calendars was really amazing, the whole town felt identiﬁed in the work they had done as a community, with the participation of everyone. Feeling identiﬁed as a community was the most important thing to me, because livestock, agro-livestock activities, and the celebrations so typical of Santa Catalina were prioritized in that calendar. And thank you, CONICET, for supporting these initiatives in communities so far away in the far north, this allows us to get to know ourselves and look at the calendar and identify as a people and a community. (E., headmistress of Santa Catalina primary school) domestic). This differentiation is also remarkable in our study. The interactions and work produced by the EE participants revealed traditional knowledge relating wild and domestic animals, expressed in a kosmos–corpus–praxis (KCP) complex, where beliefs, knowledge, and practices are indivisible (Toledo and Barrera-Bassols 2008). Wild and domestic camelids were clearly differentiated in the biodiversity collage, poems, and in the calendar, where llamas were included in the livestock ring, and vicu~nas were included in the nature ring. In November, two interesting changes were made to that calendar: Vicu~nas were included in the productive circle, as an animal captured and sheared in the chakus, and the llamas were moved to the festivities circle, as part of the Santa Catalina caravan fair. The mechanisms underlying the conservation of the wild and domestic camelids differ (Vil´a 2012). The main concern for wild vicu~nas is to prevent poaching and anthropogenic disturbance (ie being chased by dogs) and secure a space domestic). This differentiation is also remarkable in our study. The interactions and work produced by the EE participants revealed traditional knowledge relating wild and domestic animals, expressed in a kosmos–corpus–praxis (KCP) complex, where beliefs, knowledge, and practices are indivisible (Toledo and Barrera-Bassols 2008). Wild and domestic camelids were clearly differentiated in the biodiversity collage, poems, and in the calendar, where llamas were included in the livestock ring, and vicu~nas were included in the nature ring. In November, two interesting changes were made to that calendar: Vicu~nas were included in the productive circle, as an animal captured and sheared in the chakus, and the llamas were moved to the festivities circle, as part of the Santa Catalina caravan fair. The mechanisms underlying the conservation of the wild and domestic camelids differ (Vil´a 2012). BOX 1: Testimonies of participants in the development of the environmental calendar The calendar seemed super important to me because it is a work [that brought together] several communities, integrating the school is also very important for us, for the people for our region ... since there are many things we do not do together. Now, seeing the- interest of other institutions, it is good to give it to them. We are going to share this with the indigenous institutions ... Yesterday I communicated with the local radio show, they asked about this calendar because no community had ever done it, we are the ﬁrst to work on the calendar, in training, in workshops. Territorial participation of communities that can do things . . . this will help us to continue improving and keep working together, because it is hard for us to get together. While these activities and exercises of reﬂection alone might not stop the socioenvironmental deterioration of the Altiplano, we are conﬁdent that, at the very least, they could slow this process down and prepare communities for creative alternatives. In places where EE has been successfully implemented, local inhabitants are acutely aware that their camelids are valuable in many ways (including as scientiﬁc object of study) and that they have unique features (such as the single-domain antibodies in their blood). Although good news is, unfortunately, very scarce in the world of vertebrate conservation, investment in conservation measures has reduced the vulnerability and extinction risk of several species, particularly among ungulates (Waldron et al 2013; Hoffmann et al 2015). We believe that EE is a critical component of any comprehensive biodiversity conservation strategy and should not be dismissed as a secondary pursuit in conservation efforts, particularly in relation to camelids and pastoral lands. (F., president of the Athu Saphi Aboriginal Community) (F., president of the Athu Saphi Aboriginal Community) We thought it was good that our environmental calendar for Santa Catalina has be constructed. It is historical [meaning ‘‘signiﬁcant’’] that we unite everything: the culture, the customs that we have here in the puna. The work included the young and old, peasants, the whole community. This made it a more complex calendar and was beneﬁcial for us. Anyone who comes here can know the days when activities are carried out. It’s good to summon people, and for tourists and foreigners who can know what takes place here. p y p Today, South American camelids are valued as important animal genetic resources (FAO 2013). They have become progressively more relevant in the international agenda, giving them a visibility that could be a signiﬁcant driver of local development. EE can help to meet Aichi targets (CBD 2011) 12 and 13 regarding the protection of species and genetic diversity, with vicu~nas and domestic camelids as the main examples in the Altiplano. The United Nations General Assembly in 2017 declared 2024 as the International Year of Camelids: ‘‘Noting further that camelids constitute the main means of subsistence for millions of poor families that live in the most hostile ecosystems on the planet, and that they contribute to the ﬁght against hunger, the eradication of extreme poverty, the empowerment of women and the sustainable use of terrestrial ecosystems’’ and ‘‘Recalling the urgent need to raise public awareness of the importance of camelids for food security and ecosystem functions, and to promote actions that improve the management of camelids in order to contribute to the Sustainable Development Goals’’ (UN 2018). In addition, there is an international initiative promoted by the Food and Agriculture Organization of the United Nations to declare 2026 the International Year of Rangelands and Pastoralists. R E F E R E N C E S livelihoods and vicu~na management can meet most of the environmental goals set by the IPBES (2019): resource efﬁciency, circular and other economic models, environmental and social certiﬁcation of market chains, sustainable practices, and innovation. EE inﬂuences local people to conserve their territories, species, and habitats (Caro et al 2003). livelihoods and vicu~na management can meet most of the environmental goals set by the IPBES (2019): resource efﬁciency, circular and other economic models, Argueta Villamar A, Corona-M E, Alc ´antara-Salinas G, Santos-Fita D, Aldasoro Maya EM, Serrano Vel ´azquez R, Teutli Solano C, Astorga-Dom´ınguez M. 2012. Historia, situaci´on actual y perspectivas de la Etnozoolog´ıa en Mexico. Etnobiolog´ıa 10(1):15–30. environmental and social certiﬁcation of market chains, sustainable practices, and innovation. EE inﬂuences local people to conserve their territories, species, and habitats (Caro et al 2003). environmental and social certiﬁcation of market chains, sustainable practices, and innovation. EE inﬂuences local people to conserve their territories, species, and habitats (Caro et al 2003). Arzamendia Y, Baldo J, Rojo V, Samec C, Vil ´a BL. 2014. Manejo de vicu~nas silvestres en santa catalina, jujuy: Investigadores y pobladores en busqueda de la sustentabilidad y el buen vivir. Cuadernos del Instituto Nacional de Antropolog´ıa y Pensamiento Latinoamericano–Series Especiales 2 1(2):8–23. sustentabilidad y el buen vivir. Cuadernos del Instituto Nacional de Antropolog´ıa y Pensamiento Latinoamericano–Series Especiales 2 1(2):8–23. Several Sustainable Development Goals (SDGs) can be achieved in well-managed rangelands. An example is SDG 15 ‘‘Life on land’’: The sustainable use of terrestrial ecosystems via camelid management can help to reverse land degradation and biodiversity loss, and combat desertiﬁcation. Traditional knowledge innovations and practices of indigenous and local communities also help to reduce poverty, addressing SDG 1 and Aichi targets 2 and 18. Arzamendia Y, Baldo J, Vil ´a BL. 2012. Lineamientos para un plan de conservaci´on y uso sustentable de vicu~nas en Jujuy, Argentina. San Salvador de Jujuy, Argentina: Ediunju. Arzamendia Y, Bonacic C, Vil ´a BL. 2010. Behavioural and physiological consequences of capture for shearing of vicu~nas in Argentina. Applied Animal Behaviour Science 125(3–4):163–170. Arzamendia Y, Vil ´a BL. 2012. Effects of capture, shearing, and release on the ecology and behavior of wild vicu~nas. Journal of Wildlife Management 76(1):57– 64. Arzamendia Y, Vil ´a BL. 2015. Vicugna habitat use and interactions with domestic ungulates in Jujuy, northwest Argentina. Mammalia 79(3):267–278. R E F E R E N C E S Educational institutions that are deeply rooted in their surrounding community (Chapman et al 2002) can promote changes in the community’s attitude towards wildlife and nature (Uzzell 1999), which is necessary to achieve development goals. Creative, open, integrated EE that lets camelids be part of local school curricula helps to create a holistic and transformational education medium. This presents the environment in a new light, as an attractive and interesting component of the Andean ethos and network of socioecological relationships. Baldo J, Arzamendia Y, Vil ´a BL. 2013. La vicu~na. Manual para su conservaci´on y uso sustentable. 1st edition. Buenos Aires, Argentina: CONICET [Consejo Nacional de Investigaciones Cient´ıﬁcas y Tecnicas]. Boff L. 1996. Ecolog´ıa: Grito de la Tierra, grito de los pobres. San Pablo, Brazil: Editora Atica Baldo J, Arzamendia Y, Vil ´a BL. 2013. La vicu~na. Manual para su conservaci´on y uso sustentable. 1st edition. Buenos Aires, Argentina: CONICET [Consejo Nacional de Investigaciones Cient´ıﬁcas y Tecnicas]. Boff L. 1996. Ecolog´ıa: Grito de la Tierra, grito de los pobres. San Pablo, Brazil: Editora Atica. Cabrera AL. 1971. Fitogeograf´ıa de la republica Argentina. Bolet´ın de la Sociedad Argentina de Bot´anica 14:1–42. Caro T, Mulder MB, Moore M. 2003. Effects of conservation education on reasons to conserve biological diversity. Biological Conservation 114(1):143–152. CBD [Convention on Biological Diversity]. 2011. Strategic Plan 2011–2020: Aichi- Targets. https://www.cbd.int/sp/targets/; accessed on 3 March 2020. Chapman D, Barcikowski E, Sowah M, Gyamera E, Woode G. 2002. Do communities know best? Testing a premise of educational decentralization: Community members’ perceptions of their local schools in Ghana. International Journal of Educational Development 22(2):181–189. Some key messages can be derived from this work. Brief, intensive EE interventions with children can be very effective, especially on environmental ephemerides. Within a framework of acceptance and security in a playful environment, the children were actively engaged in the activities proposed by our team and were free to invent as they went along, enhancing their creativity. It was a different, thought-provoking experience within the familiar, comfortable setting of the school and its surroundings. Coppock DL, Fern ´andez-Gimenez M, Hiernaux P, Huber-Sannwald E, Schloeder C, Valdivia C, Arredondo JT, Jacobs M, Turin C, Turner M. 2017. Rangeland systems in developing nations: Conceptual advances and societal implications. In: Briske DD, editor. Rangeland Systems. Cham, Switzerland: Springer, pp 569–641. Dong S, Wen L, Liu S, Zhang X, Lassoie JP, Yi S, Li X, Li J, Li Y. 2011. R E F E R E N C E S Deﬁnici´on de ´areas de monta~na de la Republica Argentina. Buenos Aires, Argentina: Instituto Geogr´aﬁco Nacional. , g g IPBES [Intergovernmental Science-Policy Platform on Biodiversity and Ecosystem Services]. 2019. The Global Assessment Report on Biodiversity and Ecosystem Services: Summary for Policymakers [D´ıaz S, Settele J, Brond´ızio ES, Ngo HT, Gueze M, Agard J, Arneth A, Balvanera P, Brauman KA, Butchart SHM, et al, editors]. Bonn, Germany: IPBES Secretariat. https://ipbes.net/system/tdf/ipbes_global_ assessment_report_summary_for_policymakers.pdf? ﬁle¼1&type¼node&id¼35329; accessed on 2 April 2020. Leff E. 1999. La pedagog´ıa del ambiente: Educaci´on en ambiente para el desarrollo sustentable. Buenos Aires, Argentina: Confederaci´on de Trabajadores de la Educaci´on de la Republica Argentina (CTERA). IPBES [Intergovernmental Science-Policy Platform on Biodiversity and Ecosystem Services]. 2019. The Global Assessment Report on Biodiversity and Ecosystem Services: Summary for Policymakers [D´ıaz S, Settele J, Brond´ızio ES, Ngo HT, Gueze M, Agard J, Arneth A, Balvanera P, Brauman KA, Butchart SHM, et al, editors]. Bonn, Germany: IPBES Secretariat. https://ipbes.net/system/tdf/ipbes_global_ assessment_report_summary_for_policymakers.pdf? ﬁle¼1&type¼node&id¼35329; accessed on 2 April 2020. p (M., member of the local municipal government) The main concern for wild vicu~nas is to prevent poaching and anthropogenic disturbance (ie being chased by dogs) and secure a space However, recognition in international documents is meaningless if it is not supported by territorial actions that contribute to the empowerment of those responsible for the daily care of camelids. We consider EE to be a fundamental tool in achieving this goal. In situ EE helps local people, who are often part of ethnic minorities and are intrinsically less involved in national policy debates. If they are not properly informed, these people remain largely unaware of the importance of their camelids as a proﬁtable resource in the international market. In certain areas of the developed world, there is a lot of interest in incorporating these animals as ﬁber and antibody producers (Gegner 2012). Of course, EE alone is not enough, it needs support in line with the value of nature’s contributions to people. Pastoralist Mountain Research and Development https://doi.org/10.1659/MRD-JOURNAL-D-20-00009.1 D47 MountainDevelopment R E F E R E N C E S Vulnerability of worldwide pastoralism to global changes and interdisciplinary strategies for sustainable pastoralism. Ecology and Society 16(2):10. FAO [Food and Agriculture Organization of the United Nations]. 2013. In Vivo Conservation of Animal Genetic Resources. FAO Animal Production and Health Guidelines No. 14. Rome, Italy: FAO. http://www.fao.org/3/a-i3327e.pdf; accessed on 3 March 2020. g g Another key message for environmental policy and education is that, to facilitate the continuity of pastoralism, rural life must be valued from within the school. The relational aspects of people with camelids must have its own space, not only ‘‘folklorized’’ as something of the past but as a dynamic, living component that is essential to the environmental sustainability of the Andes. Flores Ochoa JA. 1974. Enqa, Enqaychu illa y Khuya Rumi: Aspectos m´agico- religiosos entre pastores. Journal de la Societe des Americanistes LXIII:246–261. Flores Ochoa JA. 1977. Pastores de Puna. Lima, Peru: Instituto de Estudios Peruanos. Flores Ochoa JA. 1974. Enqa, Enqaychu illa y Khuya Rumi: Aspectos m´agico- religiosos entre pastores. Journal de la Societe des Americanistes LXIII:246–261. Flores Ochoa JA. 1977. Pastores de Puna. Lima, Peru: Instituto de Estudios Peruanos. Freire P. 2003. El grito manso. Buenos Aires, Argentina: Siglo veintiuno editores. Gegner L. 2012. Llamas and Alpacas on the Farm. Butte, MT: ATTRA Sustainable Agriculture Program. https://www.uaex.edu/farm-ranch/animals-forages/other- livestock/llamas-alpacas/llamaalpaca_attrapub.pdf; accessed on 3 March 2020. Environmental education is a powerful tool for conservation and is the only way to translate SDG and Aichi targets from paper to the real, everyday life of people, in this case, Andean communities. ‘‘Valued from the outside, neglected from the inside’’ is a dangerous situation for any natural resource. Without active and informed local communities, the needed transformative change becomes an unattainable goal. In this sense, EE shows great potential, and it is a beautiful way to value the environment, to ‘‘feel- think’’ (sentipensar) about humans and nonhuman beings in the Altiplano. Gibbs J, Hunter M, Sterling EJ. 2008. Problem-Solving in Conservation Biology and Wildlife Management. Oxford, United Kingdom: Blackwell Publishing. Gutierrez F, Prado C. 1997. Ecopedagog´ıa y ciudadan´ıa planetaria. San Jose, Costa Rica: Editorial Pec-Heredia. Gutierrez F, Prado C. 1997. Ecopedagog´ıa y ciudadan´ıa planetaria. San Jose, Costa Rica: Editorial Pec-Heredia. Hoffmann M, Duckworth JW, Holmes K, Mallon DP, Rodrigues ASL, Stuart SN. 2015. The difference conservation makes to extinction risk of the world’s ungulates. Conservation Biology 29(5):1303–1313. IGN [Instituto Geogr ´aﬁco Nacional], SEGEMAR [Servicio Geol ´ogico Minero Argentino]. 2019. Mountain Research and Development A C K N O W L E D G M E N T S 2nd edition. Buenos Aires, Argentina: Vicu~nas, Camelidos y Ambiente (VICAM). Vil ´a BL. In press. The role of young people in the future of llama caravans to Santa Catalina. In: Clarkson P, Santoro C, editors. Global Caravans. Vol 1. Oxford, United Kingdom: Taylor & Francis. Vil ´a BL, Arzamendia Y. 2016. Construcci´on de un calendario ambiental participativo en Santa Catalina, Jujuy, Argentina. Etnobiolog´ıa 14(3):71–83. Vil ´a BL, Arzamendia Y, Rojo V. 2020. Vicu~nas (Vicugna vicugna), wild Andean altiplano camelids: Multiple valuation for their sustainable use and biocultural role in local communities. Case Studies in the Environment 4(1):1232692. https://doi.org/10.1525/cse.2020.1232692. A C K N O W L E D G M E N T S y Leff E. 1999. La pedagog´ıa del ambiente: Educaci´on en ambiente para el desarrollo sustentable. Buenos Aires, Argentina: Confederaci´on de Trabajadores de la Educaci´on de la Republica Argentina (CTERA). We thank the headmistresses and teachers of the Santa Catalina, Rodeo, and Pozuelos schools, especially Ely Funes, who always joins these initiatives. We are grateful to the Altiplano children who shared their art and knowledge. We thank Jorge Baldo, a VICAM/National University of Jujuy staff member and teacher of undergraduate students. We are grateful to the communities of Santa Catalina Canchillas, Tolamayo, Morritos, and La Cruz, and the indigenous associations Athu Saphi, Yurax Rumi, and Aucarpina Chambi, the Santa Catalina High School community, town authorities, and governmental staff of the livestock secretary, province of Jujuy. We also thank Rocio Julian, who helped us in the Biodiversity intervention. We are grateful for the valuable comments by 2 anonymous reviewers. p g ( ) Leon-Velarde C, Quiroz R, Zorogastua P, Tapia M. 2000. Sustainability concerns of livestock-based livelihoods in the Andes. In: Tulachan PM, Saleem MAM, Maki-Hokkonen J, Partap T, editors. Contribution of Livestock to Mountain Livelihoods: Research and Development Issues. 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New York, NY: UN General Assembly. http:// undocs.org/A/RES/72/210; accessed on 5 October 2020. Toledo VM, Barrera-Bassols N. 2008. La memoria biocultural: La importancia ecol´ogica de las sabidur´ıas tradicionales. Barcelona, Spain: Icaria Editorial. g p UN [United Nations]. 2018. Resolution Adopted by the General Assembly on 20 December 2017. A/RES/72/210. New York, NY: UN General Assembly. http:// undocs.org/A/RES/72/210; accessed on 5 October 2020. Vil ´a BL, Wawrzyk AC, Garcia Gomez J. 2006. La educaci´on ambiental en el proyecto MACS. In: Vil´a BL, editor. Investigaci´on, conservaci´on y manejo de vicu~nas. Buenos Aires, Argentina: Proyecto Manejo de Camelidos Silvestres (MACS) Argentina, pp 175–190. December 2017. A/RES/72/210. New York, NY: UN General Assembly. http:// undocs.org/A/RES/72/210; accessed on 5 October 2020. UNESCO [United Nations Educational, Scientiﬁc and Cultural Organization]. UNESCO [United Nations Educational, Scientiﬁc and Cultural Organization]. 1977 Intergovernmental Conference on Environmental Education Tbilisi USSR UNESCO [United Nations Educational, Scientiﬁc and Cultural Organization]. 1977. Intergovernmental Conference on Environmental Education, Tbilisi, USSR, Final Report. Paris, France: UNESCO. Waldron A, Mooers AO, Miller DC, Nibbelink N, Redding D, Kuhn TS, Roberts JT, Gittleman JL. 2013. Targeting global conservation funding to limit immediate biodiversity declines. Proceedings of the National Academy of Sciences of the United States of America 110(29):12144–12148. Uzzell D. 1999. Education for environmental action in the community: New roles and relationships. Cambridge Journal of Education 29(3):397–413. https://doi. org/10.1080/0305764990290309. Wawrzyk AC, Vil ´a BL. 2013. Dinamica de pastoreo en dos comunidades de la Puna de Jujuy, Argentina: Lagunillas del Farallon y Suripujio. Revista de Antropolog´ıa Chilena 45(2):349–362. g Vil ´a BL. 2012. Camelidos Sudamericanos. Buenos Aires, Argentina: EUDEBA. Vila BL. 2012. Camelidos Sudamericanos. Buenos Aires, Argentina: EUDEBA. Vil ´a BL. 2014. Una aproximaci´on a la etnozoolog´ıa de los camelidos andinos. Etnoecol´ogica 10(2):43–58. Vila BL. 2012. Camelidos Sudamericanos. Buenos Aires, Argentina: EUDEBA. Vil ´a BL. 2014. Una aproximaci´on a la etnozoolog´ıa de los camelidos andinos. Etnoecol´ogica 10(2):43–58. Vil ´a BL. 2018. On the brink of extinction: Llama caravans arriving at the Santa Catalina Fair, Jujuy, Argentina. Journal of Ethnobiology 38(3):372–389. Vil ´a BL. 2019. Caravanas de las alturas. Found at: https://doi.org/10.1659/MRD-JOURNAL-D-20- 00009.1.S1. Mountain Research and Development View publication stats View publication stats Supplemental material APPENDIX S1 Mountain poems in Spanish. APPENDIX S2 Biodiversity poem in Spanish. APPENDIX S3 Drawing about living in the mountains. Found at: https://doi.org/10.1659/MRD-JOURNAL-D-20- 00009.1.S1. D49 D49 https://doi.org/10.1659/MRD-JOURNAL-D-20-00009.1 Mountain Research and Development
https://openalex.org/W4256362709	https://www.uco.es/ucopress/az/index.php/az/article/download/3609/2239	Portuguese	null	Lomos de cerdo Alentejano y blanco ahumados vs curados	Archivos de zootecnia	2,018	cc-by-sa	4,520	Palavras chave adicionais Este estudo pretende avaliar as características dos dois tipos de lombo de porco, Alentejano e Branco, processados com tecnologias distintas, a fumagem, apreciada em Portugal, ao contrário dos restantes países da Europa que preferem produtos que utilizam exclusivamente a secagem para obter um produto curado. Utilizaram-se 6 porcos de Raça Alentejana, cruzados a 50% com Duroc, criados em regime extensivo, classificados como classe 2 pelo decreto-lei nº95/2014 e 6 porcos cruzados brancos, criados em regime intensivo. Com peso após o abate 120 kg e 100 kg., respectivamente. O processa mento dos lombos decorreu na Salsicharia Estremocense. A avaliação entre lombos de porco de raça “Alentejana” e brancos cruzados, fumados e secos, foi realizada com base em análises físico-químicas, (pH, aW, CRA, Humidade, Cinza, Gordura total, Proteína bruta, Fibra, ABVT e TBA), e sensorial (prova QDA). O pH, Humidade, CRA não apresentam diferenças significativas, entre os lombos elaborados com diferentes tecnologias, assim como em relação ao valor nutricional. O perfil lipídico também não apresentou diferenças significativas, nem quanto à raça nem quanto à tecnologia, no entanto devemos realçar que os lombos de porcos de raça “Alentejana” apresentam um conteúdo da gordura da ordem dos 13% enquanto os lombos de porco branco cruzado apresentam valores de 8%. O Azoto Básico Volátil Total apresenta teor mais elevado nos lombos de porco Alentejano em ambas as tecnologias, o TBA varia ao longo do processamento, mas não distingue as duas tecnologias. Verificam-se diferenças significativas entre raças e tecnologias na cor e sabor amargo. A análise sensorial apresenta maior grau de aceitação pelos provadores nos produtos fumados em ambas as raças. Análise Sensorial Ácidos gordos. TBA. NNP. Análise Sensorial Ácidos gordos. TBA. NNP. Information Cronología del artículo. Recibido/Received: 21.01.2016 Aceptado/Accepted: 08.07.2017 On-line: 15.01.2018 Correspondencia a los autores/Contact e-mail: sfpalma@ipbeja.pt Cronología del artículo. Recibido/Received: 21.01.2016 Aceptado/Accepted: 08.07.2017 On-line: 15.01.2018 Correspondencia a los autores/Contact e-mail: sfpalma@ipbeja.pt SUMMARY Additional keywords Sensory analysis. Fatty acids. TBARS. NPN. This study aim is to evaluate the characteristics of both types of pig loins from Alentejano and White pigs, manufactured with distinguish processes, taking in account that smoked technology is most appreciated in Portugal, than in the other European countries, whom preferences stands in cured pro ducts by drying technology. In the study, 6 Alentejano pigs, crossbred at 50% with Duroc and reared in free-range system, classified as class 2 by decree-law nº95/2014, and 6 crossbreed White pigs, reared in intensive system were used, weighting 120 kg and 100 kg, respectively, after slaughtering. The loins processing was in “Salsicharia Estremocense Lda”. The evaluation between Alentejano breed and White crossbreed loins, smoked and dry-cured, were made physical and chemical analysis (pH, aW, WHC, moisture content, ash, fat, protein, TBARS, TVB-N) and sensory analysis (QDA).No significant differences were showed in pH, moisture and WHC in the loins processed by the two technologies, as well as in the nutritional value. Lipid profile also had not shown significant differences, nor even due to the breed neither to the technology, however it should be highlighted that the Alentejano loins had an amount of 13% fat while the Exotic loins had shown 8% fat content. The TVB-N content was higher in Alentejano loins, for both technologies, and TBARS varies along the processing, however this parameter didn’t distinguish the different technologies.There were significant differences between the breeds and the technologies in sensory color and bitter taste. The sensory analysis heightens, in the two breeds, the smoked technology, and thus the panellists overall acceptability. Smoked vs Dry-cured Alentejano and white pig loins Silva, B.; Carvalho, M. J. and Ferro Palma, S.@ DTCA, ESA, Instituto Politécnico de Beja. Portugal. RESUMO Palavras chave adicionais Análise Sensorial Ácidos gordos. TBA. NNP. POSTER Archivos de Zootecnia Journal website: https://www.uco.es/ucopress/az/index.php/az/ POSTER Archivos de Zootecnia Journal website: https://www.uco.es/ucopress/az/index.php/az/ INTRODUCTION environmental conditions. These products respect the ecosystems, its biodiversity and the genetic heritage. Along time these products achieve a highlight position in consumer’s preference, which are in disposition to pay a fair price for the quality associated to an origin, to a way of production and to a unique sensory charac teristic (Ferro Palma, 2006). The Celtic learns the art to manufacture cured meat products and to store pork meat with the romans, and from so on this philosophy stills benefits the Por tuguese. It’s was also this civilization the responsi ble for meat industry development, with the early slaughterhouses. Arabs were responsible for spices introduction in cured meat products (Mendonça, 2012). These traditional products contributed to rural develo pment and to its valorization, conserving the natural Arch. Zootec. PROCEEDINGS IX Simposio Internacional sobre el Cerdo Mediterráneo: 221-225. 2018 MATERIAL AND METHODS MATERIAL AND METHODS Arch. Zootec. PROCEEDINGS IX Simposio Internacional sobre el Cerdo Mediterráneo: 221-225. 2018 rch. Zootec. PROCEEDINGS IX Simposio Internacional sobre el Cerdo Mediterráneo: 221-225. 2018 SILVA, CARVALHO AND FERRO PALMA In the study, 6 Alentejano pigs, crossbred at 50% x Duroc and reared in free-range system, classified as class 2 by decree-law nº95/2014, and 16 crossbred white pigs, reared in intensive system were used, weighting 120 kg and 100 kg, respectively, after slaughtering in Slaughterhouse of Alto Alentejo (Sousel). After sam pling, and in order to compare both Alentejano and crossbred white pigs breeds loins, smoked and dry- cured, physical, chemical and sensory analysis were made at 0, 40, 120 and 180 days. However sensory analysis was only made at 120 and 180 days after ma turation. The pH was determined by NP 3441:2008, moisture content by NP 1614:1979. To determine Wa ter Hold Capacity (WHC) was used the pistometric method (Grau & Hamm, 1953), the TVB-N was deter mined by NP 1848:1987, NNP by De Ketalere (1974) and the TBARS by the method describe by Tarladgis et al. (1960), fat content by NP 1613:1979 and fatty acids by gas chromatography GC-FID. In the sensory analysis was done a Quantitative Descriptive Analysis (QDA) with a scale from 1 to 9 scores. All physical, chemical and sensory parameters were statistically analyzed by SPSS Statistics version 23, and the means, standard deviation were considered by ANOVA analy sis with a significance level 0.05 and using comparison means by Scheffé test. obtained in our study. Thus the pH along the loins pro cessing with the different technologies did not presen ted significant differences between the technologies in the same breed; however, it had significant differences between the breeds. The TVB-N content dry matter (dm) in Alenteja no loins range from 9.32 mg/g to 8.89 mg/g in the smoked, and 9.97 mg/g to 11.13 mg/g in the dried one. In the White pigs’ loins, the values range from 10.12 mg/g to 12.95 mg/g for the smoked and 13.57 mg/g to 13.67 mg/g in the dried. RESULTS AND DISCUSSION The pH values in Alentejano pig range from 6.1 to 5.86 for the smoked one, and 6.01 to 5.94 for the dried one. For the white pig loin, the values range from 5.49 to 5.69 for the smoked and 5.49 to 5.55 for the dried one. This values are in agreement with a case study about the influence of the processing method on the Alentejano cured loin quality, where the values obtai ned were 6.28 to 5.71 along the smoking process, and the dried ones had pH from 6.28 to 5.76 (Ferro Palma, 2006). Another study (Lorenzo et al. 2013), obtained pH of 5.90 at 60 days, which is in accordance to those Figure 1. mg TVB-N/100g loin sample: PF (Smoked Alentejano Pig); PS (Dry-cured Alentejano Pig); BF (Smoked White Pig); e BS (Dry-cured White Pig). (P≤0,05) (mg ABVT/100g, em lombos curados nas diferentes etapas de elaboração PF (Porco Alentejano Fumado); PS (Porco Alentejano Seco); BF (Porco branco Fumado); e BS (Porco branco Seco). (P≤0,05). Fat content values are in accordance with those found in other studies related with the influence of the processing method on the Alentejano cured loin quality, where the values obtained in dry basis were between 13.15% to 13.87% during smoking process, and 13.15% to 15.90% for the dried loins (Ferro Palma, 2006). Lorenzo et al. (2013) had 3.41% of fat content at 60 days, which is in accordance with this study. The total fat content values in Alentejano pig loins range from 13.43% to 10.92% for the smoked, and from 6.65% to 12.46 % for the dried one. In the white pig loins, the values were from 8.30% to 5.48 % for the smoked and 7.28 % to 5.85% in the dried loins. The fatty acids values at 120 days in both techno logies are presented in Table I, which show that there weren’t significant differences in the majority of fatty acids in the products from the different technologies. Only two fatty acids presented significant differences, the oleic acid and linolenic acid. The oleic acid values are in accordance with a study related with the influen Figure 1. mg TVB-N/100g loin sample: PF (Smoked Alentejano Pig); PS (Dry-cured Alentejano Pig); BF (Smoked White Pig); e BS (Dry-cured White Pig). MATERIAL AND METHODS This values of TVB- N are in accordance with those found in other studies related with the influence of the processing method on the Alentejano cured loin quality, where the values obtained were 13,98 mg/g (dm) and 15.77 mg/g (dm) along the smoking stage and 13.98 mg/g (dm) to 19.92 mg/g (dm) for the dried loins TVB-N values in Alentejano pig loins range from 11.27 mg/100g to 207.32 mg/100g for the smoked and 11.27 mg/100g to 248.66 mg/100g for the dried loins. In the meanwhile, for the white pig loin the values rage from 10.76 mg/100g to 75.70 mg/100g for the smoked loins and from 10.76 mg/100g to 124.97 mg/100g for the dried loins (Figure 1). Non-Protein Nitrogen (NPN) data in Alentejano pig loins were between 6.09 mg/g and 5.94 mg/g for the smoked loins and 6.33 mg/g to 7.18 mg/g for the dried ones. In the white pig loins, the data were 5.83 mg/g and 8.00 mg/g for the smoked loins and 7.70 mg/g to 7.82 mg/g for the dried ones. In terms of data expres sed in dry matter, the Alentejano pig loins had values from 9.32 mg/g and 8.89 mg/g for the smoked loins (PF), and between 9.97 mg/g and 11.13 mg/g for the dried loins (PS). For the white pig loins, the values range from 10.12 mg/g to 12.95 mg/g for the smoked loins (BF) and 13.57 mg/g to 13.67 mg/g in the dried ones (BS). There were significant differences between the two breeds in the values expressed in dry matter at 40 and 120 days. This NPN (dm) were in accordan ce with the values found in studies related with the influence of the processing system in Alentejano cured loin quality (Ferro Palma, 2006). RESULTS AND DISCUSSION os distintos índices indicam diferentes níveis de significância (P≤0.05); as letras (a.b.c…) indicam diferenças entre colunas e os números (1.2.3…) diferenças entre filas Os produtos expressam-se por abreviaturas: PF (Porco Alentejano Fumado); PS (Porco Alentejano Seco); BF (Porco Branco Fumado); e BS (Porco Branco Seco) differences between columns The products expressed by PF (Smoked Alentejano Pig); PS (Dry-cured Alentejano Pig); BF (Smoked White Pig); e BS (Dry-cured White Pig) Os valores expressos em média ± o erro padrão. os distintos índices indicam diferentes níveis de significância (P≤0.05); as letras (a.b.c…) indicam diferenças entre colunas e os números (1.2.3…) diferenças entre filas Os produtos expressam-se por abreviaturas: PF (Porco Alentejano Fumado); PS (Porco Alentejano Seco); BF (Porco Branco Fumado); e BS (Porco Branco Seco) “White”, which are in consonance with those obtained in this study Figure 2. % Fatty acid at 120 days in dry-cured loins: PF (Smoked Alentejano Pig); PS (Dry-cured Alentejano Pig); BF (Smoked White Pig); e BS (Dry-cured White Pig). (P≤0,05) (% de ácidos gordos aos 120 dias nos lombos cu rados: PF (Porco Alentejano Fumado); PS (Porco Alentejano Seco); BF (Porco branco Fumado); e BS (Porco branco Seco) (P≤0,05). ce of the breed and feed in the intramuscular fat cha racteristics and content in Iberian pig loin, which had oleic acid values of 58.08% (Canillas, 2006); and other study obtained oleic acid between 52.13 % and 50.68 % Toribio, (2011), which is in accordance with this study. The linolenic acid values are in accord with a study related with the influence of the breed and feed in the intramuscular fat characteristics and content in Iberian pig loin, which had linolenic acid values of 0.21% to 0.27% (Canillas, 2006). The fatty acid in major amount in the all four pro ducts is the C18:1cis-9, with a percentage from 46.37% e 42.81%, followed by the C16:0, with amounts of 26.53% and 25.87%. The MUFA (Figure 2) are in higher amount in the loins, and therefore the MUFA in Alentejano pig loin are 54.45 % for the smoked and for the dried. In the white pig loin, the values are 51.75% for the smoked loins and 52.85% for the dried ones. Arch. Zootec. PROCEEDINGS IX Simposio Internacional sobre el Cerdo Mediterráneo, p. 223, 2018. RESULTS AND DISCUSSION (P≤0,05) (mg ABVT/100g, em lombos curados nas diferentes etapas de elaboração PF (Porco Alentejano Fumado); PS (Porco Alentejano Seco); BF (Porco branco Fumado); e BS (Porco branco Seco). (P≤0,05). EEDINGS IX Simposio Internacional sobre el Cerdo Mediterráneo, p. 222, 2018. Arch. Zootec. PROCEEDINGS IX Simposio Internacional sobre el Cerdo Mediterráneo, p. 222, 2018. LOMBO DE PORCO DE RAÇA ALENTEJANA E BRANCO Table I. Fatty acids percentage at 120 days in smoked and dried loins (g/100 g) (Percentagem de ácidos gordos aos 120 dias lombos fumados e secos (g/100 g). PF PS BF BS C12:0 0.08(0.01)a 0.08 (0.01) a 0.07 (0.00) a 0.07 (0.01) a C14:0 1.33(0.06)a 1.27 (0.13) a 1.18 (0.04) a 1.24 (0.08) a C16:0 26.36(1.46)a 25.87 (0.88) a 26.19 (0.21) a 26.53 (0.49) a C16:1cis-9 3.64(0.45)a 3.21 (0.64) a 2.85 (0.00) a 3.54 (0.18) a C17:0 0.13(0.03)a 0.18 (0.02) a 0.22 (0.03) a 0.19 (0.04) a C17:1cis-9 0.16(0.04)a 0.16 (0.01) a 0.21 (0.01) a 0.20 (0.04) a C18:0 11.81(1.60)a 13.54 (0.88) a 12.93 (0.02) a 11.92 (0.13) a C18:1cis-9 46.37(0.14)a 42.81 (1.24) b 45.03 (0.56) ab 44.90 (0.29) ab C18:1cis-11 4.27(0.48)a 3.93 (0.45) a 3.70 (0.05) a 4.21 (0.08) a C18:2ŋ-6 3.88(1.67)a 6.11 (2.11) a 5.08 (0.69) a 5.02 (0.63) a C18:3ŋ-3 0.73(0.06)ab 0.69 (0.06) b 0.99 (0.01) a 0.75 (0.11) ab C20:0 0.18(0.01)a 0.21 (0.01) a 0.22 (0.01) a 0.17 (0.04) a C20:3ŋ-6 0.09(0.04)a 0.16 (0.04) a 0.14 (0.02) a 0.14 (0.03) a C20:4ŋ-6 0.77(0.56)a 1.50 (0.27) a 0.88 (0.04) a 0.87 (0.22) a C22:0 0.03(0.01)a 0.04 (0.00) a 0.07 (0.02) a 0.04 (0.01) a C22:5ŋ-3 0.05(0.04)a 0.12 (0.04) a 0.08 (0.01) a 0.09 (0.03) a outros AG 0.14(0.04)a 0.17 (0.02) a 0.20 (0.04) a 0.16 (0.01) a The values expressed in means ± standard deviation. distinct index different levels of significance (P≤0.05); the letters (a.b.c. ) indicate differences between columns The products expressed by PF (Smoked Alentejano Pig); PS (Dry-cured Alentejano Pig); BF (Smoked White Pig); e BS (Dry-cured White Pig) Os valores expressos em média ± o erro padrão. RESULTS AND DISCUSSION Serna, (2013) had made a comparison between the “Murciano” pig and the “White”pig, obtaining the values of 62.02% for the “Murciano”, and 57.68% for the “White” and values of PUFA 4.7% for the “Murciano”, and 9.32% for the Figure 2. % Fatty acid at 120 days in dry-cured loins: PF (Smoked Alentejano Pig); PS (Dry-cured Alentejano Pig); BF (Smoked White Pig); e BS (Dry-cured White Pig). (P≤0,05) (% de ácidos gordos aos 120 dias nos lombos cu rados: PF (Porco Alentejano Fumado); PS (Porco Alentejano Seco); BF (Porco branco Fumado); e BS (Porco branco Seco) (P≤0,05). Arch. Zootec. PROCEEDINGS IX Simposio Internacional sobre el Cerdo Mediterráneo, p. 223, 2018. SILVA, CARVALHO AND FERRO PALMA Figure 4. Loins Overall acceptability: PF (Smoked Alentejano Pig); PS (Dry-cured Alentejano Pig); BF (Smoked White Pig); e BS (Dry-cured White Pig) (P≤0,05) (Aceitabilidade global dos lombos: PF (Porco Alentejano Fumado); PS (Porco Alentejano Seco); BF (Porco branco Fumado); e BS (Porco branco Seco). (P≤0,05). Related with TBARS in dry matter, along the sta ges of both processing technologies, this parameter didn’t distinguish the different technologies. The TBRAS values in Alentejano pig loins ranges from 0,38 µgMDA/g to 0.21 µgMDA/g for the smoked one and 0.38 µgMDA/g to 0.14 µgMDA/g for the dried loins. In the white breed, for the smoked loins the TBRAS va lues were between 0,36 µgMDA/g and 0.15 µgMDA/g and for the dried loins between 0.36 µgMDA/g and 0.19 µgMDA/g (Figure 3). The TBRAS data in dry matter are in accord with values foun in studies related with the influence of the processing method on the Alentejano cured loin quality, which obtained values from 1.85 µgMDA/g and 0.97 µgMDA/g during smoking process, and from 1.85 µgMDA/g and 5.58 µgMDA/g for the dried loins (Ferro Palma, 2006). Lorenzo et al. (2013)., obtained TBARS of 1.13 µgMDA/g at 60 days, which fact is in accordance with the values obtained in this study. Figure 4. Loins Overall acceptability: PF (Smoked Alentejano Pig); PS (Dry-cured Alentejano Pig); BF (Smoked White Pig); e BS (Dry-cured White Pig) (P≤0,05) (Aceitabilidade global dos lombos: PF (Porco Alentejano Fumado); PS (Porco Alentejano Seco); BF (Porco branco Fumado); e BS (Porco branco Seco). (P≤0,05). The TBARS value is superior at 40 days, and decrea ses along the process, possibly due to manolaldheide among other volatile compounds characteristics of the aroma and taste. RESULTS AND DISCUSSION Indeed these results were in consonance with the previous data in fat con tent, which were significantly higher in the Alentejano loins (40.72% and 31.51% for the smoked, and 18.58% and 36.55 % for the dried loins, and in the White loins, the values range from 13.91% to 9.25% for the smoked ones and from 12.80% to 10.21% in the dried ones).In a study concerning the sensory characteristics of cured Iberian pigs related with the crossbreed and the reared systems influence, had obtained scores from 1.83 to 2.27 (Ventanas, et al., 2006), which are in accordance with those obtained in this study. the flavor intensity was scored from 5.70 and 5.60 for the smoked and 6.33 and 6.00 for the dried, however in the study related with the influence of processing systems in Alentejano pig loins quality, for the smoked the score was 4.19 and on the dried loins was lower scored 3.97 (Ferro Palma, 2006). In the White loins the scores were 3.93 and 5.14 for the smoked ones and 5.43 in the dried loins were 5.14 (Figure 4). Smoke flavor in Alentejano pig loins were between 3.30 and 6.00 for the smoked and 5.00 for the dried, and in the White breed were 3.64 and 3.93 for the smoked ones and in the dried loins were 4.00 and 3.71. Rancid flavor in Alentejano pig loins were between 2.50 and 1.60 for the smoked loins and scored 1.92 and 2.08 for the dried ones, while on the White breed loins were lower as it was expected, with scores from 1.70 and 2.00 for the smoked ones, and 1.29 to 1.36 in the dried ones. Indeed these results were in consonance with the previous data in fat con tent, which were significantly higher in the Alentejano loins (40.72% and 31.51% for the smoked, and 18.58% and 36.55 % for the dried loins, and in the White loins, the values range from 13.91% to 9.25% for the smoked ones and from 12.80% to 10.21% in the dried ones).In a study concerning the sensory characteristics of cured Iberian pigs related with the crossbreed and the reared systems influence, had obtained scores from 1.83 to 2.27 (Ventanas, et al., 2006), which are in accordance with those obtained in this study. Figure 3. RESULTS AND DISCUSSION Color sensory evaluation in Alentejano pig loin sco res from 6.90 to 7.30 for the smoked and 7.42 to 6.92 for the dried loins, which were quite different from the values found in a study related with the influence of processing system in the Alentejano cured loin quality, since in these study the score for this parameter was 3.92 along the smoking process and 5.47 for dried loins (Ferro Palma, 2006). However, In the White breed, the scores reach from 3.64 to 4.21 for the smoked loins and 2.43 to 3.57 in the dried ones. Marbling in Alentejano pig loins were between 5.20 and 5.50 for the smoked and 4.17 to 4.67 for the dried, while in the White loins the scores were 5.00 and 5.50 for the smoked ones and 5.07 in the dried loins were 4.43. In Alentejano pig loins the flavor intensity was scored from 5.70 and 5.60 for the smoked and 6.33 and 6.00 for the dried, however in the study related with the influence of processing systems in Alentejano pig loins quality, for the smoked the score was 4.19 and on the dried loins was lower scored 3.97 (Ferro Palma, 2006). In the White loins the scores were 3.93 and 5.14 for the smoked ones and 5.43 in the dried loins were 5.14 (Figure 4). Smoke flavor in Alentejano pig loins were between 3.30 and 6.00 for the smoked and 5.00 for the dried, and in the White breed were 3.64 and 3.93 for the smoked ones and in the dried loins were 4.00 and 3.71. Rancid flavor in Alentejano pig loins were between 2.50 and 1.60 for the smoked loins and scored 1.92 and 2.08 for the dried ones, while on the White breed loins were lower as it was expected, with scores from 1.70 and 2.00 for the smoked ones, and 1.29 to 1.36 in the dried ones. RESULTS AND DISCUSSION Determinação do teor de azoto básico volátil total, Método das células de Conway’, Ed. 2, pp. 6. ACKOWLEGMENTS The authors are grateful to Salsicharia Estremocense The authors are grateful to Salsicharia Estremocense NP 3441 2008, ‘Norma Portuguesa, Carnes, derivados e produtos cárneos. Determinação do pH’, Ed. 1, pp.6. NP 3441 2008, ‘Norma Portuguesa, Carnes, derivados e produtos cárneos. Determinação do pH’, Ed. 1, pp.6. RESULTS AND DISCUSSION TBARS - µg MDA/g of loin sample dry mat ter: PF (Smoked Alentejano Pig); PS (Dry-cured Alen tejano Pig); BF (Smoked White Pig); e BS (Dry-cured White Pig). (P≤0,05) (TBA - µg MDA /g de resíduo seco de lombos curados fumados e lombos curados secos nos diferentes dias de elaboração PF (Porco Alentejano Fumado); PS (Porco Alentejano Seco); BF (Porco branco Fumado); e BS (Porco branco Seco). (P≤0,05) In the overall acceptability, the Alentejano pig loins scored 4.90 to 6.70 for the smoked loins, and 4.42 to 6.00 in the dried loins, while in the White pig loins, the score was 6.57 and 6.07 for the smoked ones and 5.07 to 6.07 for the dried loins. Figure 3. TBARS - µg MDA/g of loin sample dry mat ter: PF (Smoked Alentejano Pig); PS (Dry-cured Alen tejano Pig); BF (Smoked White Pig); e BS (Dry-cured White Pig). (P≤0,05) (TBA - µg MDA /g de resíduo seco de lombos curados fumados e lombos curados secos nos diferentes dias de elaboração PF (Porco Alentejano Fumado); PS (Porco Alentejano Seco); BF (Porco branco Fumado); e BS (Porco branco Seco). (P≤0,05) In conclusion, the main three goals of this study were achieved when comparing two technologies in two pigs breeds. Thus with this study it’s possible to conclude that Alentejano smoked loin and Alentejano dried loin presented physical, chemical, sensory and rch. Zootec. PROCEEDINGS IX Simposio Internacional sobre el Cerdo Mediterráneo, p. 224, 2018. LOMBO DE PORCO DE RAÇA ALENTEJANA E BRANCO Mendonça, JM, 2012, ‘Aplicação da Tecnologia de Alta Pressão na Con sevação de um Produto Cárneo Transformando em Portugal’, Lisboa: Faculdade de Medicina Veterinária da Universidade Técnica de Lisboa. NP 1613, 1979, ‘Norma Portuguesa, Carnes, derivados e produtos cárneos. Determinação do teor de matéria gorda total’, Ed. 1, pp.2. NP 1614 1979, ‘Norma Portuguesa, Carnes, derivados e produtos cárneos. Determinação da humidade’, Ed. 1, pp. 2. textural characteristics very similar, and the main di fference was related with smoke and its acceptability by the panelists. The sensory evaluation of color and hardness were the only sensory parameters with signi ficant differences either between breeds and technolo gies. The sensory analysis heightens, in the two breeds, the smoked technology, and thus the panellists overall acceptability in both smoked products with 180 days and 120 days. NP 1848 1987,’Norma Portuguesa, Carnes, derivados e produtos cárneos. Arch. Zootec. PROCEEDINGS IX Simposio Internacional sobre el Cerdo Mediterráneo, p. 225, 2018. BIBLIOGRAPHY Serna, ES, 2013, Tecnología y caracterización de productos cárnicos curados obtenidos a partir de cerdo Chato Murciano’, Murcia: Uni versidad Católica San Antonio. Serna, ES, 2013, Tecnología y caracterización de productos cárnicos curados obtenidos a partir de cerdo Chato Murciano’, Murcia: Uni versidad Católica San Antonio. Canillas, SV 2006, ‘Influencia de la Raza y de la Alimentación sobre el contenido y características de la grasa intramuscular del lomo de cerdo Ibérico: efecto sobre parámetros determinantes de la calidad’, Cáceres: Universidad de Extremadura. Canillas, SV 2006, ‘Influencia de la Raza y de la Alimentación sobre el contenido y características de la grasa intramuscular del lomo de cerdo Ibérico: efecto sobre parámetros determinantes de la calidad’, Cáceres: Universidad de Extremadura. Canillas, SV 2006, ‘Influencia de la Raza y de la Alimentación sobre el contenido y características de la grasa intramuscular del lomo de cerdo Ibérico: efecto sobre parámetros determinantes de la calidad’, Cáceres: Universidad de Extremadura. Tarladgis, BG, Watts, BM, Younathan, MT & Dugan, LR Jr 1960, ‘A dis tillation method for the quantitative determination of malonaldehyde in rancid foods’. Journal of the American Oil Chemists’ Society, vol. 37, pp. 44-48. De Ketalere, A, Demeyer, D, Vandekerckhove, P & Vervaeke, I 1974, ‘Stoichimetry of carbohydrate fermentation during dry sausage ripi ning’, Journal of.Food Science, vol. 39, 297-300. De Ketalere, A, Demeyer, D, Vandekerckhove, P & Vervaeke, I 1974, ‘Stoichimetry of carbohydrate fermentation during dry sausage ripi ning’, Journal of.Food Science, vol. 39, 297-300. Ferro Palma, S., 2006, ‘Estudio de la Influencia del Sistema de Elaboración en la Calidad del Lomo Curado Alentejano’, Tese de doutoramento, Cáceres, Universidad de Extremadura. Ferro Palma, S., 2006, ‘Estudio de la Influencia del Sistema de Elaboración en la Calidad del Lomo Curado Alentejano’, Tese de doutoramento, Cáceres, Universidad de Extremadura. Toribio, RR 2011, ‘Estudio descriptivo-comparativo de productos cárnicos asociados a la Denominación de Origen “Jamón de Teruel’, Cáceres: Universidad de Extremadura. Toribio, RR 2011, ‘Estudio descriptivo-comparativo de productos cárnicos asociados a la Denominación de Origen “Jamón de Teruel’, Cáceres: Universidad de Extremadura. Grau, R & Hamm, R 1953, ‘Eine einfache Methode zur Bestimmung der Wasserbindung im Muskel’, Naturwissenschaften, vol. 40, pp. 29. Ventanas, S, Ventanas, J & Ruiz, J 2006, Sensory characteristics of Ibe rian dry-cured loins: Influence of crossbreeding and rearing system’, Cáceres: University of Extremadura. BIBLIOGRAPHY Ventanas, S, Ventanas, J & Ruiz, J 2006, Sensory characteristics of Ibe rian dry-cured loins: Influence of crossbreeding and rearing system’, Cáceres: University of Extremadura. Ventanas, S, Ventanas, J & Ruiz, J 2006, Sensory characteristics of Ibe rian dry-cured loins: Influence of crossbreeding and rearing system’, Cáceres: University of Extremadura. Lorenzo, JM & Carballo, J 2013, ‘Changes in physico-chemical properties and volatile compounds throughout the manufacturing process of dry- cured foal loin’, Ourense: Facultad de Ciencias de Ourense.
https://openalex.org/W2138526483	https://hull-repository.worktribe.com/preview/380058/entropy-17-06397.pdf	English	null	Wavelet Entropy as a Measure of Ventricular Beat Suppression from the Electrocardiogram in Atrial Fibrillation	Entropy	2,015	cc-by	7,360	School of Engineering, University of Hull, Hull, HU6 7RX, UK; E-Mail: p.langley@hull.ac.uk; Tel.: +44-1482-466-535; Fax: +44-1482-466-664 Academic Editor: Raúl Alcaraz Martínez Academic Editor: Raúl Alcaraz Martínez Received: 16 July 2015 / Accepted: 14 September 2015 / Published: 17 September 2015 Abstract: A novel method of quantifying the effectiveness of the suppression of ventricular activity from electrocardiograms (ECGs) in atrial fibrillation is proposed. The temporal distribution of the energy of wavelet coefficients is quantified by wavelet entropy at each ventricular beat. More effective ventricular activity suppression yields increased entropies at scales dominated by the ventricular and atrial components of the ECG. Two studies are undertaken to demonstrate the efficacy of the method: first, using synthesised ECGs with controlled levels of residual ventricular activity, and second, using patient recordings with ventricular activity suppressed by an average beat template subtraction algorithm. In both cases wavelet entropy is shown to be a good measure of the effectiveness of ventricular beat suppression. Keywords: electrocardiogram; atrial fibrillation; wavelet entropy; QRST suppression entropy ISSN 1099-4300 www.mdpi.com/journal/entropy OPEN ACCESS OPEN ACCESS Entropy 2015, 17, 6397-6411; doi:10.3390/e17096397 Entropy 2015, 17, 6397-6411; doi:10.3390/e17096397 entropy ISSN 1099-4300 www.mdpi.com/journal/entropy OPEN ACCESS Philip Langley School of Engineering, University of Hull, Hull, HU6 7RX, UK; E-Mail: p.langley@hull.ac.uk; Tel.: +44-1482-466-535; Fax: +44-1482-466-664 School of Engineering, University of Hull, Hull, HU6 7RX, UK; E-Mail: p.langley@hull.ac.uk; Tel.: +44-1482-466-535; Fax: +44-1482-466-664 1. Introduction Atrial fibrillation is a cardiac arrhythmia in which the small upper chambers of the heart beat rapidly and not coordinated with the large ventricular chambers. It is the most prevalent abnormal heart rhythm in the elderly and a major health concern and drain on health service resources due to an aging population [1,2]. Atrial fibrillation is a significant risk factor for stroke, accounting for a third of all strokes and is a contributing factor for heart failure [1]. There is a range of therapeutic options for atrial fibrillation which attempt to restore the normal heart rhythm or reduce symptoms and stroke risk [1]. However, success rates of restoring sinus rhythm are relatively poor, particularly in patients with the persistent classification of the arrhythmia which is more Entropy 2015, 17 Entropy 2015, 17 6398 resilient to termination [1]. Hence the optimum treatment for a specific patient is unknown and is the focus of much clinical research. The electrocardiogram (ECG) is one of the main diagnostic tools for cardiac disease and arrhythmias. The electrocardiogram (ECG) is one of the main diagnostic tools for cardiac disease and arrhythmias. It records the electrical potentials generated by the cardiac muscles and cells that cause the heart to contract and relax. The ECG is completely non-invasive using electrodes placed on the skin at appropriate anatomical locations. The atrial and ventricular activations on the ECG are distinct but because the atrial muscle mass is much smaller than that of the ventricles the electrical signal associated with atrial activation is proportionally smaller. In the case of normal sinus rhythm, the atrial activation is seen as a distinct feature on the ECG called the P wave which, although small in amplitude, can be readily observed because it occurs when the ventricles are electrically quiescent. However, in the case of atrial fibrillation where the atria beat continuously and rapidly, there is no discrete P wave and atrial activations manifest as a low amplitude and rapidly fluctuating signal called the fibrillatory wave or f-wave. Much of the f-wave is obscured by the large QRST complex which is the manifestation of the electrical activation and recovery phases of each ventricular beat. The f-wave yields useful information about the underlying arrhythmia. 1. Introduction Wavelet analysis Entropy 2015, 17 Entropy 2015, 17 6399 is potentially well suited to quantification of residual ventricular activity after QRST suppression because of its ability to localise signal features in time and scale [27]. The aim of the present study is to demonstrate wavelet entropy as a tool to quantify the effectiveness of QRST suppression in the ECG of atrial fibrillation. Note that in this study wavelet analysis is not used to suppress the ventricular activity, the focus is on wavelet techniques for quantifying residual ventricular activity in the QRST suppressed signal and could be applied regardless of the suppression algorithm used. In this study the previously referred to ABS algorithm is used because of its simplicity and known limitations that serve to illustrate the effectiveness of the wavelet residual activity measurement. Examples to illustrate the wider applicability of the approach are also included. 1. Introduction For example, the atrial activation rate can be determined non-invasively rather than by invasive electrophysiological study [3,4], it contains information about the complexity of the underlying atrial propagation [5,6] and the effects of treatment can be quantified and predicted [7–11]. Analysis of the continuous f-wave requires suppression of the large ventricular activity from the ECG [8]. Many algorithms have been proposed to achieve this objective [12–19]. These algorithms take advantage of the disassociation of the atrial and ventricular activities to estimate the underlying atrial component during ventricular beats. One such algorithm is average beat subtraction (ABS) [20]. ABS subtracts a template QRST complex at each ventricular beat to provide an estimate of the underlying atrial signal. This template is derived from the average QRST complex from a collection of many beats from the same ECG lead. The disassociation between atrial and ventricular activities ensures there is little atrial activity in the average QRST complex. The effectiveness of the algorithm is limited because beat-by-beat QRST morphology changes due to heart rate fluctuations and respiration for example, so that the average beat cannot accurately represent in all beats the actual beat [21]. This results in residual ventricular activity remaining in the estimated atrial signal. Despite this limitation ABS remains one of the most widely used algorithms in clinical studies [22]. Several enhancements to the basic ABS algorithm have been proposed to ameliorate these limitations but all ventricular suppression algorithms have their limitations and do not completely remove the ventricular activity. This brings us to the focus of the present study which has the aim of quantifying the effectiveness of ventricular suppression from the ECG in atrial fibrillation using wavelet entropy. Despite the substantial research effort in developing ventricular suppression algorithms, little attention has been given to quantifying the effectiveness of these algorithms in terms of the residual ventricular activity. Some studies have looked at the effectiveness of suppression algorithms in terms of estimated clinical parameters, for example dominant frequency, amplitude of the estimated atrial signals or reduction in spectral power concentration [14,23–26]. Many studies have used computer simulations or synthesised ECGs with known and completely separable ventricular and atrial activities [17]. In the present study a wavelet based approach is proposed to quantify ventricular activity suppression. Wavelet analysis has been demonstrated to be useful in a range of applications related to ECG analysis [27]. 2.1. Ventricular Activity Suppression by Average Beat Subtraction The aim of ventricular activity suppression is to eliminate the components of the ECG originating from the ventricles so that only the components originating from the atria remain, allowing the analysis of the f-wave unhindered by ventricular activity [25]. The major ventricular components to be suppressed are the QRS complex corresponding to ventricular depolarisation and T wave corresponding to ventricular repolarisation. One of the simplest suppression algorithms is the ABS algorithm. Noting that during atrial fibrillation the atrial and ventricular electrical activities are disassociated enables the calculation of an average ventricular beat template that is free of any underlying atrial activity. Subtraction of the ventricular beat template at each ventricular beat then yields an estimate of the underlying f-wave. The basic ABS algorithm can be enhanced in several ways, for example by morphological clustering or separate QRS and T wave templates [28]. However, for the purpose of the present study only the basic ABS algorithm without these refinements is considered. This serves to illustrate the limitation of the algorithm with the proposed method of quantifying the residual ventricular activity. The first step of the ABS algorithm was to generate the QRST template specific to the particular ECG lead under analysis. Each ventricular beat was located using an automatic threshold based QRS detector. The locations of the R wave peaks were identified as r(j), j = 1: nb where nb is the total number of beats. Defining the beat window wb = [−w1, ... −2, −1, 0, 1, 2, ... w2] with limits −w1 and w2 which straddle the entire QRST segment relative to the R wave peak, the collection of all QRST segments was constructed X = [x1, x2,..., xnb]T (1) (1) (1) where xi is the i-th QRST segment the same length as the beat window. The average beat template, denoted qrstav, was then calculated as the mean value across all beats according to qrstav(k) = 1 nb X( j =1 nb  k, j) (2) (2) where k denotes the sample number and j the beat number. where k denotes the sample number and j the beat number. where k denotes the sample number and j the beat number. Entropy 2015, 17 6400 Having derived the average beat template it remains to subtract the template from each beat resulting in the QRST suppressed ECG. Samples outside the beat window remain unchanged. 2.2. Wavelet Analysis for Identifying Residual Ventricular Activity 2.2. Wavelet Analysis for Identifying Residual Ventricular Activity The continuous wavelet transform is the correlation of a signal with an analysing wavelet function across a range of scales and translations of the wavelet function. The mother wavelet has the form ψa,b(t) = 1 a ψ t −b a       (3) (3) and the continuous wavelet transform of the signal x(t) is defined by and the continuous wavelet transform of the signal x(t) is defined by and the continuous wavelet transform of the signal x(t) is defined by T(a,b) = 1 a x(t)ψ* t −b a       dt −∞ ∞  (4) (4) where ψ* is the complex conjugate of the wavelet function and a and b are the scale and translation parameters respectively. The transform provides a measure of similarity between the signal and wavelet function at specific scale and translation. There are numerous mother wavelet functions but an appropriate choice is one with similarity of shape to the analysed signal. In practice this choice is not critical as many wavelets have similar properties and are able to characterise similarity with good localisation in both scale and translation. In the present study the n-th order derivative of Gaussian probability density function with general form ψ(n)(t) = dn(e−t 2 2) dt n was used with n = 4 or “Gaus4” wavelet. It was anticipated that this wavelet would provide good localisation of the residual ventricular activity. Having identified a mechanism for localising the residual ventricular activity using wavelet analysis, a novel wavelet entropy method was then designed to quantify the residual activity. 2.1. Ventricular Activity Suppression by Average Beat Subtraction Inevitably the QRST suppressed ECG contains some residual ventricular activity because the average beat template is not an accurate model of the ventricular activity at every beat. Wavelet analysis was used to detect the residual ventricular activity as described in the following section. 2.2. Wavelet Analysis for Identifying Residual Ventricular Activity 2.2. Wavelet Analysis for Identifying Residual Ventricular Activity and the corresponding wavelet entropy is given by  and the corresponding wavelet entropy is given by  S(a) = − P(a,b)log(P(a,b))db  . (7) (7) In Equations (6) and (7) translation variable b is effectively the time variable, so the integral is across the duration of the signal of interest. In this case the wavelet entropy provides an indication of the temporal distribution of wavelet energy at each scale. The entropy of a scale with a broad temporal energy distribution will be greater than one with a narrow temporal distribution. With regard to the current application to QRST suppressed ECG, any residual ventricular activity will be expressed as wavelet energies which are not evenly temporally distributed but with a periodicity corresponding to the ventricular rate. Conversely, the continuous f-wave will have a relatively constant temporal energy distribution. Further, the atrial and ventricular wavelet energies will likely occur at different scales due to the different frequency content of the QRST and f-waves. Hence the temporal energy distribution may yield information about the effectiveness of ventricular suppression in the ECGs of atrial fibrillation. Optimum suppression of ventricular activity would be expected to maximise the wavelet entropies at scales corresponding to ventricular and atrial activations. To explore the measurement of effectiveness of ventricular activity suppression, three wavelet entropy based indices were defined: (i) Ventricular scale band wavelet entropy (WEvent) defined as the minimum wavelet entropy at scales corresponding to predominantly ventricular activity. Since the ventricular activity, particularly the QRS complex, contains higher frequency components than the f-wave, the frequency range 12.5 to 50 Hz with corresponding scales for the “Gaus4” wavelet of 20 to 5 respectively was used in this study. Motivation for choosing the minimum entropy in this waveband was the observation that the minimum entropy is highly influenced by the degree of QRS residual. (ii) Atrial scale band wavelet entropy (WEaf) defined as the average wavelet entropy across scales corresponding to predominantly the f-wave. The f-wave has frequencies typically in the range 3 to 10 Hz and the corresponding scales for the “Gaus4” wavelet are 80 to 25 respectively for the sample rate used in this study. (ii) (iii) Wide scale ventricular and atrial band wavelet entropy (WEva) defined as the average wavelet entropy across scales containing both the above atrial and ventricular scale bands specifically 5 (minimum scale of WEvent) to 80 (maximum scale of WEaf). and the corresponding wavelet entropy is given by  This serves to define a single index for quantifying the quality of ventricular suppression taking account of both atrial and ventricular scale wavelet entropies. 2.1. Wavelet Entropy Measure of Residual Ventricular Activity Entropy is well known for its characterisation of the information content of a signal: a highly variable signal yields greater information than one that varies little. This concept has been extended to the frequency and wavelet domains by considering the spectral and wavelet energy distributions [29]. A signal with wide variation in spectral content is considered to yield greater information content and hence has greater entropy than one with a narrow spectral distribution. As such wavelet entropy characterises the ‘organisation’ of a signal and has found use in biomedical applications such as the analysis of EEG and ECG [30,31]. g wavelet analysis of a signal, the energy at a particular scale and translation is given by Following wavelet analysis of a signal, the energy at a particular scale and translati E(a,b) = T(a,b) 2. (5) E(a,b) = T(a,b) 2. (5) Following a similar definition of the wavelet energy probability distribution by Sello [29], who calculated the distribution across scales at each translation or time point, here the distribution is 6401 Entropy 2015, 17 Entropy 2015, 17 calculated across translations at each scale according to Equation (6): calculated across translations at each scale according to Equation (6): P(a,b) = T(a,b) 2 T(a,b) 2db  P(a,b) = T(a,b) 2 T(a,b) 2db  (6) P(a,b) = T(a,b) 2 T(a,b) 2db  (6) Entropy 2015, 17 Entropy 2015, 17 which different amplitudes of residual ventricular activity were simulated. Second, a study on real ECGs of patients in atrial fibrillation to which the ABS ventricular suppression algorithm was applied. Both studies utilised the ECG recordings from 10 atrial fibrillation patients for which ethical approval was obtained and all patients provided informed consent. The ECGs were recorded at a sample rate of 500 Hz, amplitude resolution of 5 µV and bandwidth of 0.05 to 100 Hz. Lead V1 was analysed since it is the most commonly analysed lead for atrial fibrillation studies due to its high amplitude f-wave in most patients [25]. However, to show wide applicability of the technique, examples for QRST suppression in multi-lead ECGs using blind source separation are provided. 3. Experimental Studies and Discussion Application of the entropy measures of residual ventricular activity from QRST suppressed ECGs in atrial fibrillation was demonstrated in two experimental studies. First, a study with synthesised ECGs in 6402 3.1. Study on Synthesised ECGs Using the patient recordings for lead V1, for each patient a series of 10 synthesised single beat ECGs with decreasing levels of residual ventricular activity were constructed as follows. First, the longest f-wave segment free of ventricular activity was identified in each recording. Second, in each recording the average QRST complex was calculated. Third, 10 synthesised single beat ECGs were constructed by adding to the segment of ventricular free f-wave the average QRST with decreasing levels of amplitude as in Equation (8). (10 ) ( ) , 1:10 9 av i ECGsyn i fwave qrst i − = + = . (8) (8) This generated 10 synthesised ECGs for each patient, 100 ECGs in total, each simulating QRST suppressed ECGs with different levels of QRST suppression ranging from completely unsuppressed (i = 1) to completely suppressed (i = 10). Figure 1 shows the synthesised ECGs for one patient along with the results of the wavelet entropy analysis for these synthesised ECGs. From Figure 1, decreasing levels of residual ventricular activity gave rise to increasing wavelet entropy, particularly WEvent. This was a result of the decreased wavelet energies at the high frequency ventricular scales and consequently the more even energy distribution across the duration of the ECG segment as can be seen in the wavelet energy contour plots (Figure 1). This result is confirmed for all simulations in Figure 2, which shows the wavelet entropies for the synthesised ECGs constructed from all patient ECGs. Clearly, more effective ventricular activity suppression gave significantly higher ventricular and atrial scale entropies. Compared to no suppression at all (ECGsyn(1)) wavelet entropies were significant higher (all p < 0.001, paired t-test) across all levels of suppression (ECGsyn(i), i = 2: 10) with the most significant increase for complete suppression (WEvent: ECGsyn(1) = 3.45 (0.36) vs. ECGsyn(10) = 5.48 (0.24), p < 0.0000001 and WEaf: ECGsyn(1) = 5.14 (0.23) vs. ECGsyn(10) = 5.85 (0.31), p < 0.00001 and WEva: ECGsyn(1) = 4.85 (0.32) vs. ECGsyn(10) = 5.82 (0.28), p < 0.00001). The simulation study suggests that most effective ventricular suppression was achieved when WEva increased from 4.85 (0.32) (no suppression) to 5.82 (0.28) (complete suppression). Entropy 2015, 17 Entropy 2015, 17 6403 py , Figure 1. Wavelet analysis of electrocardiograms (ECGs) synthesised from one patient with decreasing levels of residual ventricular activity (columns 1 to 10). 3.1. Study on Synthesised ECGs Columns show the analysis of each synthesised ECG with column 1 having the maximum residual QRST amplitude and the final column having no residual QRST. (Row 1) Synthesised ECG segments of 600 samples (1.2 s) duration with decreasing amplitudes of residual QRST. (Row 2) Wavelet coefficients of the ECG displayed as a colour contour map. (Row 3) Wavelet energy displayed as a colour contour map. (Row 4) Wavelet entropy as a function of scale with ventricular wavelet entropy (WEvent) indicated by a red horizontal line, atrial wavelet entropy (WEaf) indicated by a black horizontal line and wide scale band wavelet entropy (WEva) indicated by a green horizontal line. Figure 1. Wavelet analysis of electrocardiograms (ECGs) synthesised from one patient with decreasing levels of residual ventricular activity (columns 1 to 10). Columns show the analysis of each synthesised ECG with column 1 having the maximum residual QRST amplitude and the final column having no residual QRST. (Row 1) Synthesised ECG segments of 600 samples (1.2 s) duration with decreasing amplitudes of residual QRST. (Row 2) Wavelet coefficients of the ECG displayed as a colour contour map. (Row 3) Wavelet energy displayed as a colour contour map. (Row 4) Wavelet entropy as a function of scale with ventricular wavelet entropy (WEvent) indicated by a red horizontal line, atrial wavelet entropy (WEaf) indicated by a black horizontal line and wide scale band wavelet ( ) i di d b h i l li Figure 1. Wavelet analysis of electrocardiograms (ECGs) synthesised from one patient with decreasing levels of residual ventricular activity (columns 1 to 10). Columns show the analysis of each synthesised ECG with column 1 having the maximum residual QRST amplitude and the final column having no residual QRST. (Row 1) Synthesised ECG segments of 600 samples (1.2 s) duration with decreasing amplitudes of residual QRST. (Row 2) Wavelet coefficients of the ECG displayed as a colour contour map. (Row 3) Wavelet energy displayed as a colour contour map. (Row 4) Wavelet entropy as a function of scale with ventricular wavelet entropy (WEvent) indicated by a red horizontal line, atrial wavelet entropy (WEaf) indicated by a black horizontal line and wide scale band wavelet entropy (WEva) indicated by a green horizontal line. Entropy 2015, 17 6404 Figure 2. Wavelet entropies WEvent, WEaf and WEva for all synthesised electrocardiograms (ECGs). (a) Bar charts of wavelet entropies for synthesied ECGs grouped by patient simulation. 3.1. Study on Synthesised ECGs For each patient the bars are ordered by increasing levels of QRST suppression (Blue: ECGsyn(1), no suppression, red: ECGsyn(10), total suppression). (b) Boxplots of wavelet entropies of synthesised ECGs, bar indicates median and boxes indicate interquartile range across patients at each suppression level. Figure 2. Wavelet entropies WEvent, WEaf and WEva for all synthesised electrocardiograms (ECGs). (a) Bar charts of wavelet entropies for synthesied ECGs grouped by patient simulation. For each patient the bars are ordered by increasing levels of QRST suppression (Blue: ECGsyn(1), no suppression, red: ECGsyn(10), total suppression). (b) Boxplots of wavelet entropies of synthesised ECGs, bar indicates median and boxes indicate interquartile range across patients at each suppression level. ure 2. Wavelet entropies WEvent, WEaf and WEva for all synthesised electrocardiograms Figure 2. Wavelet entropies WEvent, WEaf and WEva for all synthesised electrocardiograms (ECGs). (a) Bar charts of wavelet entropies for synthesied ECGs grouped by patient simulation. For each patient the bars are ordered by increasing levels of QRST suppression (Blue: ECGsyn(1), no suppression, red: ECGsyn(10), total suppression). (b) Boxplots of wavelet entropies of synthesised ECGs, bar indicates median and boxes indicate interquartile range across patients at each suppression level. 3.2. Study on Real ECGs with ABS Ventricular Suppression The aim of this study was to demonstrate the application of the method to real ECGs with ventricular beats suppressed by the ABS algorithm. It demonstrates how the “quality” of ventricular beat suppression can be quantified beat-by-beat. Lead V1 from the ECGs of the 10 patients were analysed. The ABS algorithm was used to suppress QRST at each beat as previously described. The effectiveness of ventricular suppression was quantified by the wavelet entropy measures on a beat-by-beat basis for the first 10 beats of each recording by comparing the entropies for unsuppressed and suppressed ECGs. Figure 3 illustrates the analysis for a single patient recording. It shows the unsuppressed and ABS suppressed beats along with their wavelet entropies for the first 10 beats in one patient recording. Wavelet entropy for the suppressed beats, particularly WEvent, clearly correlates with the extent of QRST suppression. Notably for example, the ABS algorithm performed very well with no visible residual activity in beat 1 for which the corresponding WEvent = 4.8, but performed poorly in beat 6 for which WEvent = 3.8. Over all analysed beats for this subject, ventricular beat suppression significantly increased 6405 Entropy 2015, 17 wavelet entropies (WEvent: 3.19 (0.10) vs. 4.35 (0.37), p < 0.000001 and WEaf: 5.03 (0.10) vs. 5.25 (0.09), p < 0.001 and WEva: 4.74 (0.09) vs. 5.17 (0.08), p < 0.000001, paired t-test). wavelet entropies (WEvent: 3.19 (0.10) vs. 4.35 (0.37), p < 0.000001 and WEaf: 5.03 (0.10) vs. 5.25 (0.09), p < 0.001 and WEva: 4.74 (0.09) vs. 5.17 (0.08), p < 0.000001, paired t-test). 0.001 and WEva: 4.74 (0.09) vs. 5.17 (0.08), p < 0.000001, paired t-test). Figure 3. Wavelet analysis of 10 beats from one patient (columns 1 to 10). Columns show the analysis of each beat. (Row 1) The unsuppressed electrocardiogram (ECG) segment of 300 samples (0.6 s) duration. (Row 2) Wavelet entropy as a function of scale for the unsuppressed ECG with ventricular wavelet entropy (WEvent) indicated by a red horizontal line, atrial wavelet entropy (WEaf) indicated by a black horizontal line and wide scale band wavelet entropy (WEva) indicated by a green horizontal line. (Row 3) The ECG segment after QRST suppression by the average beat subtraction (ABS) algorithm. 3.2. Study on Real ECGs with ABS Ventricular Suppression Wavelet entropies (WEvent, WEaf and WEva) of patient electrocardiograms (ECGs) with unsuppressed and suppressed ventricular activity with the average beat subtraction (ABS) algorithm. Boxplots show the median (red line) and interquartile range (box) of entropies over the first 10 beats. For each patient the box on the left is for the unsuppressed ECG and the box on the right is for the ABS suppressed ECG. 3.2. Study on Real ECGs with ABS Ventricular Suppression (Row 4) Wavelet entropy as a function of scale for the suppressed ECG with ventricular wavelet entropy (WEvent) indicated by a red horizontal line, atrial wavelet entropy (WEaf) indicated by a black horizontal line and wide scale band wavelet entropy (WEva) indicated by a green horizontal line. Figure 3. Wavelet analysis of 10 beats from one patient (columns 1 to 10). Columns show the analysis of each beat. (Row 1) The unsuppressed electrocardiogram (ECG) segment of 300 samples (0.6 s) duration. (Row 2) Wavelet entropy as a function of scale for the unsuppressed ECG with ventricular wavelet entropy (WEvent) indicated by a red horizontal line, atrial wavelet entropy (WEaf) indicated by a black horizontal line and wide scale band wavelet entropy (WEva) indicated by a green horizontal line. (Row 3) The ECG segment after QRST suppression by the average beat subtraction (ABS) algorithm. (Row 4) Wavelet entropy as a function of scale for the suppressed ECG with ventricular wavelet entropy (WEvent) indicated by a red horizontal line, atrial wavelet entropy (WEaf) indicated by a black horizontal line and wide scale band wavelet entropy (WEva) indicated by a green horizontal line. This result is confirmed for all the patient recordings in Figure 4. For each patient recording there is considerable variability of wavelet entropies (as indicated by the interquartile range in Figure 4) across beats. Such variability of performance is expected with the basic ABS algorithm since beat to beat QRST morphology changes are common, due for example to respiratory or postural changes. Overall across the 10 patient recordings and considering the mean wavelet entropies for each suppressed and unsuppressed recording, ABS achieved very highly significant increases in WEvent (3.48 (0.02) vs. 4.76 (0.12), p < 0.00001, two-sample t-test) and WEaf (5.06 (0.04) vs. 5.49 (0.05), p < 0.00001) and WEva (4.78 (0.04) vs. 5.41 (0.05), p < 0.00001). 6406 Entropy 2015, 17 Figure 4. Wavelet entropies (WEvent, WEaf and WEva) of patient electrocardiograms (ECGs) with unsuppressed and suppressed ventricular activity with the average beat subtraction (ABS) algorithm. Boxplots show the median (red line) and interquartile range (box) of entropies over the first 10 beats. For each patient the box on the left is for the unsuppressed ECG and the box on the right is for the ABS suppressed ECG. Figure 4. Wavelet entropies (WEvent, WEaf and WEva) of patient electrocardiograms (ECG Figure 4. 3.3. Application to Multi-Lead ECG To demonstrate the general applicability of the wavelet based indices, the application to multi-lead ECGs was considered. Multi-lead f-wave extraction techniques based on blind source separation techniques such as principal and independent component analysis have been developed [7,16]. These algorithms consider the atrial and ventricular activities of the ECG to arise from mixtures of signals from the atrial and ventricular sources. The blind source separation process aims to separate the atrial and ventricular activities into different components considering all recorded leads. Here we consider principal component analysis as the blind source separation algorithm [7,15,32]. Figure 5 shows a patient 12-lead ECG along with the first 4 principal components and their associated wavelet entropies. V1 showed the most prominent f-wave in the 12-lead ECG. The first three principal components were dominated by ventricular activities and their WEva values were 4.56, 4.78, 5.04 reflecting the progressive suppression of the ventricular activity in these components. The forth principal component contained the separated f-wave and had WEva of 5.51. The f-wave obtained by the ABS algorithm applied to V1 is also shown for comparison and its WEva was 5.46. Entropy 2015, 17 6407 Figure 5. Wavelet analysis of the 12-lead electrocardiogram (ECG) of one patient with atrial and ventricular activities separated by principal component analysis and average beat subtraction (ABS). (Row 1) The unsuppressed 12-leave ECG segment of 500 samples (1.0 s) duration. The f-wave is visible in lead V1. (Row 2) The first 4 principal components of the 12-lead ECG (PC to PC4). The first three principal components (PC1, PC2, PC3) were dominated by ventricular activity. PC4 contained the separated f-wave. The f-wave extracted from lead V1 by the ABS algorithm is also shown. (Row 3) Wavelet entropy as a function of scale for each principal component and ABS extracted f-wave with ventricular wavelet entropy (WEvent) indicated by a red horizontal line, atrial wavelet entropy (WEaf) indicated by a black horizontal line and wide scale band wavelet entropy (WEva) indicated by a green horizontal line. Figure 5. Wavelet analysis of the 12-lead electrocardiogram (ECG) of one patient with atrial and ventricular activities separated by principal component analysis and average beat subtraction (ABS). (Row 1) The unsuppressed 12-leave ECG segment of 500 samples (1.0 s) duration. The f-wave is visible in lead V1. (Row 2) The first 4 principal components of the 12-lead ECG (PC to PC4). 3.3. Application to Multi-Lead ECG The first three principal components (PC1, PC2, PC3) were dominated by ventricular activity. PC4 contained the separated f-wave. The f-wave extracted from lead V1 by the ABS algorithm is also shown. (Row 3) Wavelet entropy as a function of scale for each principal component and ABS extracted f-wave with ventricular wavelet entropy (WEvent) indicated by a red horizontal line, atrial wavelet entropy (WEaf) indicated by a black horizontal line and wide scale band wavelet entropy (WEva) indicated by a green horizontal line. 3.4. Comparison to Other Measures of the Effectiveness of Ventricular Beat Suppression Few measures to quantify the effectiveness of ventricular beat suppression have been described. In simulation scenarios where the definitive underlying f-wave signal is known indices such as cross correlation and normalised mean square error can been used [14]. It is more challenging in the case of real ECGs where the underlying f-wave is not known. Some studies have simply compared the amplitude of the f-wave in the suppressed QRST segment with the amplitude in the TQ segment where there is no ventricular activity on the reasonable assumption that any residual activity is likely to increase the signal amplitude [24]. A more robust solution was proposed by Alcaraz et al. with the ventricular residue index (VR) [14] ( ) 2 ,...., 2 1 1 ( ) max ( ) 1 ( ) i i i i r H i AA AA Q k r H r H k r H AA n VR x k x k x n Q + = − + = − = = ⋅   (9) (9) where VRi is the ventricular residue at beat i, Q the number of sample points across the beat segment, xAA is the QRST suppressed ECG, ri is the sample point of the R wave peak and H specifies a window of length 2H + 1 sample points centered on ri and encompasses the QRS interval. Entropy 2015, 17 6408 Conceptually VR can be considered similar to the ratio of the energy of the residual QRS to the energy of the entire segment [33]. Lower VR indicates more effective ventricular suppression. Implementations of this algorithm used H corresponding to 50 ms assuming a QRS interval of approximately 100 ms for all beats for all patients [14,33], presumably to avoid the difficult detection of the true onset and offset of the QRS complex and the additional computational burden. Clearly the effectiveness of the algorithm is dependent upon the specified window since choosing a window which is too small potentially misses residual activity occurring outside the window. This is most apparent because VR does not take account of any residual T wave, which may be substantial if not suppressed adequately [34]. This limitation is avoided in the wavelet entropy approach because it considers the wavelet energy across scale bands corresponding to T wave as well as QRS complex, so that residual T wave is captured by the approach. To illustrate this Figure 6 compares VR and wavelet entropy index WEva for two synthesised cases: (a) QRST suppression and (b) QRS suppression but no T wave suppression. As expected, for case (a) (QRST suppression) WEva increased and VR decreased as the residual ventricular activity reduced. However, for case (b) (QRS suppression only) where significant T wave residual existed, rather than VR being greater than case (a), it was smaller, wrongly suggesting improved ventricular suppression. The wavelet entropy measure on the other hand accurately reflected the residual T wave because WEva was smaller relative to case (a), suggesting less effective ventricular suppression. Figure 6. Comparison of wide scale band wavelet entropy (WEva) and residue index (VR) for synthesised electrocardiograms (ECGs) with decreasing levels of residual ventricular activity: (a) QRST suppression; and (b) QRS suppression but no T wave suppression. (Row 1) Synthesised ECG segments of 900 samples (1.8 s) duration with decreasing amplitudes of residual QRST. (Row 2) Same as row 1 but without T wave suppressed to simulate an f-wave with large T wave residual. (Row 3) WEva for simulated cases (a) (blue line) and (b) (green line). Note residual T wave results in lower WEva as would be expected. (Row 4) VR for simulated cases (a) (blue line) and (b) (green line). Note residual T wave results in lower VR. Figure 6. 4. Conclusions New measures to quantify the quality of ventricular activity suppression of the ECG in atrial fibrillation based on wavelet entropy have been proposed. They are based on entropy as a measure of the temporal energy distribution of wavelet coefficients of ECG beat segments. Superior suppression results in higher entropy values due to the broad temporal energy distributions at ventricular and atrial scale bands. This reflects the removal of the highly concentrated (in scale and time) energy associated with the ventricular activity. The measures have been shown to be effective on both synthesised and real ECGs. Not only do they have application in the assessment of ventricular activity suppression beat-by-beat as demonstrated in the present study, but also in comparing the effectiveness of different ventricular suppression algorithms, which will be the focus of future work. Entropy 2015, 17 Comparison of wide scale band wavelet entropy (WEva) and residue index (V Figure 6. Comparison of wide scale band wavelet entropy (WEva) and residue index (VR) for synthesised electrocardiograms (ECGs) with decreasing levels of residual ventricular activity: (a) QRST suppression; and (b) QRS suppression but no T wave suppression. (Row 1) Synthesised ECG segments of 900 samples (1.8 s) duration with decreasing amplitudes of residual QRST. (Row 2) Same as row 1 but without T wave suppressed to simulate an f-wave with large T wave residual. (Row 3) WEva for simulated cases (a) (blue line) and (b) (green line). Note residual T wave results in lower WEva as would be expected. (Row 4) VR for simulated cases (a) (blue line) and (b) (green line). Note residual T wave results in lower VR. Entropy 2015, 17 Entropy 2015, 17 6409 Acknowledgments The author would like to thank J.P. Bourke and S. King who facilitated ECG data collection at the Freeman Hospital, Newcastle upon Tyne, UK. Conflicts of Interest The author declares no conflict of interest. The author declares no conflict of interest. References 1. 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https://openalex.org/W4383551479	https://www.researchsquare.com/article/rs-3099147/latest.pdf	English	null	Examination of Medicinal Plants for Radionuclides Absorption and Their Health Implications	Research Square (Research Square)	2,023	cc-by	11,979	Examination of Medicinal Plants for Radionuclides Absorption and Their Health Implications Felix Adegoke Popoola (  felixgoke@gmail.com ) Glorious Vision University Oladotun Bamiji Isola Glorious Vision University Oluseye Daniel Fakeye University of Ibadan Tunde Ayobami Owolabi Igbinedion University Modupe Eunice Sanyaolu Redeemer’s University Sheu Isiaq Owoyemi Adeyemi Federal University of Education Isaac Oluwafemi Elijah Glorious Vision University Posted Date: July 7th, 2023 Posted Date: July 7th, 2023 DOI: https://doi.org/10.21203/rs.3.rs-3099147/v1 Research Article Keywords: radiological hazards, medicinal plants, radionuclides adsorption, health safety, gamma spectrometry DOI: https://doi.org/10.21203/rs.3.rs-3099147/v1 DOI: https://doi.org/10.21203/rs.3.rs-3099147/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. 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EXAMINATION OF MEDICINAL PLANTS FOR RADIONUCLIDES ABSORPTION 1 AND THEIR HEALTH IMPLICATIONS 2 Felix Adegoke Popoola1, Oladotun Bamiji Isola2, Oluseye Daniel Fakeye3, Tunde Ayobam 3 Owolabi4, Modupe Eunice Sanyaolu5, Sheu Isiaq Owoyemi6, Isaac Oluwafemi Elijah7 4 1Department of Mathematical & Physical Sciences, Glorious Vision University, Ogwa, 5 Edo State, Nigeria. 6 2Department of Chemical Sciences, Glorious Vision University, Ogwa, Edo State, Nigeria. 7 3Department of Zoology, University of Ibadan, Ibadan, Oyo State, Nigeria. 8 4Department of Pharmacognosy, Igbinedion University, Okada, Edo State, Nigeria. 9 5Department of Physics, Redeemer’s University, Ede, Osun State, Nigeria. 10 6Department of Integrated Science, Adeyemi Federal University of Education, Ondo, Ondo 11 State, Nigeria 12 7Department of Works & Physical Planning, Glorious Vision University, Ogwa, Edo State 13 Nigeria 14 15 Corresponding author: Felix Adegoke Popoola 16 Glorious Vision University (Formerly Samuel Adegboyega University), Ogwa, Edo State, 17 Nigeria. 18 E-mail: felixgoke@gmail.com; fpopoola@sau.edu.ng 19 ORCID: https://www.orcid.org/0000-0003-3484-6883 20 21 EXAMINATION OF MEDICINAL PLANTS FOR RADIONUCLIDES ABSORPTION 1 AND THEIR HEALTH IMPLICATIONS 2 EXAMINATION OF MEDICINAL PLANTS FOR RADIONUCLIDES ABSORPTIO 1 AND THEIR HEALTH IMPLICATIONS 2 Felix Adegoke Popoola1, Oladotun Bamiji Isola2, Oluseye Daniel Fakeye3, Tunde Ayob 3 Owolabi4, Modupe Eunice Sanyaolu5, Sheu Isiaq Owoyemi6, Isaac Oluwafemi Elijah 4 1Department of Mathematical & Physical Sciences, Glorious Vision University, Ogwa 5 Edo State, Nigeria. 6 2Department of Chemical Sciences, Glorious Vision University, Ogwa, Edo State, Nigeri 7 3Department of Zoology, University of Ibadan, Ibadan, Oyo State, Nigeria. 8 4Department of Pharmacognosy, Igbinedion University, Okada, Edo State, Nigeria. 9 5Department of Physics, Redeemer’s University, Ede, Osun State, Nigeria. 10 6Department of Integrated Science, Adeyemi Federal University of Education, Ondo, Ond 11 State, Nigeria 12 7Department of Works & Physical Planning, Glorious Vision University, Ogwa, Edo Stat 13 Nigeria 14 15 Corresponding author: Felix Adegoke Popoola 16 Glorious Vision University (Formerly Samuel Adegboyega University), Ogwa, Edo State, 17 Nigeria. 18 E-mail: felixgoke@gmail.com; fpopoola@sau.edu.ng 19 ORCID: https://www.orcid.org/0000-0003-3484-6883 20 21 22 Abstract 23 This study tests the concentration of 40K, 238U, and 232Th radionuclides and also evaluates the po 24 radiological health risks in medicinal plants found in Ewu, Edo State, Nigeria, using a NaI(Tl) ga 25 spectrometer. The six selected medicinal plants are Magnifera indica, Dacryodes edulis, Termi 26 catappa, Cymbopogon citratus, Anacardium occidentale, and Persea Americana. The r 27 showed that the activity concentrations for 40K ranged from 146.59 ± 4.81 in Persea americana to 2 28 3.42 Bq/kg in Cymbopogon citratus with a mean of 209.43 ± 5.14 Bq/kg, 238U ranged from 2.25 ± 0 29 5.57 ± 0.15 Bq/kg with a mean of 4.73 ± 0.15 Bq/kg and 232Th varied from 4.50 ± 0.35 to 12.07 ± 30 Bq/kg with a mean of 8.00 ± 0.40 Bq/kg. The maximum and minimum activity concentrations of both 31 and 232Th were found in Magnifera indica and Cymbopogon citratus respectively. The calcu 32 radiological hazards assessment due to the investigated medicinal plants is well within the internatio 33 recommended safe limits. 232Th contributes 54.91% to the total 𝐸𝐶𝐸𝐷, while 6.35% for 238U is the 34 This study tests the concentration of 40K, 238U, and 232Th radionuclides and also evaluates the possible 24 radiological health risks in medicinal plants found in Ewu, Edo State, Nigeria, using a NaI(Tl) gamma 25 spectrometer. The six selected medicinal plants are Magnifera indica, Dacryodes edulis, Terminalia 26 catappa, Cymbopogon citratus, Anacardium occidentale, and Persea Americana. EXAMINATION OF MEDICINAL PLANTS FOR RADIONUCLIDES ABSORPTION 1 AND THEIR HEALTH IMPLICATIONS 2 Human waste, animal 54 232Th exhibit a very strong, positive, and significant relationship with (𝐸𝐶𝐸𝐷 and ELCR), and it contributes 35 largely to the 𝐸𝐶𝐸𝐷 and ELCR due to ingestion of the examined herbal plant. Therefore, the use of these 36 medicinal plants is radiologically safe for human consumption. 37 232Th exhibit a very strong, positive, and significant relationship with (𝐸𝐶𝐸𝐷 and ELCR), and it contributes 35 largely to the 𝐸𝐶𝐸𝐷 and ELCR due to ingestion of the examined herbal plant. Therefore, the use of these 36 medicinal plants is radiologically safe for human consumption. 37 232Th exhibit a very strong, positive, and significant relationship with (𝐸𝐶𝐸𝐷 and ELCR), and it contributes 35 largely to the 𝐸𝐶𝐸𝐷 and ELCR due to ingestion of the examined herbal plant. Therefore, the use of these 36 medicinal plants is radiologically safe for human consumption. 37 Keywords: radiological hazards; medicinal plants; radionuclides adsorption; health safety; 38 gamma spectrometry 39 40 41 INTRODUCTION 42 Keywords: radiological hazards; medicinal plants; radionuclides adsorption; health safety; 38 gamma spectrometry 39 40 Keywords: radiological hazards; medicinal plants; radionuclides adsorption; health safety; 38 gamma spectrometry 39 40 Safety and quality of medicinal plant materials are becoming increasingly important due to global 43 demand by health authorities, pharmaceutical industries, and also the public at large (WHO, 2007). 44 Probably the oldest strategy still in use today for helping people deal with illness is the use of 45 medicinal herbs. In many traditional medical systems around the world, medicinal plants have long 46 been employed as a form of treatment. (WHO, 2007; Chandrashekara & Somashekarappa, 2016). 47 The World Health Organization (WHO) has promoted the integration of traditional medicine's 48 beneficial components into national health care systems in an effort to stimulate the use of 49 traditional medicine worldwide (Oni et al., 2011). This has served as alternative medical technique 50 although, concerns about estimated dosages and the sanitary state of the medications produced 51 using this method were addressed at various gatherings. (Oni et al., 2011). 52 Medicinal plants can be found in their natural state or processed (Saudi et al., 2022). Physical, 53 chemical, and biological pollutants can be found in herbal remedies. (WHO, 2007). Human waste, animal 54 manure, and sewage used as fertilizer can produce chemical and microbiological pollutants (WHO, 2007). EXAMINATION OF MEDICINAL PLANTS FOR RADIONUCLIDES ABSORPTION 1 AND THEIR HEALTH IMPLICATIONS 2 The results 27 showed that the activity concentrations for 40K ranged from 146.59 ± 4.81 in Persea americana to 296.08 28 3.42 Bq/kg in Cymbopogon citratus with a mean of 209.43 ± 5.14 Bq/kg, 238U ranged from 2.25 ± 0.06 to 29 5.57 ± 0.15 Bq/kg with a mean of 4.73 ± 0.15 Bq/kg and 232Th varied from 4.50 ± 0.35 to 12.07 ± 0.57 30 Bq/kg with a mean of 8.00 ± 0.40 Bq/kg. The maximum and minimum activity concentrations of both 238U 31 and 232Th were found in Magnifera indica and Cymbopogon citratus respectively. The calculated 32 radiological hazards assessment due to the investigated medicinal plants is well within the internationally 33 recommended safe limits. 232Th contributes 54.91% to the total 𝐸𝐶𝐸𝐷, while 6.35% for 238U is the least. 34 1 232Th exhibit a very strong, positive, and significant relationship with (𝐸𝐶𝐸𝐷 and ELCR), and it contributes 35 largely to the 𝐸𝐶𝐸𝐷 and ELCR due to ingestion of the examined herbal plant. Therefore, the use of these 36 medicinal plants is radiologically safe for human consumption. 37 Keywords: radiological hazards; medicinal plants; radionuclides adsorption; health safety; 38 gamma spectrometry 39 40 41 INTRODUCTION 42 Safety and quality of medicinal plant materials are becoming increasingly important due to global 43 demand by health authorities, pharmaceutical industries, and also the public at large (WHO, 2007). 44 Probably the oldest strategy still in use today for helping people deal with illness is the use of 45 medicinal herbs. In many traditional medical systems around the world, medicinal plants have long 46 been employed as a form of treatment. (WHO, 2007; Chandrashekara & Somashekarappa, 2016). 47 The World Health Organization (WHO) has promoted the integration of traditional medicine's 48 beneficial components into national health care systems in an effort to stimulate the use of 49 traditional medicine worldwide (Oni et al., 2011). This has served as alternative medical technique 50 although, concerns about estimated dosages and the sanitary state of the medications produced 51 using this method were addressed at various gatherings. (Oni et al., 2011). 52 Medicinal plants can be found in their natural state or processed (Saudi et al., 2022). Physical, 53 chemical, and biological pollutants can be found in herbal remedies. (WHO, 2007). EXAMINATION OF MEDICINAL PLANTS FOR RADIONUCLIDES ABSORPTION 1 AND THEIR HEALTH IMPLICATIONS 2 For 238U and 68 226Ra, the lungs and kidneys are typically the sites of deposition; for 232Th, the lungs, liver, or 69 bones may all accumulate; and for 40K, the body as a whole, but mostly in the muscles (Akhter et 70 al., 2007; Nahar et al., 2018; Ugbede et al., 2022). As a result, the immune system of the body 71 may weaken in the face of many illnesses with higher death rates (Nahar et al., 2018; Ugbede et 72 al., 2022). The amount of exposure to radiation depends on the intake of radionuclides, and its 73 significance depends in turn on other variables such as the age, metabolic kinetics, and weight of 74 the individual who ingests them (also known as the dose conversion factor) (WHO, 2007). The 75 level of contamination might be reduced during the manufacturing process (WHO, 2007). To 76 determine if people have been exposed to various radiation levels directly or indirectly in this 77 regard, it is crucial to test the radioactivity of the environment and native herbs. 78 Life-threatening pollution from a nuclear disaster could result from something quite different. 65 WHO has developed protocols to follow in the case of significant radionuclide contamination 66 brought on by a catastrophic nuclear accident (WHO, 2007). Actinides, activation products, and 67 long- and short-lived fission products are a few examples of these radionuclides. For 238U and 68 226Ra, the lungs and kidneys are typically the sites of deposition; for 232Th, the lungs, liver, or 69 bones may all accumulate; and for 40K, the body as a whole, but mostly in the muscles (Akhter et 70 al., 2007; Nahar et al., 2018; Ugbede et al., 2022). As a result, the immune system of the body 71 may weaken in the face of many illnesses with higher death rates (Nahar et al., 2018; Ugbede et 72 al., 2022). The amount of exposure to radiation depends on the intake of radionuclides, and its 73 significance depends in turn on other variables such as the age, metabolic kinetics, and weight of 74 the individual who ingests them (also known as the dose conversion factor) (WHO, 2007). The 75 level of contamination might be reduced during the manufacturing process (WHO, 2007). EXAMINATION OF MEDICINAL PLANTS FOR RADIONUCLIDES ABSORPTION 1 AND THEIR HEALTH IMPLICATIONS 2 55 The contamination of herbal material or products can be at various stages of the manufacturing process 56 (WHO, 2007). The consumption of medicinal plants with high levels of natural radioactivity can 57 cause health problems (Sussa et al., 2013; Kareem et al., 2016). Since most medicinal products 58 are applied topically to the skin and taken orally, radioactive substances present in large amounts 59 may expose people to radiation both internally and externally, which could harm humans (Oni et 60 Medicinal plants can be found in their natural state or processed (Saudi et al., 2022). Physical, 53 chemical, and biological pollutants can be found in herbal remedies. (WHO, 2007). Human waste, animal 54 manure, and sewage used as fertilizer can produce chemical and microbiological pollutants (WHO, 2007). 55 The contamination of herbal material or products can be at various stages of the manufacturing process 56 (WHO, 2007). The consumption of medicinal plants with high levels of natural radioactivity can 57 cause health problems (Sussa et al., 2013; Kareem et al., 2016). Since most medicinal products 58 are applied topically to the skin and taken orally, radioactive substances present in large amounts 59 may expose people to radiation both internally and externally, which could harm humans (Oni et 60 2 2 al., 2011). The particular metabolic character of plant species may lead to the accumulation of 61 radionuclides in their organs, which may depend upon the soil's physicochemical characteristics 62 (Hashim et al., 2019). An individual’s annual effective dose from ingestion increases because of 63 increased concentrations of radioactive elements in plants, increasing the risk of radiological harm. 64 radionuclides in their organs, which may depend upon the soil's physicochemical characteristics 62 (Hashim et al., 2019). An individual’s annual effective dose from ingestion increases because of 63 increased concentrations of radioactive elements in plants, increasing the risk of radiological harm. 64 Life-threatening pollution from a nuclear disaster could result from something quite different. 65 WHO has developed protocols to follow in the case of significant radionuclide contamination 66 brought on by a catastrophic nuclear accident (WHO, 2007). Actinides, activation products, and 67 long- and short-lived fission products are a few examples of these radionuclides. EXAMINATION OF MEDICINAL PLANTS FOR RADIONUCLIDES ABSORPTION 1 AND THEIR HEALTH IMPLICATIONS 2 To 76 determine if people have been exposed to various radiation levels directly or indirectly in this 77 regard, it is crucial to test the radioactivity of the environment and native herbs. 78 The radiation levels and radioactivity concentrations in some foodstuffs like vegetables, fruits, 79 tubers, maize, and meat from the oil and gas production areas in Delta State, Nigeria, as reported 80 by (Tchokossa et al., 2013) indicated that the primordial radionuclides were within the 81 internationally recommended safe limits in the food types grown in the area. Jwanbot et al., (2013) 82 obtained results that indicate that the activity concentration of the farmland soils and crops is a 83 The radiation levels and radioactivity concentrations in some foodstuffs like vegetables, fruits, 79 tubers, maize, and meat from the oil and gas production areas in Delta State, Nigeria, as reported 80 by (Tchokossa et al., 2013) indicated that the primordial radionuclides were within the 81 internationally recommended safe limits in the food types grown in the area. Jwanbot et al., (2013) 82 obtained results that indicate that the activity concentration of the farmland soils and crops is a 83 3 significant health risk for the farmers and consumers of the crops in Barkin Ladi LGA, Plateau 84 State, Nigeria. The assessment done by Sowole & Olaniyi (2018) to determine the concentrations 85 of natural radionuclides in some fruits from markets at Ijebu-Ode town in Nigeria reveals that the 86 highest concentrations of 40K, 238U and 232Th were found in pineapple (102.36 ± 10.81 Bqkg−1), 87 orange (12.18 ± 4.36 Bqkg−1) and mango (8.01 ± 3.25 Bqkg−1) respectively, with an 88 average annual committed effective dose of 0.11 mSv. yr−1, which is lower than the limit of 89 0.3 mSv. yr−1 recommended by UNSCEAR (2000) implying that the ingestion of these fruits 90 poses no radiological health hazard to consumers. Numerous authors have documented 91 radioactivity in medicinal plants in Nigeria's River State (Port Harcourt), Niger State, and Ogun 92 State (Njinga et al., 2015; Oni et al., 2011; Sowole & Olaniyi, 2018). Accordingly, the findings of 93 this study can be utilized as a baseline value for radioactivity in medicinal plants in Esan Land, 94 Edo State, Nigeria. According to Abojassim & Lawi (2018), it is crucial to assess the radionuclide 95 concentrations in medicinal plants in order to avoid subjecting consumers to radiation. EXAMINATION OF MEDICINAL PLANTS FOR RADIONUCLIDES ABSORPTION 1 AND THEIR HEALTH IMPLICATIONS 2 Therefore, 96 this research was conducted to determine the level of natural radionuclide absorption by medicinal 97 plants and their health implication using medicinal herbs grown in Ewu, Edo State, Nigeria. 98 The sampling area 100 The medicinal herbs chosen for this study originated from Ewu in Edo State's Esan-Central Local 101 Government Area (LGA). The town is located 200 feet above sea level in Edo State's plateau 102 region and is 100 kilometres north of Benin City, the state's capital. Agbede, Irrua, and Ekpoma 103 form the northern, southeast, and southwest borders of Ewu, respectively (Omonhinmin, 2017). In 104 spite of the fact that farming constitutes the majority of the population's work, occasionally 105 government employees, students, traders, and other workers also engage in it. The existence of a 106 4 4 modern herbal manufacturing company (Pax Herbals) has greatly influenced interest in the 107 cultivation of herbs for both private and commercial uses; as a result, the majority of these people 108 have extensive native knowledge of traditional medicine as it is their main source of healthcare 109 and income. 110 modern herbal manufacturing company (Pax Herbals) has greatly influenced interest in the 107 cultivation of herbs for both private and commercial uses; as a result, the majority of these people 108 have extensive native knowledge of traditional medicine as it is their main source of healthcare 109 and income. 110 Sample collection and preparation 111 Samples of six (6) different medicinal plant parts (2 kg each) commonly used and available in the 112 localities of Ewu were collected. Table 1 shows medicinal plants and their medical uses. 113 Samples of six (6) different medicinal plant parts (2 kg each) commonly used and available in the 112 localities of Ewu were collected. Table 1 shows medicinal plants and their medical uses. 113 Table 1: Name and uses of selected medicinal plants Sample Code Scientific Name Common name Uses Part used A Magnifera indica Mango tree To treat diabetes, restlessness, gall and kidney stone, respiratory problems, dysentery Leaves B Dacryodes edulis African Pear To treat wound, skin diseases, dysentery and fever Leaves C Terminalia catappa Umbrella tree Gonorrhea, ulcers, cough, catarrh, haemoptysis, cholagoghe, astringent, cardiac tonic Leaves D Cymbopogon citratus Lemmon grass For treating rheumatism, fever, high blood pressure, convulsion, stomachache, vomiting, cough Leaves E Anacardium occidentale Cashew tree Diarrhhoea, blood pressure, reducing blodd sugar level, oral ulcer, sore throat Leaves F Persea americana Avocado To improve digestion, prevent cancer, regulate blood pressure, improve vision Leaves 116 5 The fresh leaves of the selected medicinal plants were collected within the locality of Ewu town, 117 Esan LGA, Edo state, Nigeria in the month of September 2022. The plants were identified and 118 authenticated the Professor J.C. Okafor Herbarium, Pax Herbal Clinic and Research Laboratories, 119 Ewu, Nigeria, and voucher specimens were deposited. The leaves were treated separately by 120 sorting, rinsed under running water, and air-dried at room temperature to remove the surface 121 moisture for a period of 5 days. Thereafter, they were separately transferred into an oven 122 maintained at 50˚C for another 2 days before pulverizing into powder using an electric milling 123 machine (Chris Norris, England) (Owolabi et al., 2019), and 300 g of each sample was packaged 124 into a labelled and tightly sealed plastic container and kept for 1 month to ensure secular 125 equilibrium in the 238U and 232Th with their respective progeny (Popoola et al., 2019). The samples 126 were then transported to the Radiation and Health Physics Laboratory of the Ladoke Akintola 127 University of Technology, Ogbomoso, Nigeria, for gamma spectrometric measurement. 128 Gamma Spectrometry Measurement 129 A 3'' x 3'' NaI(Tl) detector made by Princeton Gamma Tech in the USA is the basis of the gamma 130 spectrometry equipment used. The detector is protected from background radiation by a cylindrical 131 lead shield. The Gamma Spectacular (type GS-2000 Pro) multichannel analyzer was connected to 132 the detector, and the computer was connected to the analyzer for display. Theremino software was 133 used to acquire data and analyze gamma-ray spectra. 134 The NaI(Tl) spectrometry system was calibrated to determine a qualitative and quantitative 135 relationship between the peak position in the spectrum and the related gamma-ray energy. With 136 the use of the RSS8 gamma source set, which can be traced to Spectrum Techniques LLC, USA, 137 the detector's energy was calibrated. It was done by examining the spectra of two-point sources 138 (Cs-137 and Co-60) that generate gamma rays with known energy. A reference source of known 139 6 radionuclide activity, including 40K (578.4 Bq/kg), 238U (20.9 Bq/kg), and 232Th (10.47 Bq/kg), 140 was used to calibrate the detector's effectiveness. The standard sources are intended for the 141 detection of radionuclides such as 40K, 238U, and 232Th in the medicinal plants. 142 radionuclide activity, including 40K (578.4 Bq/kg), 238U (20.9 Bq/kg), and 232Th (10.47 Bq/kg), 140 was used to calibrate the detector's effectiveness. The standard sources are intended for the 141 detection of radionuclides such as 40K, 238U, and 232Th in the medicinal plants. 142 The background gamma-ray distribution count was calculated by counting an empty container for 143 36000s prior to the sample measurement. Once the sealed samples had reached a state of secular 144 equilibrium, they were all sequentially put on the detector for analysis. Every sample was counted 145 for the same amount of time as the container's empty counterpart. The most notable radionuclides 146 found in the samples were identified as having energies of 1460.0 keV (40K), 1764.5 keV of 214Bi 147 (238U), and 2614.7 keV of 208Tl (232Th). Estimation of Radiological Hazard 155 After determining the values for the individual naturally occurring radionuclides' specific activity 156 concentrations in medicinal plants, the effects of radiation on people's health and the environment 157 are evaluated using the radiation hazard below. 158 After determining the values for the individual naturally occurring radionuclides' specific activity 156 concentrations in medicinal plants, the effects of radiation on people's health and the environment 157 are evaluated using the radiation hazard below. 158 Gamma Spectrometry Measurement 129 Each detected radionuclide in the sample had its activity 148 concentration A (Bqkg−1) measured by: 149 𝐴= 𝐶𝑛𝑒𝑡 𝑃𝛾× 𝜀× 𝑣× 𝑡 (1) 𝐴= 𝐶𝑛𝑒𝑡 𝑃𝛾× 𝜀× 𝑣× 𝑡 (1) 150 (1) Where Cnet is the net peak count for each radionuclide present in the sample after subtracting the 151 background count from the gross count, Pγ is the absolute gamma-ray emission probability of the 152 identified radionuclide, ε is the obtained full energy peak efficiency for each identified 153 radionuclide, v is the volume of the sample and t is the counting time. 154 i. Radium Equivalent Activity, (𝐑𝐚𝐞𝐪) 159 Absorbed Dose Rate 168 The amount of energy that ionizing radiation deposits per unit mass in a material is measured by 169 Raeq (Bqkg−1) = CU + 1.43CTh + 0.077CK (2) 165 Raeq (Bqkg−1) = CU + 1.43CTh + 0.077CK (2) 165 (2) Raeq (Bqkg−1) = CU + 1.43CTh + 0.077CK (2) 165 WhereCU, CTh and CK are the activity concentrations of 238U, 232Th and 40K, respectively 166 in Bqkg−1. 167 WhereCU, CTh and CK are the activity concentrations of 238U, 232Th and 40K, respectively 166 in Bqkg−1. 167 The amount of energy that ionizing radiation deposits per unit mass in a material is measured by 169 the absorbed dose. The dose rate, D, due to the activity concentrations of 238U, 232Th, and 40K was 170 determined in accordance with (Ofomola et al., 2023; Popoola et al., 2019; UNSCEAR, 2000): 171 D (nGy/h) = 0.462 CU + 0.604 CTh + 0.0417 CK (3) 172 The amount of energy that ionizing radiation deposits per unit mass in a material is measured by 169 the absorbed dose. The dose rate, D, due to the activity concentrations of 238U, 232Th, and 40K was 170 determined in accordance with (Ofomola et al., 2023; Popoola et al., 2019; UNSCEAR, 2000): 171 D (nGy/h) 0 462 C + 0 604 C + 0 0417 C (3) 172 (3) D (nGy/h) = 0.462 CU + 0.604 CTh + 0.0417 CK (3) 172 Where CU, CTh and CK are the activity concentrations of 238U, 232Th and 40K, respectively, and 173 coefficients are dose rate conversion factors. 174 Where CU, CTh and CK are the activity concentrations of 238U, 232Th and 40K, respectively, and 173 coefficients are dose rate conversion factors. 174 iii. Hazard Indices (𝐇𝐞𝐱 and 𝐇𝐢𝐧) 175 iii. i. Radium Equivalent Activity, (𝐑𝐚𝐞𝐪) 159 The radium equivalent (Raeq) activity is used to compare the activities of 238U, 232Th and 40K in 160 samples by a single quantity (Ugbede, 2020). It is the weighted sum of activities of 238U, 232Th and 161 The radium equivalent (Raeq) activity is used to compare the activities of 238U, 232Th and 40K in 160 samples by a single quantity (Ugbede, 2020). It is the weighted sum of activities of 238U, 232Th and 161 7 7 40K. It is calculated that 1 Bq/kg of 238U, 0.7 Bq/kg of 232Th and 13 Bq/kg of 40K provide th 162 same radiation dosage. The radium equivalent activity index was computed using (Ononugbo e 163 al., 2017; Sultana et al., 2020). 164 Raeq (Bqkg−1) = CU + 1.43CTh + 0.077CK (2 165 WhereCU, CTh and CK are the activity concentrations of 238U, 232Th and 40K, respectivel 166 in Bqkg−1. 167 ii. Absorbed Dose Rate 168 The amount of energy that ionizing radiation deposits per unit mass in a material is measured b 169 the absorbed dose. The dose rate, D, due to the activity concentrations of 238U, 232Th, and 40K wa 170 determined in accordance with (Ofomola et al., 2023; Popoola et al., 2019; UNSCEAR, 2000): 171 D (nGy/h) = 0.462 CU + 0.604 CTh + 0.0417 CK (3 172 Where CU, CTh and CK are the activity concentrations of 238U, 232Th and 40K, respectively, an 173 coefficients are dose rate conversion factors. 174 iii. Hazard Indices (𝐇𝐞𝐱 and 𝐇𝐢𝐧) 175 Indicators for external hazard, Hex and the internal hazard, H𝑖𝑛 were estimated using (UNSCEAR 176 2000; Ononugbo et al., 2017; Ugbede, 2020): 177 𝐻𝑒𝑥= 𝐶𝑈 370 + 𝐶𝑇ℎ 259 + 𝐶𝐾 4810 ≤ 1 (4 178 same radiation dosage. The radium equivalent activity index was computed using (Ononugbo et 163 al., 2017; Sultana et al., 2020). 164 Raeq (Bqkg−1) = CU + 1.43CTh + 0.077CK (2) 165 WhereCU, CTh and CK are the activity concentrations of 238U, 232Th and 40K, respectively 166 in Bqkg−1. 167 ii. i. Radium Equivalent Activity, (𝐑𝐚𝐞𝐪) 159 Hazard Indices (𝐇𝐞𝐱 and 𝐇𝐢𝐧) 175 Indicators for external hazard, Hex and the internal hazard, H𝑖𝑛 were estimated using (UNSCEAR, 176 Indicators for external hazard, Hex and the internal hazard, H𝑖𝑛 were estimated using (UNSCEAR, 176 Indicators for external hazard, Hex and the internal hazard, H𝑖𝑛 were estimat 176 Indicators for external hazard, Hex and the internal hazard, H𝑖𝑛 were estimated using (UNSCEAR, 176 2000; Ononugbo et al., 2017; Ugbede, 2020): 177 2000; Ononugbo et al., 2017; Ugbede, 2020): 177 2000; Ononugbo et al., 2017; Ugbede, 2020): 177 𝐻𝑒𝑥= 𝐶𝑈 370 + 𝐶𝑇ℎ 259 + 𝐶𝐾 4810 ≤ 1 (4) 178 𝐻𝑖𝑛= 𝐶𝑈 185 + 𝐶𝑇ℎ 259 + 𝐶𝐾 4810 < 1 (5) 179 𝐻𝑒𝑥= 𝐶𝑈 370 + 𝐶𝑇ℎ 259 + 𝐶𝐾 4810 ≤ 1 (4) 178 (4) 𝐻𝑖𝑛= 𝐶𝑈 185 + 𝐶𝑇ℎ 259 + 𝐶𝐾 4810 < 1 (5) 179 𝐻𝑖𝑛= 𝐶𝑈 185 + 𝐶𝑇ℎ 259 + 𝐶𝐾 4810 < 1 (5) 179 (5) Where CU, CTh and CK are the activity concentrations of 238U, 232Th and 40K, respectively 180 Where CU, CTh and CK are the activity concentrations of 238U, 232Th and 40K, respectively 180 Where CU, CTh and CK are the activity concentrations of 238U, 232Th and 40K, respectively 180 iv. Annual Gonadal Equivalent Dose (AGED) 181 iv. Annual Gonadal Equivalent Dose (AGED) 181 iv. Annual Gonadal Equivalent Dose (AGED) 181 8 8 The Annual Gonadal Equivalent Dose (AGED) is the risk radiation poses to susceptible cells, such 182 as the gonads, bone marrow, and surface cells. It has been observed that an increase in AGED 183 affects the bone marrow, causing the destruction of red blood cells and their replacement by white 184 blood cells. This syndrome manifests itself as leukemia, a deadly type of blood cancer. (Ononugbo 185 et al., 2017). The equation (Avwiri et al., 2014; Saudi et al., 2022) is used to determine AGED 186 given activity concentrations of 238U, 232Th and 40K: 187 The Annual Gonadal Equivalent Dose (AGED) is the risk radiation poses to susceptible cells, such 182 as the gonads, bone marrow, and surface cells. It has been observed that an increase in AGED 183 affects the bone marrow, causing the destruction of red blood cells and their replacement by white 184 blood cells. This syndrome manifests itself as leukemia, a deadly type of blood cancer. (Ononugbo 185 et al., 2017). i. Radium Equivalent Activity, (𝐑𝐚𝐞𝐪) 159 The equation (Avwiri et al., 2014; Saudi et al., 2022) is used to determine AGED 186 given activity concentrations of 238U, 232Th and 40K: 187 𝐴𝐺𝐸𝐷 (𝑆𝑣𝑦𝑟 ⁄ ) = 3.09 𝐶𝑈 + 4.18 𝐶𝑇ℎ + 0.314 𝐶𝐾 (6) 188 𝐴𝐺𝐸𝐷 (𝑆𝑣𝑦𝑟 ⁄ ) = 3.09 𝐶𝑈 + 4.18 𝐶𝑇ℎ + 0.314 𝐶𝐾 (6) 188 𝐴𝐺𝐸𝐷 (𝑆𝑣𝑦𝑟 ⁄ ) = 3.09 𝐶𝑈 + 4.18 𝐶𝑇ℎ + 0.314 𝐶𝐾 (6) 188 (6) Where CU, CTh, and CK are the activity concentrations of 238U, 232Th and 40K respectively. The 189 standard UNSCEAR value for AGED is 300 mSv.yr-1. 190 Where CU, CTh, and CK are the activity concentrations of 238U, 232Th and 40K respectively. The 189 standard UNSCEAR value for AGED is 300 mSv.yr-1. 190 Where CU, CTh, and CK are the activity concentrations of 238U, 232Th and 40K respectively. The 189 standard UNSCEAR value for AGED is 300 mSv.yr-1. 190 v. Representative Gamma Index (𝑰𝜸): 191 This is done to determine the gamma radiation hazards connected to the radionuclides in the 192 samples of medicinal plants under investigation. The representative gamma index is given as 193 follows (Ofomola et al., 2023; Ononugbo et al., 2017): 194 𝐼𝛾= 𝐶𝑈 150 + 𝐶𝑇ℎ 100 + 𝐶𝐾 1500 (7) 195 𝐼𝛾= 𝐶𝑈 150 + 𝐶𝑇ℎ 100 + 𝐶𝐾 1500 (7) 𝐼𝛾= 𝐶𝑈 150 + 𝐶𝑇ℎ 100 + 𝐶𝐾 1500 (7) 195 (7) Values of 𝐼𝛾≤ 1 indicate an annual effective dose of less than or equal to 1 mSv, whereas 𝐼𝛾 ≤ 196 0.5 indicates an annual effective dose less than or equal to 0.3 mSv (Ugbede, 2020). 197 𝛾≤ q , 𝛾≤ 0.5 indicates an annual effective dose less than or equal to 0.3 mSv (Ugbede, 2020). 197 0.5 indicates an annual effective dose less than or equal to 0.3 mSv (Ugbede, 2020). 197 vi. i. Radium Equivalent Activity, (𝐑𝐚𝐞𝐪) 159 (2022), it is calculated 215 as follows: 216 𝐸𝐿𝐶𝑅= 𝐸𝐶𝐸𝐷 × 𝐷𝐿 × 𝑅𝐹 (9) 217 Where 𝐸𝐶𝐸𝐷 is the average annual committed effective dose equivalent, RF is the fatal risk factor 218 and DL is the duration of life. The RF to the public is taken to be 0.05 Sv/y (Popoola et al., 2019) 219 and DL is 54.5 years, the life expectancy at birth in Nigeria (WHO, 2015). 220 i. Radium Equivalent Activity, (𝐑𝐚𝐞𝐪) 159 Average annual committed effective dose (𝐸𝐶𝐸𝐷) 198 The 𝐸𝐶𝐸𝐷 for ingestion of NORMS in medicinal plants was calculated using the expression in 199 equation (8) (Saudi et al 2022; Tettey larbi et al 2013): 200 The 𝐸𝐶𝐸𝐷 for ingestion of NORMS in medicinal plants was calculated using the expression in 199 equation (8) (Saudi et al., 2022; Tettey-larbi et al., 2013): 200 𝐸𝐶𝐸𝐷= 𝐴𝑐∗𝐷𝐶𝐹𝑖𝑛𝑔∗𝐶𝑟 (8) 201 𝐸𝐶𝐸𝐷= 𝐴𝑐∗𝐷𝐶𝐹𝑖𝑛𝑔∗𝐶𝑟 (8) 201 𝐸𝐶𝐸𝐷= 𝐴𝑐∗𝐷𝐶𝐹𝑖𝑛𝑔∗𝐶𝑟 (8) (8) where 𝐸𝐶𝐸𝐷 is the average annual committed effective dose, 𝐴𝑐 is the activity concentration of 202 each radionuclide in the plant sample, 𝐷𝐶𝐹𝑖𝑛𝑔 is the dose conversion coefficient for ingestion for 203 where 𝐸𝐶𝐸𝐷 is the average annual committed effective dose, 𝐴𝑐 is the activity concentration of 202 each radionuclide in the plant sample, 𝐷𝐶𝐹𝑖𝑛𝑔 is the dose conversion coefficient for ingestion for 203 9 each radionuclide (i.e. 6.2 × 10−9 mSv/Bq, 4.5 × 10−8 mSv/Bq, and 2.3 × 10−7 mSv/Bq for 204 40K, 238U, and 232Th respectively for an adult (UNSCEAR 2000), and 𝐶𝑟 is the rate of consumption 205 rate from intake of NORMS in medicinal plants. 206 each radionuclide (i.e. 6.2 × 10−9 mSv/Bq, 4.5 × 10−8 mSv/Bq, and 2.3 × 10−7 mSv/Bq for 204 40K, 238U, and 232Th respectively for an adult (UNSCEAR 2000), and 𝐶𝑟 is the rate of consumption 205 rate from intake of NORMS in medicinal plants. 206 Although there is no set dosage for medicinal plants in Nigeria, it was assumed that a patient 207 requires 100 mL/day of herbal preparation or product to successfully treat a common condition 208 (Njinga et al., 2015). As a result, it was assumed that the medicinal plants used in the present study 209 had a NORM consumption rate of 1 kg/yr (Alade et al., 2020; Njinga et al., 2015). 210 vii. Excess Lifetime Cancer Risk (ELCR) 211 This has to do with the potential for a lifelong case of cancer at a specific level of exposure. It is 212 expressed as a number that indicates the number of extra cancers that could be detected in a 213 specified number of individuals. This is following exposure to a carcinogen at a particular dose. 214 According to Orosun et al. (2018), Popoola et al. (2019), and Saudi et al. vii. Excess Lifetime Cancer Risk (ELCR) 211 This has to do with the potential for a lifelong case of cancer at a specific level of exposure. It is 212 expressed as a number that indicates the number of extra cancers that could be detected in a 213 specified number of individuals. This is following exposure to a carcinogen at a particular dose. 214 According to Orosun et al. (2018), Popoola et al. (2019), and Saudi et al. (2022), it is calculated 215 as follows: 216 𝐸𝐿𝐶𝑅= 𝐸 × 𝐷𝐿× 𝑅𝐹 (9) 𝐸𝐿𝐶𝑅= 𝐸𝐶𝐸𝐷 × 𝐷𝐿 × 𝑅𝐹 (9) (9) Where 𝐸𝐶𝐸𝐷 is the average annual committed effective dose equivalent, RF is the fatal risk factor 218 and DL is the duration of life. The RF to the public is taken to be 0.05 Sv/y (Popoola et al., 2019) 219 and DL is 54.5 years, the life expectancy at birth in Nigeria (WHO, 2015). 220 Where 𝐸𝐶𝐸𝐷 is the average annual committed effective dose equivalent, RF is the fatal risk factor 218 and DL is the duration of life. The RF to the public is taken to be 0.05 Sv/y (Popoola et al., 2019) 219 and DL is 54.5 years, the life expectancy at birth in Nigeria (WHO, 2015). 220 Table 2: Activity concentration (Bq/L) of natural radionuclides in the medicinal plant samples 223 Sample Code Scientific Name 40K 238U 232Th A Magnifera indica 241.39 ± 6.16 5.57 ± 0.15 12.07 ± 0.57 Table 2: Activity concentration (Bq/L) of natural radionuclides in the medicinal plant samples 223 Sample Code Scientific Name 40K 238U 232Th A Magnifera indica 241.39 ± 6.16 5.57 ± 0.15 12.07 ± 0.57 10 B Dacryodes edulis 202.14 ± 5.64 4.97 ± 0.29 7.76 ± 0.18 C Terminalia catappa 185.67 ± 5.41 5.31 ± 0.09 9.19 ± 0.50 D Cymbopogon citratus 296.08 ± 3.41 2.25 ± 0.06 4.50 ± 0.35 E Anacardium occidentale 184.71 ± 5.40 5.06 ± 0.08 9.51 ± 0.59 F Persea americana 146.59 ± 4.81 5.21 ± 0.25 4.97 ± 0.19 Min 146.59 ± 4.81 2.25 ± 0.06 4.50 ± 0.35 Max 296.08 3.42 5.57 ± 0.15 12.07 ± 0.57 Mean 209.43 ± 5.14 4.73 ± 0.15 8.00 ± 0.40 224 The results of activity concentrations of 40K, 238U and 232Th obtained for the medicinal plants 225 samples in Ewu is presented in Table 2. vii. Excess Lifetime Cancer Risk (ELCR) 211 The average activity concentrations in the medicinal 226 plants’ samples for 40K ranged from 146.59 ± 4.81 (in Persea americana) to 296.08 3.42 Bq/kg 227 (in Cymbopogon citratus) with a mean of 209.43 ± 5.14 Bq/kg; for 238U, it ranged from 2.25 ± 228 0.06 to 5.57 ± 0.15 Bq/kg with a mean of 4.73 ± 0.15 Bq/kg and 232Th varied from 4.50 ± 0.35 to 229 12.07 ± 0.57 Bq/kg with a mean of 8.00 ± 0.40 Bq/kg. The maximum and minimum activity 230 concentration of both 238U and 232Th were found in Magnifera indica and Cymbopogon citratus 231 respectively. As can be noted, the radionuclides were present in all the examined samples, though 232 at varying concentrations. Variances in the geological location and radiochemical makeup of the 233 soils where medicinal plants are produced cause variations in natural radionuclide activity 234 concentrations (Tettey-larbi et al., 2013). 235 The mean concentrations of 40K, 238U, and 232Th radionuclides in the examined medicinal plants 236 are lower than the worldwide average of 400 Bq/kg for 40K, 33 Bq/kg for 238U, and 45 Bq/kg for 237 232Th (UNSCEAR, 2000). It is obvious that the activity concentration of 40K was the highest of all 238 the samples (Fig. 1). 239 The mean concentrations of 40K, 238U, and 232Th radionuclides in the examined medicinal plants 236 are lower than the worldwide average of 400 Bq/kg for 40K, 33 Bq/kg for 238U, and 45 Bq/kg for 237 232Th (UNSCEAR, 2000). It is obvious that the activity concentration of 40K was the highest of all 238 the samples (Fig. 1). 239 11 240 241 242 243 Fig.1: Activity concentration in selected samples of medicinal plants. 244 245 0 50 100 150 200 250 300 350 A B C D E F K-40 U-238 Th-232 243 Fig.1: Activity concentration in selected samples of medicinal plants. 244 245 According to their metabolic needs and potential regional differences, plants may absorb varied 246 amounts of potassium from soil (Kolapo & Omoboyede, 2018). Popoola et al., (2019) reported 247 radionuclide concentration of 57.80 ± 1.7 Bq/Kg and 30.19 ± 1.22 Bq/Kg in the soil of higher 248 educational institutions in Ogwa and Igueben, respectively. The results of 40K in this study imply 249 that the area under study has experienced some 40K enrichment. vii. Excess Lifetime Cancer Risk (ELCR) 211 These plants have a high potassium 250 activity concentration because they can absorb potassium from the soil more readily than other 251 elements (Tettey-larbi et al., 2013) and maybe as a result of fertilizer application by farmers to 252 increase agricultural yields. When compared to the concentration reported by (Oni et al., 2011) for 253 Ugheli and Ogbomoso, the 40K concentrations for Cymbopogon citratus obtained in this study are 254 significantly higher. Similar to this, Terminalia catappa had 40K concentrations that were greater 255 than the 145.59 ± 7.19 Bq/kg reported by (Njinga et al., 2015) but with elevated concentration of 256 According to their metabolic needs and potential regional differences, plants may absorb varied 246 amounts of potassium from soil (Kolapo & Omoboyede, 2018). Popoola et al., (2019) reported 247 radionuclide concentration of 57.80 ± 1.7 Bq/Kg and 30.19 ± 1.22 Bq/Kg in the soil of higher 248 educational institutions in Ogwa and Igueben, respectively. The results of 40K in this study imply 249 that the area under study has experienced some 40K enrichment. These plants have a high potassium 250 activity concentration because they can absorb potassium from the soil more readily than other 251 elements (Tettey-larbi et al., 2013) and maybe as a result of fertilizer application by farmers to 252 increase agricultural yields. When compared to the concentration reported by (Oni et al., 2011) for 253 Ugheli and Ogbomoso, the 40K concentrations for Cymbopogon citratus obtained in this study are 254 significantly higher. Similar to this, Terminalia catappa had 40K concentrations that were greater 255 than the 145.59 ± 7.19 Bq/kg reported by (Njinga et al., 2015) but with elevated concentration of 256 12 12 232Th. Magnifera indica’s activity concentration of 40K for this investigation is lower than the 257 389.63 ± 34.42 Bq/kg reported by (Alade et al., 2020). The high activity concentration of 40K in 258 leaves may be due to the significant function that potassium plays in plant photosynthesis, which 259 causes it to accumulate more in leaves where photosynthesis occurs most frequently. 260 (Saenboonruang et al., 2018). 261 Table 3: Comparison of activity concentration (Bq/L) of natural radionuclides in medicinal plant 62 samples. vii. Excess Lifetime Cancer Risk (ELCR) 211 63 Activity concentration (Bq.𝐤𝐠−𝟏) References Country 40K 238U 232Th Nigeria 209.43 4.73 8.00 This study Bangladesh 661.10 12.65 7.38 (Sultana et al., 2020) Iraq 219.1 4.68 2.91 (Kareem et al., 2016) Turkey 1910 - 5.67 (Cengiz & Çağlar, 2019) Egypt 471.4 7.25 7.78 (Saudi et al., 2022) India 230.00 - 36.00 (Monica et al., 2020) Uganda 359.59 - 9.65 (Biira et al., 2021) Nigeria 171.72 - 35.09 (Njinga et al., 2015) Nigeria 630.02 5.79 4.13 (Alade et al., 2020) Ghana 839.80 31.78 56.16 (Tettey-larbi et al., 2013) World 400 33 45 UNSCEAR, 2000 64 265 13 266 Fig. 2: Comparison of the mean activity concentration (Bq/L) of 40K, 238U and, 232Th radionuclides 267 in medicinal plant samples with other parts of the world. 268 0 200 400 600 800 1000 1200 1400 1600 1800 2000 40K 238U 232Th Fig. 2: Comparison of the mean activity concentration (Bq/L) of 40K, 238U and, 232Th radionuclides 267 in medicinal plant samples with other parts of the world. 268 A comparison of the mean activity concentration of 40K, 238U and, 232Th radionuclides in the 270 examined medicinal plant to other studies in other parts of the world is presented in Table 3 and 271 illustrated in Fig. 2. There is no doubt that 40K is prevalent in medicinal plants around the world, 272 as can be seen from the chart. 273 A comparison of the mean activity concentration of 40K, 238U and, 232Th radionuclides in the 270 examined medicinal plant to other studies in other parts of the world is presented in Table 3 and 271 illustrated in Fig. 2. There is no doubt that 40K is prevalent in medicinal plants around the world, 272 as can be seen from the chart. 273 A comparison of the mean activity concentration of 40K, 238U and, 232Th radionuclides in the 270 examined medicinal plant to other studies in other parts of the world is presented in Table 3 and 271 illustrated in Fig. 2. There is no doubt that 40K is prevalent in medicinal plants around the world, 272 as can be seen from the chart. 273 The activity concentration of 40K in this study is higher than the results reported by (Njinga et al., 274 2015) in Lapai, Niger State, Nigeria, and slightly below the results found in Iraq (Kareem et al., 275 2016) and India (Monica et al., 2020). vii. Excess Lifetime Cancer Risk (ELCR) 211 Our results demonstrate that the 280 level of 238U in medicinal plant samples is comparable to that reported by Kareem et al. (2016) in 281 Iraq but lower than that Saudi et al. (2022) in Egypt, Sultana et al. (2010) in Bangladesh, Alade et 282 al. (2010) in Ibadan, Nigeria, and Ghana (Tettey-larbi et al. (2013). The findings of the 232Th study 283 demonstrate that our findings are comparable to those of studies conducted in Egypt and 284 Bangladesh, respectively (Saudi et al., 2022) and Bangladesh (Sultana et al., 2020). Also, 232Th is 285 higher than the results found in Iraq (Kareem et al., 2016), Ibadan, Nigeria (Alade et al., 2020), 286 and Turkey (Cengiz & Çağlar, 2019) and lower than the results found in India (Monica et al., 287 2020), Uganda (Biira et al., 2021), Lapai, Nigeria (Njinga et al., 2015), and Ghana (Tettey-Larbi 288 et al., 2013). It's possible that variances in geography, geology, and the mineral makeup of 289 different sample locations are accountable for the discrepancy in natural radioactivity 290 concentrations across the various sampling sites. 291 vii. Excess Lifetime Cancer Risk (ELCR) 211 However, it is more than nine times lower than that found 276 in Turkey (Cengiz & Çağlar, 2019), and four times lower than that recorded in Ghana (Tettey-larbi 277 et al., 2013). It is lower than that of Uganda (Biira et al., 2021) and is more than three times lower 278 than that recorded in Bangladesh (Sultana et al., 2020), and Ibadan (Alade et al., 2020), but more 279 The activity concentration of 40K in this study is higher than the results reported by (Njinga et al., 274 2015) in Lapai, Niger State, Nigeria, and slightly below the results found in Iraq (Kareem et al., 275 2016) and India (Monica et al., 2020). However, it is more than nine times lower than that found 276 in Turkey (Cengiz & Çağlar, 2019), and four times lower than that recorded in Ghana (Tettey-larbi 277 et al., 2013). It is lower than that of Uganda (Biira et al., 2021) and is more than three times lower 278 than that recorded in Bangladesh (Sultana et al., 2020), and Ibadan (Alade et al., 2020), but more 279 14 than twice lower than that obtained in Egypt (Saudi et al., 2022). Our results demonstrate that the 280 level of 238U in medicinal plant samples is comparable to that reported by Kareem et al. (2016) in 281 Iraq but lower than that Saudi et al. (2022) in Egypt, Sultana et al. (2010) in Bangladesh, Alade et 282 al. (2010) in Ibadan, Nigeria, and Ghana (Tettey-larbi et al. (2013). The findings of the 232Th study 283 demonstrate that our findings are comparable to those of studies conducted in Egypt and 284 Bangladesh, respectively (Saudi et al., 2022) and Bangladesh (Sultana et al., 2020). Also, 232Th is 285 higher than the results found in Iraq (Kareem et al., 2016), Ibadan, Nigeria (Alade et al., 2020), 286 and Turkey (Cengiz & Çağlar, 2019) and lower than the results found in India (Monica et al., 287 2020), Uganda (Biira et al., 2021), Lapai, Nigeria (Njinga et al., 2015), and Ghana (Tettey-Larbi 288 et al., 2013). It's possible that variances in geography, geology, and the mineral makeup of 289 different sample locations are accountable for the discrepancy in natural radioactivity 290 concentrations across the various sampling sites. 291 than twice lower than that obtained in Egypt (Saudi et al., 2022). Radiological hazard indices for medicinal plants 292 Table 4 presents the estimated radiological hazard indices in medicinal plant samples. The samples' 293 results for radium equivalent activity ranged from 23.6 ± 0.89 to 41.42 ± 1.44 Bq/kg, with a mean 294 of 32.29 ± 1.12 Bq/kg. The obtained mean value was less than the Ibadan finding (Alade et al., 295 2020) and the 370 Bq/kg allowable global threshold (UNSCEAR, 2000). Additionally, it was 296 presented that the absorbed dose rate varied from 11.52 ± 0.43 to 19.93 ± 0.67 nGy/h with a mean 297 of 15.75 ± 0.52 nGy/h which is lower than the public exposure rate of 84 nGy/h (UNSCEAR, 2000). 298 Also, the estimated values of H𝑒𝑥 and H𝑖𝑛 are less than unity, with corresponding mean values of 299 0.087 ± 0.003 and 0.100 ± 0.003 respectively in the samples. These values do not exceed the 1 upper 300 maximum that is allowed. The values of H𝑒𝑥 and H𝑖𝑛 concur with (Saudi et al., 2022). The annual 301 gonadal equivalent dose (AGED) was also assessed and can be found in Table 4. AGED values have 302 15 a minimum value of 82.90 ± 3.08 Sv/yr and a maximum of 143.46 ± 4.78 with an average of 113.81 303 ± 3.75 Sv/yr. All values are below the stated value of (Saudi et al., 2022) and are within the range 304 of the 300 Sv/yr global recommendation (UNSCEAR, 2000). Another index evaluated is the gamma 305 representative index, Iγ which is a screening parameter for medicinal plants of possible radiological 306 health burden. The minimum, maximum and mean values are 0.18 ± 0.007, 0.32 ± 0.011 and 0.250 307 ± 0.008. the mean value compares well with (Saudi et al., 2022). All samples of medicinal plants 308 had Iγ values below 1, as observed, suggesting that they pose no radiological risk and may not have 309 any adverse effects. 310 a minimum value of 82.90 ± 3.08 Sv/yr and a maximum of 143.46 ± 4.78 with an average of 113.81 303 ± 3.75 Sv/yr. All values are below the stated value of (Saudi et al., 2022) and are within the range 304 of the 300 Sv/yr global recommendation (UNSCEAR, 2000). Another index evaluated is the gamma 305 representative index, Iγ which is a screening parameter for medicinal plants of possible radiological 306 health burden. Radiological hazard indices for medicinal plants 292 The minimum, maximum and mean values are 0.18 ± 0.007, 0.32 ± 0.011 and 0.250 307 ± 0.008. the mean value compares well with (Saudi et al., 2022). All samples of medicinal plants 308 had Iγ values below 1, as observed, suggesting that they pose no radiological risk and may not have 309 any adverse effects. Radiological hazard indices for medicinal plants 292 310 16 311 13 Sample Code Scientific Name 𝐑𝐚𝐞𝐪 (Bq/kg) D (nGy/h) 𝐇𝒆𝒙 𝐇𝒊𝒏 AGED mSv/y Gamma Index A Magnifera indica 41.42 ± 1.44 19.93 ± 0.67 0.112 ± 0.004 0.127 ± 0.004 143.46 ± 4.78 0.32 ± 0.011 B Dacryodes edulis 31.63 ± 0.98 15.41 ± 0.48 0.085 ± 0.003 0.099 ± 0.003 111.27 ± 3.42 0.25 ± 0.007 C Terminalia catappa 32.75 ± 1.22 15.75 ± 0.57 0.088 ± 0.003 0.103 ± 0.004 113.12 ± 4.07 0.25 ± 0.009 D Cymbopogon citratus 31.48 ± 0.82 16.10 ± 0.38 0.085 ± 0.002 0.091 ± 0.002 118.73 ± 2.72 0.26 ± 0.006 E Anacardium occidentale 32.88 ± 1.34 15.78 ± 0.62 0.089 ± 0.004 0.102 ± 0.004 113.39 ± 4.41 0.25 ± 0.010 F Persea americana 23.6 ± 0.89 11.52 ± 0.43 0.064 ± 0.002 0.078 ± 0.003 82.90 ± 3.08 0.18 ± 0.007 Minimum 23.6 ± 0.89 11.52 ± 0.43 0.064 ± 0.002 0.078 ± 0.003 82.90 ± 3.08 0.18 ± 0.007 Maximum 41.42 ± 1.44 19.93 ± 0.67 0.112 ± 0.004 0.127 ± 0.004 143.46 ± 4.78 0.32 ± 0.011 Mean 32.29 ± 1.12 15.75 ± 0.52 0.087 ± 0.003 0.100 ± 0.003 113.81 ± 3.75 0.250 ± 0.008 14 17 Table 5: Radiological hazard indices in medicinal plant samples 319 320 Sample Code Scientific Name 𝐸𝐶𝐸𝐷 (mSv/yr) 𝐸𝐶𝐸𝐷 (𝜇Sv/yr) ELCR (× 10−3) A Magnifera indica 0.00452 ± 0.00018 4.52 ± 0.176 0.01233 ± 0.00048 B Dacryodes edulis 0.00326 ± 0.00009 3.26 ± 0.089 0.00889 ± 0.00024 C Terminalia catappa 0.00350 ± 0.00015 3.50 ± 0.153 0.00955 ± 0.00024 D Cymbopogon citratus 0.00297 ± 0.00010 2.97 ± 0.104 0.00810 ± 0.00028 E Anacardium occidentale 0.00356 ± 0.00017 3.56 ± 0.173 0.00970 ± 0.00047 F Persea americana 0.00229 ± 0.00009 2.29 ± 0.085 0.00623 ± 0.00023 Minimum 0.00229 ± 0.00009 2.29 ± 0.085 0.00623 ± 0.00023 Maximum 0.00452 ± 0.00018 4.52 ± 0.176 0.01233 ± 0.00048 Mean 0.00335 ± 0.00013 3.35 ± 0.130 0.00913 ± 0.00035 321 Table 5: Radiological hazard indices in medicinal plant samples 319 The calculated average annual collective dose equivalent, 𝐸𝐶𝐸𝐷 for examined medicinal plants 322 have been presented in Table 5. The values vareied from 2.29 ± 0.085 to 4.52 ± 0.176 𝜇Sv/yr, with 323 3.35 ± 0.130 𝜇Sv/yr as the mean. Compared to the comparable global value of 1 mSv/yr, the 𝐸𝐶𝐸𝐷 324 values are less significant. Radiological hazard indices for medicinal plants 292 335 18 Overall, Sample D, Cymbopogon citratus, had the greatest value of all the medicinal plants under 336 examination (40K), whereas Sample A, Magnifera indica, had the highest value of all the hazard 337 indices. This is so because Magnifera indica, contains the greatest concentration of 232Th and 238U 338 out of all the samples, and 232Th is what mostly contributes to the estimated radiological risk. 339 Overall, Sample D, Cymbopogon citratus, had the greatest value of all the medicinal plants under 336 examination (40K), whereas Sample A, Magnifera indica, had the highest value of all the hazard 337 indices. This is so because Magnifera indica, contains the greatest concentration of 232Th and 238U 338 out of all the samples, and 232Th is what mostly contributes to the estimated radiological risk. 339 Radiological hazard indices for medicinal plants 292 According to UNSCEAR (2000), the general populace receives an 325 annual effective dose of NORMs from medicinal plants of 𝐸𝐶𝐸𝐷 = 0.3 mSv/yr. 232Th contributes 326 54.91% to the total average annual collective equivalent dose 𝐸𝐶𝐸𝐷, followed by 40K with 38.75%, 327 while 238U adds 6.35% to have the least impact on the 𝐸𝐶𝐸𝐷. In comparison to Alade et al. (2020), 328 Njinga et al. (2015), and Tettey-larbi et al. (2013), our findings for 𝐸𝐶𝐸𝐷 are of lesser value. The 329 consumers' health may not be affected by the yearly effective dosage of the therapeutic herbs in 330 this investigation. The minimum, maximum, and mean values of the estimated ELCR for the 331 medicinal plants are 0.00623 ± 0.00023, 0.01233 ± 0.00048 and 0.00913 ± 0.00035 respectively. 332 Since the results are lower than the UNSCEAR (2000) global average of 0.29 × 10−3, there is 333 extremely little probability of acquiring stomach cancer when these plant samples are used as 334 medications. 335 The calculated average annual collective dose equivalent, 𝐸𝐶𝐸𝐷 for examined medicinal plants 322 have been presented in Table 5. The values vareied from 2.29 ± 0.085 to 4.52 ± 0.176 𝜇Sv/yr, with 323 3.35 ± 0.130 𝜇Sv/yr as the mean. Compared to the comparable global value of 1 mSv/yr, the 𝐸𝐶𝐸𝐷 324 values are less significant. According to UNSCEAR (2000), the general populace receives an 325 annual effective dose of NORMs from medicinal plants of 𝐸𝐶𝐸𝐷 = 0.3 mSv/yr. 232Th contributes 326 54.91% to the total average annual collective equivalent dose 𝐸𝐶𝐸𝐷, followed by 40K with 38.75%, 327 while 238U adds 6.35% to have the least impact on the 𝐸𝐶𝐸𝐷. In comparison to Alade et al. (2020), 328 Njinga et al. (2015), and Tettey-larbi et al. (2013), our findings for 𝐸𝐶𝐸𝐷 are of lesser value. The 329 consumers' health may not be affected by the yearly effective dosage of the therapeutic herbs in 330 this investigation. The minimum, maximum, and mean values of the estimated ELCR for the 331 medicinal plants are 0.00623 ± 0.00023, 0.01233 ± 0.00048 and 0.00913 ± 0.00035 respectively. 332 Since the results are lower than the UNSCEAR (2000) global average of 0.29 × 10−3, there is 333 extremely little probability of acquiring stomach cancer when these plant samples are used as 334 medications. 3.2 Correlation Analysis 340 Pearson’s correlation analysis was conducted to ascertain the degree of association between the 341 examined radiological parameters in the medicinal plants. The matrix of established correlation 342 coefficient (r-values) is shown in Tables 6. Some of the highest correlations observed are 343 discussed. The analysis’s findings showed that 238U exhibited significantly strong negative 344 relationships with 40K contents in the dry season (r = -0.75, p= 0.0859). This result show that the 345 decrease in 238U values leads to an increase in the concentration of 40K. The association between 346 40K with (D, AGED and 𝐼𝛾) is strong and not statistically significant, according to the data. 347 The correlation matrix showed that 232Th exhibited a very strong and significant positive 348 correlation with H𝑖𝑛 (r= 0.83, p= 0.0410), with H𝑖𝑛 (0.92, p= 0.0095), with 𝐸𝐶𝐸𝐷 (r= 0.92, 349 p= 0.0087), ELCR (r= 0.92, p= 0.0087) in the medicinal plants. This could be as a result of the 350 fact that 232Th makes up the majority of the annual effective dose. The 𝐸𝐶𝐸𝐷 perfectly connected 351 to the internal exposure H𝑖𝑛 and ELCR. Additionally, 𝐸𝐶𝐸𝐷 has a very strong and near perfect 352 positive and significant relationship with Raeq (r= 0.98, p= 0.0006), H𝑒𝑥 (r= 0.98, p= 353 0.0006), AGED (r= 0.92, p= 0.0088), and 𝐼𝛾 (r= 0.93, p= 0.0071). Like the association of 354 𝐸𝐶𝐸𝐷 with the other radiological parameter, a similar trend was observed for ELCR. This implies 355 that there is a direct relationship between the 𝐸𝐶𝐸𝐷 and ELCR. 356 19 Table 6: Correlation coefficient matrix for all radiological variables 357 The average concentrations of 209.43 ± 5.14 378 Bq/kg, 4.73 ± 0.15 Bq/kg, and 8.00 ± 0.40 Bq/kg for 40K, 238U and 232Th, respectively are below 379 the global recommended limit set by UNSCEAR (2000) and all predicted radiological hazards fall 380 within the boundaries that have been established globally. The relationship between 232Th and 381 (𝐸𝐶𝐸𝐷 and ELCR) was shown to be extremely strong, positive, and significant. This indicates that 382 the 232Th radionuclide has a significant role in both the increased lifetime cancer risk brought on 383 by ingesting the studied herbal plant and the average annual collective effective dose, 𝐸𝐶𝐸𝐷. 384 Because of this, there is extremely little risk to customers' health and very little natural radiation 385 exposure associated with ingesting these therapeutic plants. This study can therefore be used as 386 baseline values for radioactivity of medicinal plants in Esan Land, Edo State, Nigeria and may 387 help develop standards and guidelines for medicinal plants as well as radiation protection rules 388 and regulations. 389 Table 6: Correlation coefficient matrix for all radiological variables 357 357 358 8 40K 238U 232Th Raeq (Bq/kg) D (nGy/h) H𝑒𝑥 H𝑖𝑛 AGED mSv/y Gamma Index 𝐼𝛾 𝐸𝐶𝐸𝐷 ELCR 40K 1 238U -0.75 1 232Th -0.06 0.66 1 Raeq (Bq/kg) 0.50 0.17 0.83** 1 D (nGy/h) 0.62 0.03 0.75* 0.99* 1 H𝑒𝑥 0.50 0.17 0.83 1.00* 0.99* 1 H𝑖𝑛 0.32 0.37 0.92 0.98* 0.94 0.98* 1 AGED mSv/y 0.67 -0.03 0.70 0.98* 1.00* 0.98* 0.92 1 Gamma Index, 𝐼𝛾 0.65 0.00 0.72 0.98* 1.00* 0.98* 0.93 1.00* 1 𝐸𝐶𝐸𝐷 0.33 0.34 0.92 0.98* 0.94 0.98* 1.00* 0.92 0.93 1 ELCR 0.33 0.34 0.92 0.98* 0.95 0.98* 1.00* 0.92 0.93 1.00* 1 9 r-values significant p<0.1; p<0.05; **p<0.001 0 1 20 CONCLUSION 374 375 Natural radionuclide activity concentrations of six (6) different samples of medicinal plants used 376 in the manufacture of herbal products were examined using the gamma spectrometric method. 377 40K, 238U, and 232Th were detected in all samples. The average concentrations of 209.43 ± 5.14 378 Bq/kg, 4.73 ± 0.15 Bq/kg, and 8.00 ± 0.40 Bq/kg for 40K, 238U and 232Th, respectively are below 379 the global recommended limit set by UNSCEAR (2000) and all predicted radiological hazards fall 380 within the boundaries that have been established globally. The relationship between 232Th and 381 (𝐸𝐶𝐸𝐷 and ELCR) was shown to be extremely strong, positive, and significant. This indicates that 382 the 232Th radionuclide has a significant role in both the increased lifetime cancer risk brought on 383 by ingesting the studied herbal plant and the average annual collective effective dose, 𝐸𝐶𝐸𝐷. 384 Because of this, there is extremely little risk to customers' health and very little natural radiation 385 exposure associated with ingesting these therapeutic plants. This study can therefore be used as 386 baseline values for radioactivity of medicinal plants in Esan Land, Edo State, Nigeria and may 387 help develop standards and guidelines for medicinal plants as well as radiation protection rules 388 and regulations. 389 CONCLUSION 374 375 Natural radionuclide activity concentrations of six (6) different samples of medicinal plants used 376 in the manufacture of herbal products were examined using the gamma spectrometric method. 377 40K, 238U, and 232Th were detected in all samples. Acknowledgement 390 https://doi.org/10.9790/5736- 425 1301010107 426 Avwiri G.O, Ononugbo C.P, & Nwokeoji I.E. (2014). Radiation Hazard Indices and Excess 427 Lifetime Cancer Risk in Soil Sediment and Water Around Mini Okoro/Oginigba Creek Port 428 Authors’ Contribution Statement 398 Authors’ Contribution Statement 398 Felix Adegoke Popoola conceptualized and proposed the project, created the study strategy, 399 laboratory analysis, and preliminary drafting. Oladotun Bamiji Isola was responsible for the 400 manuscript's validation, conclusion, text editing, and critical revision. Tunde Ayobami Owolabi 401 collected and prepared field samples, assisted with article composition, and edited the manuscript. 402 Oluseye Daniel Fakeye participated in developing the study plan and interpretation of data. Isaac 403 Oluwafemi Elijah and Sheu Isiaq Owoyemi conducted the search for relevant literature and 404 contributed to data acquisition. Modupe Eunice Sanyaolu performed the statistical analysis and 405 contributed to the article drafting. The article was revised and approved for submission by all 406 authors. 407 Felix Adegoke Popoola conceptualized and proposed the project, created the study strategy, 399 laboratory analysis, and preliminary drafting. Oladotun Bamiji Isola was responsible for the 400 manuscript's validation, conclusion, text editing, and critical revision. Tunde Ayobami Owolabi 401 collected and prepared field samples, assisted with article composition, and edited the manuscript. 402 Oluseye Daniel Fakeye participated in developing the study plan and interpretation of data. Isaac 403 Oluwafemi Elijah and Sheu Isiaq Owoyemi conducted the search for relevant literature and 404 contributed to data acquisition. Modupe Eunice Sanyaolu performed the statistical analysis and 405 contributed to the article drafting. The article was revised and approved for submission by all 406 authors. 407 Acknowledgement 390 The authors would like to appreciate the Management of Pax Herbal Clinic and laboratories fo 391 the assistance in the processing and preparation of plant materials used in this study. 392 Competing interest 393 The authors hereby declare that the study presented in this article is original and that no an 394 conflicting interest exist between us as regard to the study. 395 396 397 The authors would like to appreciate the Management of Pax Herbal Clinic and laboratories for 391 the assistance in the processing and preparation of plant materials used in this study. 392 The authors hereby declare that the study presented in this article is original and that no any 394 conflicting interest exist between us as regard to the study. 395 21 21 Authors’ Contribution Statement 398 Felix Adegoke Popoola conceptualized and proposed the project, created the study strategy, 399 laboratory analysis, and preliminary drafting. Oladotun Bamiji Isola was responsible for the 400 manuscript's validation, conclusion, text editing, and critical revision. Tunde Ayobami Owolabi 401 collected and prepared field samples, assisted with article composition, and edited the manuscript. 402 Oluseye Daniel Fakeye participated in developing the study plan and interpretation of data. Isaac 403 Oluwafemi Elijah and Sheu Isiaq Owoyemi conducted the search for relevant literature and 404 contributed to data acquisition. Modupe Eunice Sanyaolu performed the statistical analysis and 405 contributed to the article drafting. The article was revised and approved for submission by all 406 authors. 407 408 Availability of data and materials 409 The datasets and other relevant materials developed during the study can be made accessible by 410 the corresponding author upon reasonable request. 411 Funding 412 No funding was obtained for this study. 413 414 415 REFERENCES 416 417 Abojassim, A. A.; Lawi, D. 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https://openalex.org/W3041512673	https://acp.copernicus.org/articles/20/8083/2020/acp-20-8083-2020.pdf	English	null	Seasonal impact of biogenic very short-lived bromocarbons on lowermost stratospheric ozone between 60° N and 60° S during the 21st century	Atmospheric chemistry and physics	2,020	cc-by	17,695	orrespondence: Alfonso Saiz-Lopez (a.saiz@csic.es) and Rafael Pedro Fernandez (rpfernandez@m Received: 26 November 2019 – Discussion started: 24 January 2020 Revised: 7 May 2020 – Accepted: 13 May 2020 – Published: 13 July 2020 Abstract. Biogenic very short-lived bromocarbons (VSLBr) currently represent ∼25 % of the total stratospheric bromine loading. Owing to their much shorter lifetime compared to anthropogenic long-lived bromine (e.g. halons) and chlorine (e.g. chloroﬂuorocarbons), the impact of VSLBr on ozone peaks in the lowermost stratosphere, which is a key climatic and radiative atmospheric region. Here we present a mod- elling study of the evolution of stratospheric ozone and its chemical loss within the tropics and at mid-latitudes dur- ing the 21st century. Two different experiments are explored: considering and neglecting the additional stratospheric injec- tion of 5 ppt biogenic bromine naturally released from the ocean. Our analysis shows that the inclusion of VSLBr results in a realistic stratospheric bromine loading and improves the agreement between the model and satellite observations of the total ozone column (TOC) for the 1980–2015 period at mid-latitudes. We show that the overall ozone response to VSLBr at mid-latitudes follows the stratospheric evolu- tion of long-lived inorganic chlorine and bromine through- out the 21st century. Additional ozone loss due to VSLBr is maximized during the present-day period (1990–2010), with TOC differences of −8 DU (−3 %) and −5.5 DU (−2 %) for the Southern Hemisphere and Northern Hemisphere mid- latitudes (SH-MLs and NH-MLs), respectively. Moreover, the projected TOC differences at the end of the 21st century are ∼50 % lower than the values found for the present-day period. We ﬁnd that seasonal VSLBr impact on lowermost strato- spheric ozone at mid-latitude is inﬂuenced by the seasonal- ity of the heterogeneous inorganic-chlorine reactivation pro- cesses on ice crystals. Indeed, due to the more efﬁcient re- activation of chlorine reservoirs (mainly ClONO2 and HCl) within the colder SH-ML lowermost stratosphere, the sea- sonal VSLBr impact shows a small but persistent hemispheric asymmetry through the whole modelled period. Our results indicate that, although the overall VSLBr-driven ozone de- struction is greatest during spring, the halogen-mediated (Halogx-Loss) ozone loss cycle in the mid-latitude lower- most stratosphere during winter is comparatively more ef- ﬁcient than the HOx cycle with respect to other seasons. Indeed, when VSLBr are considered, Halogx-Loss dominates wintertime lowermost stratospheric ozone loss at SH-MLs between 1985 and 2020, with a contribution of inter-halogen ClOx–BrOx cycles to Halogx-Loss of ∼50 %. orrespondence: Alfonso Saiz-Lopez (a.saiz@csic.es) and Rafael Pedro Fernandez (rpfernandez@m Within the tropics, a small (< −2.5 DU) and relatively constant (∼−1 %) ozone depletion mediated by VSLBr is closely related to their ﬁxed emissions throughout the mod- elled period. By including the VSLBr sources, the seasonal Atmos. Chem. Phys., 20, 8083–8102, 2020 https://doi.org/10.5194/acp-20-8083-2020 © Author(s) 2020. This work is distributed under the Creative Commons Attribution 4.0 License. Seasonal impact of biogenic very short-lived bromocarbons on lowermost stratospheric ozone between 60◦N and 60◦S during the 21st century Javier Alejandro Barrera1, Rafael Pedro Fernandez1,2,3, Fernando Iglesias-Suarez2, Carlos Alberto Cuevas2, Jean-Francois Lamarque4, and Alfonso Saiz-Lopez2 1Institute for Interdisciplinary Science, National Research Council (ICB-CONICET), FCEN-UNCuyo, Mendoza, 5500, Argentina 2Department of Atmospheric Chemistry and Climate, Institute of Physical Chemistry Rocasolano, CSIC, Madrid, 28006, Spain 3Atmospheric and Environmental Studies Group (GEAA), UTN-FRM, Mendoza, 5500, Argentina 4Atmospheric Chemistry, Observations & Modelling Laboratory, National Center for Atmospheric Research, Boulder, CO 80301, USA Correspondence: Alfonso Saiz-Lopez (a.saiz@csic.es) and Rafael Pedro Fernandez (rpfernandez@mendoza-conicet.gob.ar) Received: 26 November 2019 – Discussion started: 24 January 2020 Revised: 7 May 2020 – Accepted: 13 May 2020 – Published: 13 July 2020 Atmos. Chem. Phys., 20, 8083–8102, 2020 https://doi.org/10.5194/acp-20-8083-2020 © Author(s) 2020. This work is distributed under the Creative Commons Attribution 4.0 License. Seasonal impact of biogenic very short-lived bromocarbons on lowermost stratospheric ozone between 60◦N and 60◦S during the 21st century Javier Alejandro Barrera1, Rafael Pedro Fernandez1,2,3, Fernando Iglesias-Suarez2, Carlos Alberto Cuevas2, Jean-Francois Lamarque4, and Alfonso Saiz-Lopez2 1Institute for Interdisciplinary Science, National Research Council (ICB-CONICET), FCEN-UNCuyo, Mendoza, 5500, Argentina 2Department of Atmospheric Chemistry and Climate, Institute of Physical Chemistry Rocasolano, CSIC, Madrid, 28006, Spain 3Atmospheric and Environmental Studies Group (GEAA), UTN-FRM, Mendoza, 5500, Argentina 4Atmospheric Chemistry, Observations & Modelling Laboratory, National Center for Atmospheric Research, Boulder, CO 80301, USA 1Institute for Interdisciplinary Science, National Research Council (ICB-CONICET), FCEN-UNCuyo, Mendoza, 5500, Argentina 2Department of Atmospheric Chemistry and Climate, Institute of Physical Chemistry Rocasolano CSIC, Madrid, 28006, Spain 3Atmospheric and Environmental Studies Group (GEAA), UTN-FRM, Mendoza, 5500, Argentina 4Atmospheric Chemistry, Observations & Modelling Laboratory, National Center for Atmospheric Research, Boulder, CO 80301, USA Correspondence: Alfonso Saiz-Lopez (a.saiz@csic.es) and Rafael Pedro Fernandez (rpfernandez@mendoza-conicet.gob.ar) Correspondence: Alfonso Saiz-Lopez (a.saiz@csic.es) and Rafael Pedro Fernandez (rpfernandez@mendoza-conicet.gob.ar) 1 Introduction The role of bromine in stratospheric ozone depletion has been discussed in several studies (Wofsy et al., 1975; Prather and Watson, 1990; Daniel et al., 1999; Dvortsov et al., 1999; Solomon, 1999). Although bromine is much less abundant than chlorine in the atmosphere, it is known to deplete strato- spheric ozone 45 to 69 times more efﬁciently on a per atom basis (Daniel et al., 1999; Sinnhuber et al., 2009). More- over, the ozone depletion efﬁciency of active bromine (Br and BrO) is strongly related to the available amount of ac- tivated chlorine (mainly Cl and ClO radicals) in the atmo- sphere (McElroy et al., 1986; Solomon et al., 1999, and refer- ences therein) as well as to enhanced sulfate aerosol loading via heterogeneous reactions (Salawitch et al., 2005). Con- sequently, even low concentrations of bromine have a rela- tively large impact on stratospheric ozone. In addition to an- thropogenic long-lived chlorine (LLCl) and bromine (LLBr) substances such as chloroﬂuorocarbons (CFCs), chlorocar- bons, methyl bromide, and bromoﬂuorocarbons (halon ﬁre suppressants), other substances with photochemical lifetimes shorter than 6 months, often referred to as very short-lived (VSL) substances, have the potential to transport a signiﬁ- cant amount of reactive halogens into the stratosphere. Ow- ing to their short lifetimes, the impact of VSL substances on stratospheric ozone peaks in the lowermost stratosphere (Salawitch et al., 2005; Feng et al., 2007; Sinnhuber et al., 2009; Sinnhuber and Meul, 2015; Yang et al., 2014; Hos- saini et al., 2015a; Falk et al., 2017), which is an important atmospheric region because surface temperature and climate are most sensitive to ozone perturbations (Riese et al., 2012; Hossaini et al., 2015b; Iglesias-Suarez et al., 2018). In fact, Hossaini et al. (2015b) found that very short-lived bromo- carbons (VSLBr) exert an ozone radiative effect (normalized by halogen content) that is 3.6 times larger than that arising from long-lived substances. The total stratospheric chlorine and bromine budgets de- rived for 2016 were 3.29 ppb Cl and 19.60 ppt Br, respec- tively (WMO, 2018), and are expected to return to their 1980 values, an arbitrary reference date before the discov- ery of the Antarctic ozone hole, around the middle of this century (Dhomse et al., 2018). J. A. Barrera et al.: Seasonal impact of VSL bromocarbons on lowermost stratospheric ozone J. A. Barrera et al.: Seasonal impact of VSL bromocarbons on lowermost stratospheric ozone 8084 lace, 2003). The most abundant VSLBr released to the at- mosphere are bromoform (CHBr3) and dibromomethane (methylene dibromide, CH2Br2), followed by a minor (but non-negligible) contribution of inter-halogen species (bromochloromethane, CH2BrCl; dibromochloromethane, CHBr2Cl; and bromodichloromethane, CHBrCl2). Alto- gether, VSLBr contribute ∼5 (3–7) ppt to stratospheric bromine, which accounted for about 25 % of total strato- spheric bromine in 2016 (WMO, 2018). Moreover, this addi- tional input of bromine is required to reconcile current strato- spheric bromine trends (Salawitch et al., 2010; WMO, 2018). Halogx-Loss contribution to lowermost stratospheric ozone loss is practically dominated by the BrOx cycle, reﬂect- ing the low sensitivity of very short-lived (VSL) bromine to background halogen abundances to drive tropical strato- spheric ozone depletion. We conclude that the link between biogenic bromine sources and seasonal changes in heteroge- neous chlorine reactivation is a key feature for future projec- tions of mid-latitude lowermost stratospheric ozone during the 21st century. p Production of anthropogenic LLCl and LLBr substances has been restricted by the Montreal Protocol and its subse- quent amendments and adjustments (Solomon, 1999; WMO, 2018). After application, the stratospheric LLCl and LLBr load peaked at the end of the 20th century and has been decreasing at a rate that depends on their respective atmo- spheric lifetimes. This implies, for example, that hydrogen chloride (HCl) shows a long-term decrease at a rate of about 0.5 % yr−1 in the middle stratosphere between 60◦N and 60◦S, while total stratospheric bromine decreased at a rate of about 0.75 % yr−1 from 2004 to 2014 (WMO, 2018). Ac- cordingly, stratospheric ozone is expected to recover from the effects of anthropogenic halogen-induced loss on a sim- ilar timescale, which is already detectable in the Antarc- tic and the upper stratosphere (e.g. Solomon et al., 2016a; Chipperﬁeld et al., 2017; Dhomse et al., 2018; Strahan and Douglass, 2008, and references therein). Hossaini et al. (2015a) quantiﬁed the stratospheric injection of organic and inorganic chlorine from anthropogenic VSLCl sources such as chloroform (CHCl3), dichloromethane (CH2Cl2), tetra- chloroethene (C2Cl4), trichloroethene (C2HCl3), and 1,2- dichloroethane (CH2ClCH2Cl), which are not controlled by the Montreal Protocol. From their results, the total strato- spheric chlorine load from VSLCl inferred for 2013 is 123 ppt, with a stratospheric injection dominated by source gases (∼83 %). J. A. Barrera et al.: Seasonal impact of VSL bromocarbons on lowermost stratospheric ozone Moreover, the stratospheric injection of VSLCl increased by ∼52 % between 2005 and 2013, mainly due to recent and ongoing growth in anthropogenic CH2Cl2 emissions. In fact, Hossaini et al. (2017) showed that the impact of CH2Cl2 on stratospheric ozone has increased markedly in recent years, and if these increases continue into the future, the recovery of the Earth’s ozone layer could be delayed even further, offsetting some of the gains achieved by the Montreal Protocol. Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. J. A. Barrera et al.: Seasonal impact of VSL bromocarbons on lowermost stratospheric ozone VSLBr contribution to total stratospheric bromine into the fu- ture. Thus, understanding the role of natural VSLBr sources is a key issue for chemistry–climate projections. However, neither of the previous studies had distinguished the contribution to ozone depletion – including its seasonal variability – that arises from VSLBr with respect to LLCl and LLBr throughout the 21st century. y y p j The impact of additional bromine is closely related to the heterogeneous chemistry of chlorine (Solomon, 1999; Salawitch et al., 2005; Müller, 2012). The inﬂuence of tem- perature and water vapour in the heterogeneous conversion processes of inorganic-chlorine reservoirs (mostly HCl and ClONO2) into active radicals on sulfate aerosols has been re- viewed elsewhere (e.g. Anderson et al., 2012, 2017; Drdla and Müller, 2012; Anderson and Clapp, 2018). In particu- lar, Drdla and Müller (2012) highlighted that an increase in water vapour above background values would allow chlorine activation at higher temperatures than those observed in po- lar regions, which led to the hypothesis that chlorine reacti- vation and subsequent ozone loss could occur in the lower stratosphere at mid-latitudes during summer (Robrecht et al., 2019). Indeed, the spatial and seasonal distributions of chlo- rine monoxide and chlorine nitrate in the monsoon regions provide indicators of heterogeneous chlorine processing in the tropical and subtropical lowermost stratosphere of the Northern Hemisphere (Solomon et al., 2016b). Moreover, heterogeneous chorine reactivation has also been observed on ice particles in cirrus clouds located near the tropopause (Borrman et al., 1996, 1997; Solomon et al., 1997; Bregman et al., 2002; Thornton et al., 2003). The occurrence of cirrus clouds was reported in the lowermost stratosphere of north- ern high-mid-latitudes (40–75◦N; Spang et al., 2015), with summertime clouds located up to ∼3 km (or 40–50 K of po- tential temperature) above the tropopause (Dessler, 2009). Furthermore, von Hobe et al. (2011) suggested that cirrus ice particles and/or liquid aerosol at low temperatures may promote a signiﬁcant activation of heterogeneous chlorine in the tropical upper troposphere and lower stratosphere. Fi- nally, satellite observations indicate that chlorine activation also occurs in the Antarctic and Arctic sub-vortex regions, where processed air dispersing from the decaying vortex in spring induces rapid changes in extra-vortex trace gas abun- dances (Santee et al., 2011). J. A. Barrera et al.: Seasonal impact of VSL bromocarbons on lowermost stratospheric ozone g y In this work, using the CAM-chem model, we present a coupled chemistry–climate modelling study from 1960 to 2100 with and without the contribution from VSLBr sources. We focus on natural VSLBr-driven impacts on the temporal evolution of stratospheric ozone and the total ozone column (TOC) in the tropics and at mid-latitudes, distin- guishing both the long-term seasonal change and the re- sulting hemispheric asymmetries. Additionally, we present a timeline assessment of the individual contribution from an- thropogenic (LLCl and LLBr) and biogenic (VSLBr) sources to stratospheric halogen loading throughout the 21st cen- tury while also recognizing the VSLBr contribution to overall halogen-catalysed ozone loss in the lowermost stratosphere. The layout of the paper is as follows: Sect. 2 describes the main characteristics and conﬁguration of the CAM-chem model as well as the observation data sets that are used for the evaluation of the CAM-chem performance. Section 3 presents a quantitative comparison between the model and observations of stratospheric chlorine and bromine loading (Sect. 3.1) and the TOC (Sect. 3.2) as well as discusses long-term seasonal impacts mediated by VSLBr on the TOC (Sect. 3.2) and the lowermost stratospheric ozone budget (Sect. 3.3). Changes in the seasonal evolution of halogen- driven ozone loss rates due to VSLBr are shown in Sect. 3.4. The ﬁnal concluding remarks are summarized in Sect. 4. 1 Introduction Although we still lack scien- tiﬁc consensus with respect to the future evolution of VSLBr oceanic source strength and stratospheric injection (WMO, 2014; Lennartz et al., 2015; Ziska et al., 2017), the contin- uous decrease in LLBr leads to an increase in the relative VSLBr are produced in the ocean via the metabolism of marine organisms such as phytoplankton and macroal- gae (e.g. Carpenter et al., 2007; Quack et al., 2007; Leed- ham Elvidge et al., 2015), even in sea ice (e.g. Abra- hamsson et al., 2018). Due to their high volatility, they are transferred into the marine boundary layer through air– sea exchange (Carpenter and Liss, 2000; Quack and Wal- Atmos. Chem. Phys., 20, 8083–8102, 2020 https://doi.org/10.5194/acp-20-8083-2020 8085 https://doi.org/10.5194/acp-20-8083-2020 Atmos. Chem. Phys., 20, 8083–8102, 2020 J. A. Barrera et al.: Seasonal impact of VSL bromocarbons on lowermost stratospheric ozone The number of stratospheric levels changes depending on the latitudinal region: within the tropics, there are eight levels above the tropopause (∼100 hPa), with a mean thickness of 1.25 km (15.5 hPa) for the lower stratospheric levels and 5.2 km (3.8 hPa) between the two highest levels, while for the mid-latitudes, the tropopause is located at approximately ∼200 hPa, and the stratosphere contains up to 12 levels. To ensure a consistent dynamical description of the stratosphere within the relatively low model top of CAM-chem (i.e. grav- ity wave dragging and the Brewer–Dobson circulation), the integrated momentum that would have been deposited above the model top is speciﬁed by an upper boundary condition (Lamarque et al., 2008). A similar procedure is applied to the altitude-dependent photolysis rate computations, which include an upper boundary condition that considers the ozone column fraction prevailing above the model top. The CAM4- chem version used in this work includes a non-orographic gravity wave scheme based on the inertia–gravity wave pa- rameterization and an observation-based implementation of stratospheric aerosol and surface area density. The quasi- biennial oscillation is imposed in the model by relaxing equa- torial zonal winds between 90 and 3 hPa to the observed in- terannual variability (see Tilmes et al., 2016, for more de- tails). Two ensembles of independent modelled experiments were performed, each with three individual simulations from 1960 to 2100, considering approximately 10 years of spin-up to allow stabilization of the stratospheric circulation. The individual simulations 001, 002, and 003 started in 1950, 1951, and 1952, respectively, with initial conditions taken from the equivalent years of the CESM1 WACCM (Whole Atmosphere Community Climate Model) 20th-century en- semble for CMIP5 (Marsh et al., 2013). The baseline simula- tion set-up of the ﬁrst experiment considered only the halo- gen LLCl and LLBr contribution from anthropogenic chlo- roﬂuorocarbons, hydrochloroﬂuorocarbons, halons, methyl chloride, and methyl bromide, while the simulation set-up of the second experiment included the background biogenic contribution from VSLBr oceanic sources in addition to the anthropogenic sources above. The mean ensemble of each experiment (i.e. with and without the VSLBr sources) was calculated as the average of the three individual simulations. Differences between these two mean ensembles allow quan- tiﬁcation of the overall impact of VSLBr on stratospheric ozone as well as the determination of their relative contri- butions to the halogen-mediated catalytic ozone-depleting families. J. A. Barrera et al.: Seasonal impact of VSL bromocarbons on lowermost stratospheric ozone 8086 Even though the interactive ocean coupling would have al- lowed us to compute the evolution of VSLBr ocean source emissions throughout the modelled period, we still lack a considerable understanding of the seasonal processes that dominate VSLBr ocean emissions in both the present and the future, which is combined with a limited observation data set of VSL bromocarbons (WMO, 2014, 2018). Therefore, in this work, we forced the model with annually cycled VSLBr ﬂuxes, replicating the Ordoñez et al. emission inventory for all years between 1950 and 2100. This procedure aims to re- duce the uncertainties associated with the unknown evolu- tion of both biogeochemical production and oceanic emis- sions of VSLBr into the future (Lennartz et al., 2015; Ziska et al., 2017). and large-scale transport – has been given elsewhere (Or- dóñez et al., 2012; Fernandez et al., 2014). Monthly and sea- sonally varying lower boundary conditions were considered for long-lived chlorine (LLCl = CH3Cl, CH3CCl3, CCl4, CFC-11, CFC-12, CFC-113, HCFC-22, CFC-114, CFC- 115, HCFC-141b, and HCFC-142b) and long-lived bromine (LLBr = CH3Br, H-1301, H-1211, H-1202, and H-2402) fol- lowing the A1 halogenated ozone-depleting substance emis- sions scenario (WMO, 2011). The surface concentrations of CO2, CH4, H2, and N2O were speciﬁed following the moder- ate Representation Concentration Pathway 6.0 (RCP6.0) sce- nario (Meinshausen et al., 2011; Eyring et al., 2013). and large-scale transport – has been given elsewhere (Or- dóñez et al., 2012; Fernandez et al., 2014). Monthly and sea- sonally varying lower boundary conditions were considered for long-lived chlorine (LLCl = CH3Cl, CH3CCl3, CCl4, CFC-11, CFC-12, CFC-113, HCFC-22, CFC-114, CFC- 115, HCFC-141b, and HCFC-142b) and long-lived bromine (LLBr = CH3Br, H-1301, H-1211, H-1202, and H-2402) fol- lowing the A1 halogenated ozone-depleting substance emis- sions scenario (WMO, 2011). The surface concentrations of CO2, CH4, H2, and N2O were speciﬁed following the moder- ate Representation Concentration Pathway 6.0 (RCP6.0) sce- nario (Meinshausen et al., 2011; Eyring et al., 2013). The CAM-chem conﬁguration used in this work extends from the surface to approximately 40 km (3.5 hPa in the up- per stratosphere), with 26 vertical levels, and includes a hor- izontal resolution of 1.9◦latitude by 2.5◦longitude. 2 Methods: model description The Community Earth System Model (CESM1) with the Community Atmospheric Model including interactive chem- istry (CAM4-chem version; Lamarque et al., 2012; Tilmes et al., 2016) has been used to explore VSLBr-driven strato- spheric ozone loss in three latitude bands: Southern Hemi- sphere mid-latitudes (SH-MLs; 60–35◦S), tropics (25◦N– 25◦S), and Northern Hemisphere mid-latitudes (NH-MLs; 35–60◦N). The model set-up follows the Chemistry–Climate Model Initiative (CCMI) REFC2 conﬁguration described in detail by Tilmes et al. (2016), with the exception that in this work we consider a full halogen chemistry mechanism and oceanic emission – seasonally dependent and geograph- ically distributed – of six bromocarbons (VSLBr = CHBr3, CH2Br2, CH2BrCl, CHBrCl2, CHBr2Cl, and CH2IBr) from the Ordóñez et al. (2012) emission inventory. This emis- sion inventory is based on a monthly varying satellite chl a climatology, which allows the introduction of a complete seasonal cycle to the emission strength and spatial distri- bution of oceanic bromocarbons. A full description of the VSLBr mechanism implemented in CAM-chem – including both biogenic and anthropogenic sources, heterogeneous re- cycling reactions, dry and wet deposition, convective uplift, Previous chemistry–climate modelling studies consider- ing VSLBr chemistry have mainly focused on improving the model vs. observed mid-latitude ozone trends at the end of the 20th century (Feng et al., 2007; Sinnhuber et al., 2009). More recently, Sinnhuber and Meul (2015) have shown that the model simulations including VSLBr resulted in a much greater ozone decrease in the lowermost stratosphere dur- ing the 1979–1995 period and a faster ozone increase dur- ing the 1996–2005 period, which is in better agreement with observed ozone trends. Moreover, Falk et al. (2017) have re- ported that the projected additional VSLBr impact on ozone at the end of the 21st century is reduced compared to present day. These results are in agreement with those of Yang et al. (2014), who performed time slice simulations to ad- dress the sensitivity of stratospheric ozone to a speculative doubling of VSLBr sources under different LLCl scenarios. https://doi.org/10.5194/acp-20-8083-2020 Atmos. Chem. Phys., 20, 8083–8102, 2020 3.1 Contribution of LLCl, LLBr, and VSLBr to stratospheric inorganic-halogen loading Total stratospheric Bry trend reported in the latest WMO Ozone Assessment Reports (WMO, 2014, 2018), which shows changes in total Bry between 1991 and 2012 derived from stratospheric BrO observations by balloon-borne (Dorf et al., 2006) and ground-based UV–visible (at Harestua, 60◦N, and Lauder, 45◦S) measurements (Hendrick et al., 2007, 2008); ii. Total stratospheric Bry trend reported in the latest WMO Ozone Assessment Reports (WMO, 2014, 2018), which shows changes in total Bry between 1991 and 2012 derived from stratospheric BrO observations by balloon-borne (Dorf et al., 2006) and ground-based UV–visible (at Harestua, 60◦N, and Lauder, 45◦S) measurements (Hendrick et al., 2007, 2008); iii. The Solar Backscatter Ultraviolet (SBUV) merged total ozone column data set (MOD; Frith et al., 2014, 2017) from 1980 to 2015. An important point to highlight about the SBUV MOD (and its uncertainties) is that it was constructed from ozone pro- ﬁles measured by individual SBUV instruments and retrieved using the Version 8.6 algorithm (Bhartia et al., 2013; Frith et al., 2014, 2017). To maintain consistency over the entire time series, the individual instrument records were analysed with respect to each other, and absolute calibration adjustments were applied as needed based on comparison of radiance measurements during periods of instrument overlap (DeLand et al., 2012; Weber et al., 2018). During the overlap periods, the records of the involved SBUV instruments were com- bined using a simple average. Frith et al. (2014) have esti- mated the potential errors in the SBUV MOD through Monte Carlo model simulations, taking into account the range of offsets and drifts observed during the overlap periods be- tween individual SBUV instruments and between SBUV and Dobson ground-based measurements. The authors also as- sessed the extent to which these potential errors might affect the long-term variability of ozone in a multiple-regression model. See Frith et al. (2014) for a detailed description of the data used and their associated uncertainties in the SBUV MOD. In the lower stratosphere, at 50 hPa, BrLL y abundances re- turn to 1980 levels before ∼2080 and ∼2050 for the mid- latitudes and tropics, respectively, while the ClLL y return date remains unaltered (see Fig. 1b–d). Furthermore, the contribu- tion from VSLBr to mid-latitude total inorganic bromine also reaches ∼5 ppt throughout the modelled period, while in the tropics there prevails a small carbon-bonded organic fraction that has not been converted to the reactive inorganic form. Falk et al. 3 Results and discussion dinal bands deﬁned above. To highlight seasonal changes, we computed the average for the months of December–January– February (DJF), March–April–May (MAM), June–July– August (JJA), and September–October–November (SON). Ozone loss trends due to VSLBr between 2000 and 2100 were calculated on the basis of the least-squares linear trends, while the trend errors were estimated as twice the statistical deviation stemming from the least-squares ﬁt. 3.1 Contribution of LLCl, LLBr, and VSLBr to stratospheric inorganic-halogen loading J. A. Barrera et al.: Seasonal impact of VSL bromocarbons on lowermost stratospheric ozone 8087 J. A. Barrera et al.: Seasonal impact of VSL bromocarbons on lowermost stratospheric ozon J. A. Barrera et al.: Seasonal impact of VSL bromocarbons on lowermost stratospheric ozone In addition, from this analysis it is also possible to distinguish the contributions of both LLBr and VSLBr to the inorganic fraction of stratospheric bromine (BrLL+VSL y = BrLL y +BrVSL y = Br+BrO+HBr+BrONO2+BrCl+HOBr+ 2Br2 + BrNO2), whereas the inorganic fraction of chlorine (ClLL y = ClO+Cl+HOCl+2Cl2O2+2Cl2+OClO+HCl+ ClONO2+BrCl+ClONO2) arises only from the degradation of LLCl, which is identical for both experiments. Equivalent CAM-chem conﬁgurations, such as the one im- plemented in this work, have been used for the CCMVal-2 (Chemistry–Climate Model Validation 2) and CMIP5 (Cou- pled Model Intercomparison Project, Phase 5) activities in or- der to represent trends in the ozone evolution and to estimate the return dates, which lie in the middle of the multi-model range (Eyring et al., 2010, 2013). Moreover, our model con- ﬁguration uses a fully coupled Earth system model approach, i.e. the ocean and sea ice are explicitly computed (Neale et al., 2013). This implies that instead of isolating the chemi- cal impact mediated by VSLBr on the climatological ozone budget (as would be the case using a speciﬁed dynamic ap- proach), the chemical interaction between VSL bromine and ozone in the lowermost stratosphere is affected by dynamic feedbacks (i.e. temperature, radiation, etc.). For the case of vertical distributions and latitudinal varia- tions, the zonal mean of each mean ensemble was computed from the monthly output before processing the data, while a locally weighted scatter plot smoothing (LOWESS) ﬁlter with a 0.2 fraction was applied to all long-term time series to smooth the data. Most of the ﬁgures and values in the Re- sults section include geographically averaged quantities for the present day (deﬁned here as the 1990–2010 mean, nomi- nally year 2000) and the end of the 21st century (deﬁned here as the 2080–2100 mean, nominally year 2100) over the latitu- Atmos. Chem. Phys., 20, 8083–8102, 2020 https://doi.org/10.5194/acp-20-8083-2020 3.1 Contribution of LLCl, LLBr, and VSLBr to stratospheric inorganic-halogen loading The evolution (1960–2100) of the modelled annual mean abundances of inorganic chlorine (Cly) and bromine (Bry) as well as the VSLBr in the global upper stratosphere (3.5 hPa) and lower stratosphere (50 hPa) of the tropics and mid- latitudes is shown in Fig. 1. In addition, Fig. 1 includes the total Bry trends (WMO, 2014, 2018) and the MLS obser- vations (HCl + ClO). The values of bias, normalized mean bias (NMB), and normalized mean error (NME) used to eval- uate the agreement between the modelled Cly and obser- vations for each of the regions under study are shown in Table S1 in the Supplement. Clearly, the temporal evolu- tion of ClLL y and BrLL y shows a pronounced peak at the end of the 20th century and beginning of the 21st century, re- spectively, after which the abundance of both halogens de- clines. Within the A1 halogen emission scenario considered in this work, the ClLL y abundance in our model returns to its past 1980 levels just before ∼2060 in the global upper stratosphere, whereas the prevailing BrLL y abundance for the year 1980 is not recovered even by the end of the 21st cen- tury (see Fig. 1a). In comparison, the evolution of BrVSL y abundances remains constant over time, resulting in an addi- tional and time-independent ﬁxed contribution of ∼5 ppt to- tal bromine injection, which leads to maximum global upper- stratosphere BrLL+VSL y abundances of up to ∼20.5 ppt at the beginning of the 21st century. This is in agreement with previous studies performed only for present-day conditions (Fernandez et al., 2014). Furthermore, note that the modelled global BrLL+VSL y abundances are in good agreement with the observations within reported errors, highlighting the impor- tance of considering the additional contribution from VSLBr to determine the overall evolution of stratospheric ozone dur- ing the 21st century. To provide a basic evaluation of the model performance, the modelled results of the TOC and the inorganic-halogen abundances were compared with the following selected ob- servation data sets: i. Microwave Limb Sounder (MLS) observations of the annual mean volume mixing ratio of HCl + ClO (Wa- ters et al., 2006; Livesey et al., 2018), which provides a lower limit of the Cly abundance in the stratosphere from 2005 to 2015; ii. Atmos. Chem. Phys., 20, 8083–8102, 2020 https://doi.org/10.5194/acp-20-8083-2020 3.1 Contribution of LLCl, LLBr, and VSLBr to stratospheric inorganic-halogen loading (2017) showed an increase in VSLBr of about 0.5 ppt in the tropical lowermost stratosphere by the end of the century compared to present day under the RCP6.0 sce- nario and assuming constant VSLBr ﬂuxes, following sce- nario ﬁve of Warwick et al. (2006). This increase in the stratospheric VSLBr attributed to enhanced vertical transport in the tropics is counteracted by a decrease in the BrVSL y injected into the stratosphere so that the total stratospheric VSL bromine remains unchanged between the two peri- ods. In this work, the stratospheric injection of VSLBr and https://doi.org/10.5194/acp-20-8083-2020 Atmos. Chem. Phys., 20, 8083–8102, 2020 J. A. Barrera et al.: Seasonal impact of VSL bromocarbons on lowermost stratospheric ozon J. A. Barrera et al.: Seasonal impact of VSL bromocarbons on lowermost stratospheric ozone 8088 Figure 1. Temporal evolution (1960–2100) of the modelled annual mean abundances of VSLBr and inorganic halogen (ClLL y and BrLL+VSL y ): (a) global upper stratosphere (3.5 hPa); lower stratosphere (50 hPa) in the (b) tropics and at (c) SH-MLs and (d) NH-MLs. The BrLL+VSL y abundance was split into long-lived (BrLL y ) and very short-lived (BrVSL y ) contributions. The black squares (ﬁlled and open) and orange triangles (ﬁlled and open) of panel (a) show the total stratospheric Bry trends from 1991 to 2012 (WMO, 2014, 2018). The red triangles show the mean annual mixing ratios (HCl + ClO) from the Microwave Limb Sounder (MLS) data (Waters et al., 2006; Livesey et al., 2018) from 2005 to 2015. The dashed horizontal lines indicate the modelled ClLL y and BrLL y abundances for 1980. Note that both ClLL y values and (HCl + ClO) MLS data were divided by 100. Figure 1. Temporal evolution (1960–2100) of the modelled annual mean abundances of VSLBr and inorganic halogen (ClLL y and BrLL+VSL y Figure 1. Temporal evolution (1960–2100) of the modelled annual mean abundances of VSLBr and inorganic halogen (ClLL y and BrLL+VSL y ): (a) global upper stratosphere (3.5 hPa); lower stratosphere (50 hPa) in the (b) tropics and at (c) SH-MLs and (d) NH-MLs. The BrLL+VSL y abundance was split into long-lived (BrLL y ) and very short-lived (BrVSL y ) contributions. The black squares (ﬁlled and open) and orange triangles (ﬁlled and open) of panel (a) show the total stratospheric Bry trends from 1991 to 2012 (WMO, 2014, 2018). J. A. Barrera et al.: Seasonal impact of VSL bromocarbons on lowermost stratospheric ozone In the low- ermost stratosphere, the available inorganic fraction of both chlorine and bromine rapidly increases with altitude as halo- gen atoms are released by the photolysis and oxidation from all LLCl, LLBr, and VSLBr species. In contrast, within the upper stratosphere no major changes in inorganic-halogen abundances are observed since the conversion from their or- ganic sources is nearly complete. Although our model shows a symmetric hemispheric distribution in stratospheric Cly and Bry abundances, there is a marked difference in the ClOx/Cly ratio. In fact, the experiments capture a local in- crease in the ClOx/Cly ratio at 15◦N and ∼100 hPa as a re- sult of a large increase in heterogeneous reactivation of Cly species due to enhanced vertical transport during the Indian summer monsoon (Solomon et al., 2016b). Along the same lines, the higher ClOx/Cly values modelled in the lowermost stratosphere at SH-MLs compared to NH-MLs are mainly due to the enhancement in the heterogeneous chlorine re- activation processes on ice crystals (i.e. HCl and ClONO2 reactivation; Santee et al., 2011; Solomon, 1999, and ref- erences therein), inﬂuenced mainly by the proximity of the Antarctic polar vortex edge and the lower temperatures pre- vailing at high- and mid-latitudes of the Southern Hemi- sphere (see Fig. S2). Additionally, the favourable conditions Figure 2. Annual zonal mean distribution of (a) ClLL y , (b) BrLL+VSL y , and (c) BrLL y during the present-day period. The colour scale represents volume mixing ratios (ppb or ppt), while black contour lines show the contribution percentage of ClOx to Cly and BrOx to Bry. The lower solid white line indicates the loca- tion of the tropopause (chemical deﬁnition of 150 ppb ozone level from the experiment without VSLBr sources). Figure 2. Annual zonal mean distribution of (a) ClLL y , (b) B LL+VSL d ( ) B LL d i th t d i d Th (b) BrLL+VSL y , and (c) BrLL y during the present-day period. The colour scale represents volume mixing ratios (ppb or ppt), while black contour lines show the contribution percentage of ClOx to Cly and BrOx to Bry. The lower solid white line indicates the loca- tion of the tropopause (chemical deﬁnition of 150 ppb ozone level from the experiment without VSLBr sources). J. A. Barrera et al.: Seasonal impact of VSL bromocarbons on lowermost stratospheric ozone J. A. Barrera et al.: Seasonal impact of VSL bromocarbons on lowermost stratospheric ozon 8089 Figure 2. Annual zonal mean distribution of (a) ClLL y , (b) BrLL+VSL y , and (c) BrLL y during the present-day period. The colour scale represents volume mixing ratios (ppb or ppt), while black contour lines show the contribution percentage of ClOx to Cly and BrOx to Bry. The lower solid white line indicates the loca- tion of the tropopause (chemical deﬁnition of 150 ppb ozone level from the experiment without VSLBr sources). uncertainty that prevents a reliable model-observation com- parison (Santee et al., 2011). p The stratospheric BrVSL y injection remains constant dur- ing the whole modelled period, representing ∼25 % of BrLL+VSL y in the global upper stratosphere for the year 2000 and increasing up to ∼40 % by the end of the 21st century. These results are in agreement with those reported by Fer- nandez et al. (2017). Due to the much shorter lifetimes of VSLBr with respect to LLBr, the contribution percentage is larger in the lower stratosphere. For example, the BrVSL y rela- tive contribution to BrLL+VSL y for the year 2000 at 50 hPa rep- resents ∼30 % and ∼45 % for the mid-latitudes and tropics, respectively. Furthermore, at mid-latitudes, BrVSL y represents up to 45 % of BrLL+VSL y both at the end of the 21st century and in the years prior to 1980, while in the tropics BrVSL y represents up to 65 % for these time periods. Consequently, these changes in the relative contribution of VSLBr to total inorganic bromine with respect to time, region, and altitude in the presence of different inorganic-chlorine abundances lead to relatively different ozone loss within those regions and altitude regimes, as described in detail below. Figure 2 shows the annual zonal mean distribution of inorganic-chlorine and inorganic-bromine abundances as well as the corresponding ClOx/Cly and BrOx/Bry percent- age ratios (black contour lines) for the present day. Note that the ClOx/Cly and BrOx/Bry ratios reﬂect the changes in the contribution of the active-chlorine (ClOx = ClO + Cl + HOCl+2Cl2O2+OClO+2Cl2) and active-bromine (BrOx = BrO + Br) fractions relative to their total inorganic abun- dances. Equivalent results are presented for the end of the 21st century in the Supplement (see Fig. S1). 3.1 Contribution of LLCl, LLBr, and VSLBr to stratospheric inorganic-halogen loading The red triangles show the mean annual mixing ratios (HCl + ClO) from the Microwave Limb Sounder (MLS) data (Waters et al., 2006; Livesey et al., 2018) from 2005 to 2015. The dashed horizontal lines indicate the modelled ClLL y and BrLL y abundances for 1980. Note that both ClLL y values and (HCl + ClO) MLS data were divided by 100. ison between modelled ClLL y abundances and MLS observa- tions is about 3.5 % and 19 % at 50 hPa for the mid-latitudes and tropics, respectively, while the NME is about 2 % for the global mean at 3.5 hPa (see Table S1). This relatively good agreement between the model and MLS observation occurs even without consideration of the recent contribution of anthropogenic VSLCl sources (i.e. Hossaini et al., 2015b, 2017). Moreover, the inter-annual variation in MLS obser- vations at 50 hPa occurs mainly due to heterogeneous chlo- rine reactivation, which can be inﬂuenced by the polar vortex dynamics at mid-latitudes (Dhomse et al., 2018). Note that although we processed output at 120 hPa in the mid-latitude lowermost stratosphere in Sect. 3.4, we did not compare ClLL y at this altitude level. This is because below 50 hPa, the frac- tional conversion of organic chlorine to ClLL y is very small, and thus the inferred HCl + ClO MLS data have a very large BrVSL y between 2000 and 2100 remains unchanged. Based on the limited observational data, it is uncertain whether any trend in the stratospheric injection of organic and inor- ganic VSL bromine would be expected, and thus no con- clusive statement can be made. Nevertheless, our present- day stratospheric injection of VSLBr (∼2 ppt) and BrVSL y (∼3 ppt) is in perfect accordance with that reported in the latest WMO Ozone Assessment Report (WMO, 2018). Fur- ther studies are needed to evaluate the uncertain evolution of the stratospheric injection of organic and inorganic species from VSLBr throughout the 21st century. The modelled inorganic-chlorine abundance shows a good agreement with the (HCl + ClO) MLS observations for the 2005–2015 period mainly in the upper stratosphere, where most chlorine has already been photochemically converted to ClLL y . For example, the normal mean error of the compar- Atmos. Chem. Phys., 20, 8083–8102, 2020 https://doi.org/10.5194/acp-20-8083-2020 Atmos. Chem. Phys., 20, 8083–8102, 2020 J. A. Barrera et al.: Seasonal impact of VSL bromocarbons on lowermost stratospheric ozone 2b, c and S1b, c), highlighting that this ratio is nearly independent of the total inorganic bromine abundance. for each of the regions under study. Based on the comparison of the TOC results over the different latitudinal bands, the following features can be described: i. The constant emission of VSLBr introduces a continu- ous reduction in the TOC that exceeds the mean ensem- ble variability between the experiments (see Fig. S5). Moreover, the inclusion of VSLBr sources improves the overall agreement between the model and observations, with a reduction of 1 % and 0.9 % in NME for SH-MLs and NH-MLs, respectively (see Table S2). However, the inclusion of VSLBr sources leads to a slight increase of 0.2 % in NME for the tropics. Note that the statistical analysis presented in Table S2 does not take into ac- count the uncertainties that may arise from the merging of the individual SBUV instruments in the SBUV MOD (see Sect. 2). J. A. Barrera et al.: Seasonal impact of VSL bromocarbons on lowermost stratospheric ozone J. A. Barrera et al.: Seasonal impact of VSL bromocarbons on lowermost stratospheric ozon J. A. Barrera et al.: Seasonal impact of VSL bromocarbons on lowermost stratospheric ozone 8090 Figure 3. Temporal evolution of the annual mean total ozone column (TOCLL+VSL and TOCLL) (a) at SH-MLs, (b) in the tropics, and (c) at NH-MLs as well as the corresponding absolute (DU; d–f) and relative (%; g–i) TOC difference (1TOC). TOC values of the mean ensemble (thin lines) and the smoothed time series via LOWESS ﬁltering (0.2 fractions; thick lines) are shown in blue for TOCLL+VSL and black for TOCLL. The red lines and symbols show the observations from the Solar Backscatter Ultraviolet (SBUV) merged total ozone column data set (Frith et al., 2014, 2017) within the same spatial and temporal mask as the model output. Figure 3. Temporal evolution of the annual mean total ozone column (TOCLL+VSL and TOCLL) (a) at SH-MLs, (b) in the tropics, and (c) at NH-MLs as well as the corresponding absolute (DU; d–f) and relative (%; g–i) TOC difference (1TOC). TOC values of the mean ensemble (thin lines) and the smoothed time series via LOWESS ﬁltering (0.2 fractions; thick lines) are shown in blue for TOCLL+VSL and black for TOCLL. The red lines and symbols show the observations from the Solar Backscatter Ultraviolet (SBUV) merged total ozone column data set (Frith et al., 2014, 2017) within the same spatial and temporal mask as the model output. HBr and BrONO2 reactivation; Solomon, 1999, and refer- ences therein), which show neither any hemispheric asym- metry nor seasonal changes in the reactivation processes (see Fig. S4). Thus, even when the inclusion of VSLBr sources increases the total stratospheric Bry abundance and therefore its active fraction (BrOx), the BrOx/Bry ratio remains un- changed in the stratosphere during the whole modelled pe- riod (see Figs. 2b, c and S1b, c), highlighting that this ratio is nearly independent of the total inorganic bromine abundance. HBr and BrONO2 reactivation; Solomon, 1999, and refer- ences therein), which show neither any hemispheric asym- metry nor seasonal changes in the reactivation processes (see Fig. S4). Thus, even when the inclusion of VSLBr sources increases the total stratospheric Bry abundance and therefore its active fraction (BrOx), the BrOx/Bry ratio remains un- changed in the stratosphere during the whole modelled pe- riod (see Figs. J. A. Barrera et al.: Seasonal impact of VSL bromocarbons on lowermost stratospheric ozone for isentropic exchange of young subtropical air with ex- tremely old winter polar vortex air masses at SH-MLs (Spang et al., 2015; Rolf et al., 2015) could drive additional hetero- geneous chlorine reactivation and thus enhance hemispheric asymmetry. Moreover, the increase in the ClOx abundance and ClOx/Cly ratio is highest during winter and spring for both SH-MLs and NH-MLs, which is consistent with the sea- sonal changes of the lowermost stratospheric temperatures that affect chlorine reactivation on ice crystals (see Fig. S3). In contrast, a symmetric hemispheric distribution of the BrOx/Bry ratio is modelled in the mid-latitude lowermost stratosphere and is associated with heterogeneous bromine reactivation on sulfate aerosols instead of ice crystals (i.e. https://doi.org/10.5194/acp-20-8083-2020 Atmos. Chem. Phys., 20, 8083–8102, 2020 3.2 Impact of VSLBr on the seasonal evolution of the total ozone column (TOC) Annual and seasonal TOC changes (1TOC) mediated by VSLBr at mid-latitudes and in the tropics for the present day and the end of the 21st century as well as the 1TOC trends (% per decade) over the century. Table 1. Annual and seasonal TOC changes (1TOC) mediated by VSLBr at mid-latitudes and in the tropics for the present day and the end of the 21st century as well as the 1TOC trends (% per decade) over the century. as the 1TOC trends (% per decade) over the century. Season Region 1TOC2000,a 1TOC2100,a 1TOC trendsb DU (%) DU (%) (% per decade) Annual mean NH-MLs −5.5 ± 0.6 −2.7 ± 0.2 0.10 ± 0.03 (−1.6 ± 0.6) (−0.8 ± 0.2) Tropics −2.1 ± 0.3 −1.5 ± 0.1 0.03 ± 0.01 (−0.8 ± 0.2) (−0.6 ± 0.1) SH-MLs −8.0 ± 0.8 −3.9 ± 0.4 0.15 ± 0.04 (−2.5 ± 0.3) (−1.2 ± 0.3) DJF NH-MLs −6.2 ± 0.1 −3.2 ± 0.3 0.11 ± 0.03 (−1.7 ± 0.6) (−0.8 ± 0.2) Tropics −2.3 ± 0.3 −1.8 ± 0.1 0.02 ± 0.01 (−0.9 ± 0.1) (−0.7 ± 0.1) SH-MLs −6.9 ± 0.6 −4.5 ± 0.2 0.10 ± 0.03 (−2.4 ± 0.4) (−1.5 ± 0.1) MAM NH-MLs −7.7 ± 0.9 −3.7 ± 0.3 0.14 ± 0.04 (−2.0 ± 0.6) (−0.9 ± 0.2) Tropics −2.1 ± 0.2 −1.5 ± 0.1 0.02 ± 0.01 (−0.8 ± 0.2) (−0.6 ± 0.1) SH-MLs −6.2 ± 0.5 −4.2 ± 0.2 0.11 ± 0.03 (−2.2 ± 0.3) (−1.4 ± 0.1) JJA NH-MLs −4.1 ± 0.6 −2.3 ± 0.2 0.09 ± 0.03 (−1.3 ± 0.2) (−0.7 ± 0.1) Tropics −2.0 ± 0.3 −1.6 ± 0.1 0.02 ± 0.01 (−0.8 ± 0.2) (−0.6 ± 0.1) SH-MLs −8.0 ± 0.9 −3.2 ± 0.4 0.19 ± 0.06 (−2.5 ± 0.2) (−0.9 ± 0.1) SON NH-MLs −3.8 ± 0.4 −1.6 ± 0.1 0.10 ± 0.03 (−1.2 ± 0.1) (−0.5 ± 0.1) Tropics −1.9 ± 0.2 −1.5 ± 0.1 0.01 ± 0.01 (−0.7 ± 0.1) (−0.6 ± 0.1) SH-MLs −10.0 ± 1.2 −5.7 ± 0.4 0.20 ± 0.06 (−3.0 ± 0.8) (−1.6 ± 0.8) a Absolute (DU) and relative (%; in brackets) TOC changes during the present-day period (1TOC2000) and the end of the 21st century (1TOC2100) were computed considering the mean ensemble of each experiment at mid-latitudes (SH-MLs and NH-MLs) and in the tropics. Annual and seasonal 1TOC errors were estimated from standard mean errors. 3.2 Impact of VSLBr on the seasonal evolution of the total ozone column (TOC) b Trend errors were estimated as twice the statistical deviation stemming from the least-squares ﬁt. a Absolute (DU) and relative (%; in brackets) TOC changes during the present-day period (1TOC2000) and the end of the 21st century (1TOC2100) were computed considering the mean ensemble of each experiment at mid-latitudes (SH-MLs and NH-MLs) and in the tropics. Annual and seasonal 1TOC errors were estimated from standard mean errors. b Trend errors were estimated as twice the statistical deviation stemming from the least-squares ﬁt. for the present day, which is in line with the projected 1TOC trends of 0.15 % and 0.10 % per decade for SH- MLs and NH-MLs, respectively. TOCLL (i.e. towards where the inorganic-chlorine peak is located in the year 1995), which is in agreement with observations. for the present day, which is in line with the projected 1TOC trends of 0.15 % and 0.10 % per decade for SH- MLs and NH-MLs, respectively. iii. Within the tropics, the inclusion of VSLBr leads to small impacts on the TOC compared to impacts observed at mid-latitudes. The TOC differences due to VSLBr are close to −1.5 DU (< −1 %) during practically the entire modelled period, with a maximum 1TOC2000 reach- ing −2.1 DU (< −1 %). This is in line with a projected 1TOC trend of 0.03 % per decade. Moreover, even though VSLBr slightly worsen the agreement between the modelled TOCLL and observations, the minimum TOCLL+VSL is shifted to ∼5 years earlier compared to iii. Within the tropics, the inclusion of VSLBr leads to small impacts on the TOC compared to impacts observed at mid-latitudes. The TOC differences due to VSLBr are close to −1.5 DU (< −1 %) during practically the entire modelled period, with a maximum 1TOC2000 reach- ing −2.1 DU (< −1 %). This is in line with a projected 1TOC trend of 0.03 % per decade. Moreover, even though VSLBr slightly worsen the agreement between the modelled TOCLL and observations, the minimum TOCLL+VSL is shifted to ∼5 years earlier compared to iv. Overall, our model results show much higher ozone de- pletion due to VSLBr at mid-latitudes compared to the tropics. iv. Overall, our model results show much higher ozone de- pletion due to VSLBr at mid-latitudes compared to the tropics. 3.2 Impact of VSLBr on the seasonal evolution of the total ozone column (TOC) The temporal evolution (1960–2100) of the modelled annual mean TOC at mid-latitudes and in the tropics, along with SBUV MOD observations, is illustrated in Fig. 3. Equivalent results for the temporal evolution of the individual simula- tions of each experiment are shown in Fig. S5. Table 1 shows the annual and seasonal mean values of the absolute and rel- ative TOC differences (1TOC) between the experiments for the present day and the end of the 21st century as well as the 1TOC trends (% per decade) over the century. In ad- dition, Table S2 shows the values of bias, normalized mean bias (NMB), and normalized mean error (NME) to evaluate the agreement between the modelled TOC and observations ii. At mid-latitudes, the additional ozone loss due to VSLBr peaks during the present-day period, coinciding with the temporal location of the minimum TOC for both ex- periments and reaching a 1TOC2000 of approximately −8 DU (∼−2.5 %) and −5.5 DU (∼−1.6 %) for SH- MLs and NH-MLs, respectively (see Table 1). More- over, VSLBr impacts on the TOC by the end of the 21st century are ∼50 % lower than the values found ii. At mid-latitudes, the additional ozone loss due to VSLBr peaks during the present-day period, coinciding with the temporal location of the minimum TOC for both ex- periments and reaching a 1TOC2000 of approximately −8 DU (∼−2.5 %) and −5.5 DU (∼−1.6 %) for SH- MLs and NH-MLs, respectively (see Table 1). More- over, VSLBr impacts on the TOC by the end of the 21st century are ∼50 % lower than the values found https://doi.org/10.5194/acp-20-8083-2020 Atmos. Chem. Phys., 20, 8083–8102, 2020 J. A. Barrera et al.: Seasonal impact of VSL bromocarbons on lowermost stratospheric ozone Table 1. Annual and seasonal TOC changes (1TOC) mediated by VSLBr at mid-latitudes and in the tropics for the pr of the 21st century as well as the 1TOC trends (% per decade) over the century. 3.2 Impact of VSLBr on the seasonal evolution of the total ozone column (TOC) Season Region 1TOC2000,a 1TOC2100,a 1TOC trendsb DU (%) DU (%) (% per decade) Annual mean NH-MLs −5.5 ± 0.6 −2.7 ± 0.2 0.10 ± 0.03 (−1.6 ± 0.6) (−0.8 ± 0.2) Tropics −2.1 ± 0.3 −1.5 ± 0.1 0.03 ± 0.01 (−0.8 ± 0.2) (−0.6 ± 0.1) SH-MLs −8.0 ± 0.8 −3.9 ± 0.4 0.15 ± 0.04 (−2.5 ± 0.3) (−1.2 ± 0.3) DJF NH-MLs −6.2 ± 0.1 −3.2 ± 0.3 0.11 ± 0.03 (−1.7 ± 0.6) (−0.8 ± 0.2) Tropics −2.3 ± 0.3 −1.8 ± 0.1 0.02 ± 0.01 (−0.9 ± 0.1) (−0.7 ± 0.1) SH-MLs −6.9 ± 0.6 −4.5 ± 0.2 0.10 ± 0.03 (−2.4 ± 0.4) (−1.5 ± 0.1) MAM NH-MLs −7.7 ± 0.9 −3.7 ± 0.3 0.14 ± 0.04 (−2.0 ± 0.6) (−0.9 ± 0.2) Tropics −2.1 ± 0.2 −1.5 ± 0.1 0.02 ± 0.01 (−0.8 ± 0.2) (−0.6 ± 0.1) SH-MLs −6.2 ± 0.5 −4.2 ± 0.2 0.11 ± 0.03 (−2.2 ± 0.3) (−1.4 ± 0.1) JJA NH-MLs −4.1 ± 0.6 −2.3 ± 0.2 0.09 ± 0.03 (−1.3 ± 0.2) (−0.7 ± 0.1) Tropics −2.0 ± 0.3 −1.6 ± 0.1 0.02 ± 0.01 (−0.8 ± 0.2) (−0.6 ± 0.1) SH-MLs −8.0 ± 0.9 −3.2 ± 0.4 0.19 ± 0.06 (−2.5 ± 0.2) (−0.9 ± 0.1) SON NH-MLs −3.8 ± 0.4 −1.6 ± 0.1 0.10 ± 0.03 (−1.2 ± 0.1) (−0.5 ± 0.1) Tropics −1.9 ± 0.2 −1.5 ± 0.1 0.01 ± 0.01 (−0.7 ± 0.1) (−0.6 ± 0.1) SH-MLs −10.0 ± 1.2 −5.7 ± 0.4 0.20 ± 0.06 (−3.0 ± 0.8) (−1.6 ± 0.8) a Absolute (DU) and relative (%; in brackets) TOC changes during the present-day period (1TOC2000) and the end of the 21st century (1TOC2100) were computed considering the mean ensemble of each experiment at mid-latitudes (SH-MLs and NH-MLs) and in the tropics. Annual and seasonal 1TOC errors were estimated from standard mean errors. b Trend errors were estimated as twice the statistical deviation stemming from the least-squares ﬁt. 8091 J. A. Barrera et al.: Seasonal impact of VSL bromocarbons on lowermost stratospheric ozone J. A. Barrera et al.: Seasonal impact of VSL bromocarbons on lowermost stratospheric ozone 8091 Table 1. Annual and seasonal TOC changes (1TOC) mediated by VSLBr at mid-latitudes and in the tropics for the present day and the end of the 21st century as well as the 1TOC trends (% per decade) over the century. Table 1. J. A. Barrera et al.: Seasonal impact of VSL bromocarbons on lowermost stratospheric ozone A. Barrera et al.: Seasonal impact of VSL bromocarbons on lowermost stratospheric ozone 8092 8092 J. A. Barrera et al.: Seasonal impact of VSL bromocarbons on lowermost stratospheric ozone Figure 4. Temporal evolution of total ozone column difference (1TOC) between the experiments. Panels (a–c) show the annual and seasonal mean absolute 1TOC (DU) at SH-MLs, in the tropics, and at NH-MLs, respectively, while panels (d–f) show the corresponding relative 1TOC (%). Figure 4. Temporal evolution of total ozone column difference (1TOC) between the experiments. Panels (a–c) show the annual and seasonal mean absolute 1TOC (DU) at SH-MLs, in the tropics, and at NH-MLs, respectively, while panels (d–f) show the corresponding relative 1TOC (%). Feng et al. (2007), the model conﬁgurations included the di- rect supply of tropospheric Bry as a lower condition in addi- tion to a VSL substance tracer. Furthermore, our results are in accord with the future projection trends in Falk et al. (2017), although their analysis on annual mean ozone loss due to VSLBr only focused on the late 21st century (2075–2100). elled in this work on projected ozone loss trends throughout the current century. The largest mid-latitude TOC differences between exper- iments occur during spring, when the maximum seasonal TOC levels are found, followed by the winter. The maxi- mum springtime 1TOC2000 reaches −10 DU (∼−3 %) and −7.7 DU (∼−2 %) for SH-MLs and NH-MLs, respectively (see Table 1 and Fig. 4). In contrast, during summer and au- tumn, the maximum 1TOC2000 values remain below −6.8 (∼−2.4 %) and −4 DU (∼−4.1 %) for SH-MLs and NH- MLs, respectively. This is in line with the ozone loss me- diated by VSLBr within the southern polar cap reported by Fernandez et al. (2017), who showed a maximum ozone de- pletion of up to −15 DU (∼−14 % of TOC) during Oc- tober at the beginning of the century. Moreover, as men- tioned above, the projected seasonal VSLBr impact on the TOC signiﬁcantly decreases towards the end of the century, with springtime 1TOC trends of up to 0.20 % and 0.14 % per decade for SH-MLs and NH-MLs, respectively (see Ta- ble 1). In comparison, the projected 1TOC trends for sum- mer and autumn reach approximately 0.10 % per decade at mid-latitudes. Atmos. Chem. Phys., 20, 8083–8102, 2020 3.2 Impact of VSLBr on the seasonal evolution of the total ozone column (TOC) Our results of the mid-latitude 1TOC due to VSLBr lie within the lower range of previous modelling studies over the 1960–2005 (Sinnhuber and Meul, 2015) and 1980–2005 (Feng et al., 2007) periods. This is expected as the detailed treatment of VSLBr sources in Sinnhuber and Meul (2015) results in an additional BrVSL y injection of 6 ppt, while in https://doi.org/10.5194/acp-20-8083-2020 Atmos. Chem. Phys., 20, 8083–8102, 2020 J. A. Barrera et al.: Seasonal impact of VSL bromocarbons on lowermost stratospheric ozone 8093 Figure 5. Latitude distributions of total ozone column difference (1TOC) between the experiments. Panels (a) and (b) show the annual and seasonal mean absolute 1TOC (DU) for the present day and the end of 21st century, respectively, while panels (c) and (d) show the corresponding relative 1TOC (%). The corresponding horizontal lines shown in panel (d) indicate where the relative 1TOC values between the present day and the end of the 21st century are statistically signiﬁcant with a 95 % conﬁdence interval using a two-tailed Student’s t test. Figure 5. Latitude distributions of total ozone column difference (1TOC) between the experiments. Panels (a) and (b) show the annual and seasonal mean absolute 1TOC (DU) for the present day and the end of 21st century, respectively, while panels (c) and (d) show the corresponding relative 1TOC (%). The corresponding horizontal lines shown in panel (d) indicate where the relative 1TOC values between the present day and the end of the 21st century are statistically signiﬁcant with a 95 % conﬁdence interval using a two-tailed Student’s t test. that is in line with its ﬁxed emissions considered throughout the whole modelled period. local 1O3(z) trends of up to 1 % and 0.5 % per decade at SH-MLs and NH-MLs, respectively (see Fig. 6f). The largest O3(z) reductions are located precisely at the same altitudes and latitudes where the ClOx/Cly ratio peak is modelled within the lowermost stratosphere at SH-MLs (see Fig. 2), which explains the hemispheric asymmetry in the VSLBr impacts. This highlights that the enhancement in local ozone depletion mediated by VSLBr is largest in the context of maximum background active-chlorine abundance since they provide an additional partner for chlorine species in- volved in the ozone-depleting inter-halogen chemical reac- tions (e.g. ClOx–BrOx-Loss; see Sect. 3.4). These results are in agreement with Yang et al. (2014), who determined that VSLBr have a signiﬁcantly larger ozone impact in the pres- ence of a high stratospheric chlorine background than a low chlorine background using time slice sensitivity simulations with a variable and speculative VSLBr contribution on differ- ent background stratospheric Cly abundances. J. A. Barrera et al.: Seasonal impact of VSL bromocarbons on lowermost stratospheric ozone Note also that the changes in the magnitudes of these seasonal 1TOC trends are mainly attributed to a marked seasonal difference of ozone loss mediated by VSLBr during the period of highest background halogen abundance (i.e. present day). These seasonal differences are reduced to- wards the end of the 21st century due to the decline in long- lived inorganic-halogen abundance. y y ( ) Figure 4 shows the temporal evolution (1960–2100) of the annual and seasonal 1TOC between the experiments for SH- MLs, the tropics, and NH-MLs. In addition, an analysis of 1TOC as a function of latitude for the present day and the end of the 21st century is shown in Fig. 5. The inclusion of VSLBr leads to a continuous reduction in the TOC at all latitudes, with a larger ozone destruction efﬁciency moving from the tropics to the high latitudes. Moreover, note that when comparing the seasonal relative 1TOC between both periods, a statistically signiﬁcant difference (here deﬁned as p < 0.05) is only observed at mid-latitudes, as shown by the horizontal lines at the bottom of Fig. 5d. This highlights that the VSLBr-driven ozone depletion efﬁciency changes signif- icantly over the century even though the contribution from VSLBr to bromine stratospheric injection remains constant. Since gas-phase and heterogeneous inter-halogen reactions involving both bromine and chlorine play a fundamental role in stratospheric ozone loss (McElroy et al., 1986; Solomon, 1999; Salawitch et al., 2005), the VSL bromine efﬁciency in ozone depletion is primarily linked to the background inorganic-halogen abundance, which shows a continuous de- cline over the course of the 21st century due to the A1 halo- gen emission scenario considered in this work. This is in line with the ﬁndings of Yang et al. (2014). Therefore, an addi- tional stratospheric chlorine and bromine load from natural or anthropogenic substances not regulated by the Montreal Protocol will induce an increase in the VSLBr impacts mod- Within the tropics, no signiﬁcant changes in the TOC loss due to VSLBr are projected, though the model captures a slightly major TOC depletion during the 1990s. Therefore, VSLBr-driven ozone depletion is less sensitive to background halogen abundances, introducing an almost constant impact Atmos. Chem. Phys., 20, 8083–8102, 2020 https://doi.org/10.5194/acp-20-8083-2020 J. A. Barrera et al.: Seasonal impact of VSL bromocarbons on lowermost stratospheric ozone J. A. Barrera et al.: Seasonal impact of VSL bromocarbons on lowermost stratospheric ozone A. Barrera et al.: Seasonal impact of VSL bromocarbons on lowermost stratospheric ozone J. A. Barrera et al.: Seasonal impact of VSL bromocarbons on lowermost stratospheric ozone 8094 Figure 6. Temporal evolution of the annual mean ozone difference (1O3) between experiments as a function of altitude (a) at SH-MLs, (b) in the tropics, and (c) at NH-MLs. The right panels show the zonal mean 1O3 distributions for the (d) present day and (e) the end of the 21st century as well as the (f) 1O3 trends (% per decade) over the century. The absolute (colour scale) and relative (contour line) ozone differences were calculated considering the ozone number densities (i.e. molecules cm−3) of each experiment. The masked regions in panel (f) indicate where the relative 1O3(z) values between the present day and the end of the 21st century are statistically signiﬁcant with a 95 % conﬁdence interval using a two-tailed Student’s t test. Figure 6. Temporal evolution of the annual mean ozone difference (1O3) between experiments as a function of altitude (a) at SH-MLs, (b) in the tropics, and (c) at NH-MLs. The right panels show the zonal mean 1O3 distributions for the (d) present day and (e) the end of the 21st century as well as the (f) 1O3 trends (% per decade) over the century. The absolute (colour scale) and relative (contour line) ozone differences were calculated considering the ozone number densities (i.e. molecules cm−3) of each experiment. The masked regions in panel (f) indicate where the relative 1O3(z) values between the present day and the end of the 21st century are statistically signiﬁcant with a 95 % conﬁdence interval using a two-tailed Student’s t test. until the end of the century, they could lead to signiﬁcantly different seasonal perturbations in the radiative effects medi- ated by ozone. Future studies are needed to explore the po- tential VSLBr-mediated seasonal effects on the atmosphere’s radiative balance. heterogeneous bromine reactivation occurs mainly in sulfate aerosols and therefore lacks seasonal changes (see Figs. S3 and S4). Consequently, since the seasonal changes in ozone loss are maximized during the present-day period, the largest local 1O3(z) trends are also projected for spring and win- ter, with springtime 1O3(z) trends reaching up to 1.6 % and 0.7 % per decade for SH-MLs and NH-MLs, respectively (see Fig. S6b and d). 3.3 VSLBr impact on vertical ozone distributions Timeline analysis of the annual mean stratospheric ozone difference between the experiments as a function of al- titude at mid-latitudes and in the tropics is presented in Fig. 6. The inclusion of VSLBr produces a reduc- tion in ozone concentrations (i.e. O3(z) number densi- ties) throughout most of the stratosphere during the whole modelled period. The largest O3(z) reductions occur dur- ing the present-day period in the mid-latitude lowermost stratosphere, reaching ozone differences (1O3(z)) of up to −8 % (−0.25 × 1012 molecules cm−3) and −5 % (−0.14 × 1012 molecules cm−3) for SH-MLs and NH-MLs, respec- tively. Above 50 hPa (∼20 km), 1O3(z) values are prac- tically constant at around −1 % for all latitudes, which is linked to the lesser role played by VSL bromine compared to catalytic ozone loss by hydrogen (HOx = OH+HO2) and nitrogen (NOx = NO+NO2) oxides as well as by long-lived reactive halogens (Brasseur and Solomon, 2005; Salawitch et al., 2005; Müller, 2012). y Figure 7 shows the seasonal zonal mean distribution of the 1O3(z) during the present-day period. The corresponding 1O3(z) trends (% per decade) over the century are shown in Fig. S6. The largest contributions to the annual mean ozone loss in the mid-latitude lowermost stratosphere correspond to spring, followed by the preceding winter and the posterior summer in each hemisphere, with springtime 1O3(z) reach- ing up to −10 % and −7 % for SH-MLs and NH-MLs, re- spectively (see Fig. 7b and d). This seasonal enhancement in VSLBr-mediated local ozone depletion is inﬂuenced by the seasonal changes in the heterogeneous chlorine reactiva- tion processes on lowermost-stratospheric ice crystals since Our modelled 1O3(z) latitudinal distribution is in agree- ment with those reported by Sinnhuber and Meul et al. (2015) during the 1980–2005 period, with a larger percentage of ozone loss deepening around the mid-latitude tropopause. In addition, O3(z) reduction due to VSLBr in the mid- latitude lowermost stratosphere is about 50 % smaller by the end of the 21st century compared to the present-day period (see Fig. 6d and e). This is in line with projected https://doi.org/10.5194/acp-20-8083-2020 Atmos. Chem. Phys., 20, 8083–8102, 2020 J. A. Barrera et al.: Seasonal impact of VSL bromocarbons on lowermost stratospheric ozone 8095 Figure 7. Seasonal zonal mean 1O3 distributions during the present-day period. Figure 7. Seasonal zonal mean 1O3 distributions during the present-day period. tury between 50 and 300 hPa (see Figs. 6f and S6a–d) even though the contribution percentage from BrVSL y to BrLL+VSL y increases towards the end of the century. Moreover, the pro- jected seasonal and annual 1O3(z) trends peak precisely at the same altitudes where the modelled relative 1O3(z) val- ues are signiﬁcant (see Figs. 6f and S6a–d). These results highlight the clear dependence of VSLBr impacts on the tem- poral evolution of background halogen abundances and the processes of heterogeneous halogen reactivation, which in turn depend on temperature as well as the formation and dis- tribution of ice crystals and stratospheric sulfate aerosols. In fact, the projected impacts on vertical ozone distribution at the end of the century are similar to those prevailing be- fore 1980 for both SH-MLs and NH-MLs since the back- ground halogen abundances are similar between these peri- ods. Thus, potential future changes in the lowermost strato- spheric temperature (therefore inﬂuencing ice crystal forma- tion), geoengineering of climate via injection of stratospheric sulfate, and the formation and distribution of stratospheric aerosol (e.g. future volcanic eruptions) will directly inﬂuence the processes of heterogeneous halogen reactivation and con- sequently VSLBr-driven ozone depletion efﬁciency (Tilmes et al., 2008, 2012; Banerjee et al., 2016; Klobas et al., 2017; Heckendorn et al., 2009). family considering the independent contributions of oxy- gen (Ox-Loss), hydrogen (HOx-Loss), nitrogen (NOx-Loss), and halogen (Halogx-Loss; Brasseur and Solomon, 2005; Saiz- Lopez et al., 2014). Moreover, we discriminate between the individual contributions of pure bromine (BrOx-Loss) and chlorine (ClOx-Loss) cycles and the inter-halogen (ClOx– BrOx-Loss) cycle to the halogen family (Halogx-Loss = BrOx-Loss +ClOx-Loss +ClOx −BrOx-Loss). The odd-oxygen loss rate equations for the ozone-depleting families consid- ered in this work are presented in Table S3. tury between 50 and 300 hPa (see Figs. 6f and S6a–d) even though the contribution percentage from BrVSL y to BrLL+VSL y Figure 8 shows the temporal evolution of the annual con- tribution percentage of each ozone-depleting family to the total ozone loss rate in the lowermost stratosphere at SH- MLs (∼120 hPa) for both experiments as well as the sea- sonal evolution of HOx-Loss and Halogx-Loss contributions in each experiment. J. A. Barrera et al.: Seasonal impact of VSL bromocarbons on lowermost stratospheric ozone The corresponding ﬁgures for the lower- most stratosphere at NH-MLs (∼120 hPa) and in the trop- ics (∼50 hPa) are shown in Figs. S7 and S8, respectively. Halogx-Loss represents the second most important contribu- tion to the total ozone loss rate after HOx-Loss (∼80 %) at mid-latitudes in both experiments (see Figs. 8a and S7a), while within the tropics it represents the third family af- ter HOx-Loss and NOx-Loss (see Fig. S8a). This partially ex- plains the smaller modelled VSL bromine impact on ozone within the tropics as HalogLL+VSL x-Loss represents at most 10 % of the total ozone destruction over this region. Moreover, the enhancement in the HalogLL+VSL x-Loss contribution with re- spect to HalogLL x-Loss by including VSLBr is mostly compen- sated by a decrease in the HOLL+VSL x-Loss contribution at mid- latitudes. For example, for the present day, HalogLL+VSL x-Loss shows an increase of ∼7 % with respect to HalogLL x-Loss at SH-MLs, which is compensated by a reduction of ∼6 % in J. A. Barrera et al.: Seasonal impact of VSL bromocarbons on lowermost stratospheric ozone Near the tropical tropopause, the O3(z) reductions due to VSLBr are practically constant during the whole mod- elled period, with 1O3(z) of approximately −2 % (−0.06 × 1012 molecules cm−3) at 50 hPa (see Fig. 6b). Even with an increase in the contribution of BrVSL y to BrLL+VSL y of up to 65 % by the end of century – due to the implementa- tion of the Montreal Protocol (WMO, 2014, 2018) and the ﬁxed stratospheric BrVSL y injection – 1O3(z) changes in the tropical lowermost stratosphere are not signiﬁcant over the century (see Fig. 6f). This is in line with the modelled im- pacts on the TOC. In contrast, at mid-latitudes, the seasonal VSLBr impacts on ozone signiﬁcantly decrease over the cen- Compared to the annual mean, springtime absolute O3(z) reductions along mid-latitudes are deepened over the ﬁrst ∼5 km above the tropopause during the present-day period, whereas these reductions are located at higher altitudes (be- tween 2 and 5 km above the tropopause) and over a narrower geographic area, mainly at NH-MLs, during summer and au- tumn. Therefore, VSL bromine introduces seasonal changes not only in overall O3(z) reduction but also in vertical O3(z) distribution. Moreover, since seasonal VSLBr impacts persist Atmos. Chem. Phys., 20, 8083–8102, 2020 https://doi.org/10.5194/acp-20-8083-2020 ra et al.: Seasonal impact of VSL bromocarbons on lowermost stratospheric ozone J. A. Barrera et al.: Seasonal impact of VSL bromocarbons on lowermost stratospheric ozone 3.4 Changes in the long-term seasonal evolution of halogen-driven ozone loss rate due to VSLBr In order to determine the drivers controlling the VSLBr- driven seasonal impact on ozone in the lowermost strato- sphere at mid-latitudes and in the tropics, we compute the odd-oxygen chemical loss from each ozone-depleting https://doi.org/10.5194/acp-20-8083-2020 Atmos. Chem. Phys., 20, 8083–8102, 2020 A. Barrera et al.: Seasonal impact of VSL bromocarbons on lowermost stratospheric ozone J. A. Barrera et al.: Seasonal impact of VSL bromocarbons on lowermost stratospheric ozone 8096 Figure 8. Temporal evolution of the contribution percentages from different ozone-depleting families (Ox-Loss, NOx-Loss, HOx-Loss, Halogx-Loss) to the total odd-oxygen loss rate in the lowermost stratosphere (120 hPa) at SH-MLs. Panel (a) shows the annual mean contribution of each ozone-depleting family for the experiments with (solid line; ensLL+VSL) and without (dashed line; ensLL) VSLBr sources. Panels (b) and (c) show the seasonal mean contri- butions of both HOx-Loss and Halogx-Loss for the experiments with and without VSLBr sources, respectively. time). In fact, the enhancement in the HalogLL+VSL x-Loss contri- bution due to VSLBr makes it the dominant ozone-depleting family during winter between 1985 and 2020 at SH-MLs, surpassing in importance the role played by the otherwise dominant HOx-Loss. In contrast, no seasonal variability of Halogx-Loss contribution is modelled within the tropics in ei- ther experiment (see Fig. S8b and c), and thus the seasonal VSLBr impact on ozone remains approximately constant dur- ing the whole modelled period around the annual mean. g p Figure 9 shows the long-term evolution of the contribu- tion percentage of BrOx-Loss, ClOx-Loss, and ClOx–BrOx-Loss to the Halogx-Loss as a function of months and years in the lowermost stratosphere at SH-MLs (120 hPa) for both exper- iments. Equivalent results are presented for NH-MLs and the tropics in Figs. S9 and S10, respectively. For any ﬁxed year in both experiments, the BrOx-Loss contribution peaks during late summer and early autumn, while its minimum contri- bution occurs during the austral winter. In contrast, both the ClOx-Loss and ClOx–BrOx-Loss reach their maximum contri- bution during winter and early spring. Consequently, the in- clusion of VSLBr increases the BrOLL+VSL x-Loss contribution in all seasons while also increasing the ClOx–BrOLL+VSL x-Loss con- tribution mainly in winter. This is at the expense of a de- crease in the ClOLL+VSL x-Loss contribution. 3.4 Changes in the long-term seasonal evolution of halogen-driven ozone loss rate due to VSLBr In addition, the win- tertime ClOx–BrOLL+VSL x-Loss contribution reaches up to 50 % between 1990 and 2020, decreasing towards the end of the century as ClLL y and BrLL y abundances decrease within the A1 emission scenario. The decrease in both ClOLL+VSL x-Loss and ClOx–BrOLL+VSL x-Loss over time is offset by an increase in the BrOLL+VSL x-Loss contribution. Accordingly, the inclusion of VSLBr produces similar changes in the evolution of the con- tributions of each of the cycles that compose Halogx-Loss at NH-MLs, although the seasonal BrOx-Loss contribution is greater in both experiments compared to SH-MLs during the entire modelled period (see Fig. S9). Figure 8. Temporal evolution of the contribution percentages from different ozone-depleting families (Ox-Loss, NOx-Loss, HOx-Loss, Halogx-Loss) to the total odd-oxygen loss rate in the lowermost stratosphere (120 hPa) at SH-MLs. Panel (a) shows the annual mean contribution of each ozone-depleting family for the experiments with (solid line; ensLL+VSL) and without (dashed line; ensLL) VSLBr sources. Panels (b) and (c) show the seasonal mean contri- butions of both HOx-Loss and Halogx-Loss for the experiments with and without VSLBr sources, respectively. Figure 8. Temporal evolution of the contribution percentages from p ( g ) In summary, the HalogLL+VSL x-Loss contribution to the to- tal ozone loss rate in summer and autumn is almost en- tirely dominated by BrOLL+VSL x-Loss . In the winter months, when the seasonal HalogLL+VSL x−Loss contribution to the total ozone loss rate is comparatively higher, a transition from the ClOxBrOLL+VSL x-Loss contribution (∼50 %) at the begin- ning of the century towards BrOLL+VSL x-Loss at the end of the century is modelled. Thus, the halogen-driven ozone loss projected towards the end of the 21st century is mostly dominated by BrOx-Loss, leading to a less marked seasonal VSLBr impact on both vertical ozone distribution and the TOC, in accordance with the modelled results. On the other hand, within the tropics, BrOLL+VSL x-Loss dominates the seasonal HalogLL+VSL x-Loss contribution for practically the entire mod- elled period (see Fig. S10), while ClOLL+VSL x-Loss represents a maximum contribution of up to ∼35 % during the end of the 20th century. The seasonal increase from BrOLL x-Loss to BrOLL+VSL x-Loss by including VSLBr is well marked and is prac- HOLL+VSL x-Loss . 3.4 Changes in the long-term seasonal evolution of halogen-driven ozone loss rate due to VSLBr In particular, the interaction between different catalytic ozone-depleting cycles involving OH and halogen radicals as well as the inﬂuence of reactive halogens in alter- ing the OH/HO2 ratio has been described elsewhere (Bloss et al., 2005; Saiz-Lopez and von Glasow, 2012). Even though the additional seasonal ozone depletion due to VSLBr peaks during spring followed by winter at mid- latitudes, the largest Halogx-Loss contribution to the total ozone loss rate occurs during winter in both experiments, presenting a much smaller contribution during the rest of the year (see Figs. 8b, c and S7b, c). Indeed, this marked seasonal behaviour prevails both during boreal winter at NH-MLs and during the austral winter at SH-MLs. This is explained by considering the strong seasonal increase in active-chlorine abundance (driven by heterogeneous reactivation on ice crys- tals) as well as the winter decline in HOx abundance (driven by the decrease in solar radiation with respect to summer- https://doi.org/10.5194/acp-20-8083-2020 Atmos. Chem. Phys., 20, 8083–8102, 2020 A. Barrera et al.: Seasonal impact of VSL bromocarbons on lowermost stratospheric ozone J. A. Barrera et al.: Seasonal impact of VSL bromocarbons on lowermost stratospheric ozone 8097 igure 9. Evolution of the (a, b) BrOx-Loss, (c, d) ClOx–BrOx-Loss, and (e, f) ClOx-Loss contribution percentages to Halogx-Loss as a unction of the year and month in the lowermost stratosphere (120 hPa) at SH-MLs for the experiments (a, c, e) with and (b, d, f) without SLBr sources. Figure 9. Evolution of the (a, b) BrOx-Loss, (c, d) ClOx–BrOx-Loss, and (e, f) ClOx-Loss contribution percentages to Halogx-Loss as a function of the year and month in the lowermost stratosphere (120 hPa) at SH-MLs for the experiments (a, c, e) with and (b, d, f) without VSLBr sources. tically offset by a reduction in the ClOLL+VSL x-Loss contribution. Furthermore, unlike mid-latitudes, ClOx–BrOLL+VSL x-Loss has a lower contribution to the tropical HalogLL+VSL x-Loss , showing that VSLBr-driven topical ozone loss is independent of changes in the background halogen abundance throughout the modelled period. considers – in addition to long-lived sources – the contri- butions of VSLBr oceanic sources. 3.4 Changes in the long-term seasonal evolution of halogen-driven ozone loss rate due to VSLBr We have evaluated an- nual and seasonal mean changes in vertical stratospheric ozone distribution and the TOC as well as examined the pro- jected ozone depletion (1O3 and 1TOC) trends over the century under a scenario in which both the relative contribu- tion of VSLBr to total inorganic-bromine and the background inorganic-chlorine abundances shift towards the future. Fi- nally, we have also assessed the long-term seasonal contribu- tion changes of halogen-mediated ozone loss (Halogx-Loss) due to VSL bromine, focusing on the lowermost stratosphere and distinguishing the role of pure (BrOx-Loss, ClOx-Loss) and inter-halogen (ClOx–BrOx-Loss) cycles that compose Halogx-Loss. The results presented here highlight the impor- tance of considering natural sources of halocarbons to de- 4 Conclusions This study has explored the impact of VSLBr on the tempo- ral evolution of stratospheric ozone both in the late 20th cen- tury and in the course of the 21st century at mid-latitudes and in the tropics. The analysis compares two indepen- dent experiments, one of which considers only the anthro- pogenic LLCl and LLBr source contributions, while the other Atmos. Chem. Phys., 20, 8083–8102, 2020 Atmos. Chem. Phys., 20, 8083–8102, 2020 J. A. Barrera et al.: Seasonal impact of VSL bromocarbons on lowermost stratospheric ozone Even though the contribution of VSL bromine to total stratospheric bromine reaches almost 50 % by the end of the 21st century, our simulations show a statistically signiﬁcant smaller VSLBr impact on lowermost stratospheric ozone as we move into the future. This result highlights that VSLBr-driven ozone loss is closely linked to the temporal evolution of LLCl and LLBr abundances regulated by the Montreal Protocol, as suggested by Yang et al. (2014). Along these same lines, Hossaini et al. (2015a) demonstrated that the chlorine load in the lowermost stratosphere has increased as a consequence of a recent and ongoing growth in emis- sions from anthropogenic VSLCl sources (mainly CH2Cl2) not controlled by the Montreal Protocol. If this VSLCl emis- sions trend continues into the future, additional studies will be required to quantify the increase in VSLBr impact mod- elled in this work on the projected ozone loss trends over the century considering the additional inorganic chlorine from VSLCl. Within the tropics, although our model captures a slightly greater TOC depletion associated with VSLBr dur- ing the 1990s, no signiﬁcant change is projected by the end of the 21st century. Halogx-Loss represents the third family in the contribution to the total ozone loss rate after HOx-Loss and NOx-Loss in the tropical lowermost stratosphere, with an al- most constant seasonal contribution throughout the modelled period. BrOLL+VSL x-Loss practically dominates the contribution to HalogLL+VSL x-Loss , with a maximum ClOx–BrOLL+VSL x-Loss contribu- tion of up to ∼30 % by the end of the 20th century, reﬂecting the low sensitivity of VSL bromine to background halogen abundances to drive stratospheric ozone depletion. Data availability. Computing resources, support, and data storage are provided and maintained by the Computational and Information System Laboratory from the National Center of Atmospheric Re- search (CISL, 2017). The code of the CAM-chem model can be downloaded from the website (https://www2.acom.ucar.edu/gcm/ cam-chem (last access: 10 March 2018) (Atmospheric Chemistry Observations and Modelling Division repository, 2018). Data that support the ﬁndings of this study can be downloaded from the AC2 web page (https://ac2.iqfr.csic.es/en/publications, last access: 25 June 2020) (Atmospheric Chemistry and Climate Department, 2020). The largest TOC depletion associated with VSLBr corre- sponds to the spring months followed by winter, with maxi- mum springtime 1TOC2000 reaching −10 DU (∼−3 %) and −7.7 DU (∼−2 %) for SH-MLs and NH-MLs, respectively. J. A. Barrera et al.: Seasonal impact of VSL bromocarbons on lowermost stratospheric ozone J. A. Barrera et al.: Seasonal impact of VSL bromocarbons on lowermost stratospheric ozon 8098 termine the overall evolution of stratospheric ozone in the tropics and at mid-latitudes during the 21st century. erogeneous chlorine reactivation efﬁciency in the lowermost stratosphere at SH-MLs compared to NH-MLs leads to hemi- spheric asymmetry in the VSLBr-driven ozone reductions. Our analysis shows that the inclusion of VSLBr results in a realistic stratospheric bromine loading that improves the agreement between the model and observations of the TOC at mid-latitudes, highlighting the need to consider these ad- ditional natural emissions to determine the overall evolution of ozone during the 21st century. Our results are in accor- dance with previous modelling studies that included natural bromocarbons and explored stratospheric ozone in the recent past (Salawitch et al., 2005; Feng et al., 2007; Sinnhuber et al., 2015) as well as by the end of the 21st century (Falk et al., 2017). However, unlike these previous works, we have explored seasonal ozone depletion arising from VSLBr with respect to the background stratospheric LLCl and LLBr abun- dances between 1960 and 2100. Even though the additional seasonal ozone depletion due to VSLBr peaks during spring followed by winter, the largest HalogLL+VSL x-Loss contribution to the total ozone loss rate oc- curs during winter, with a much smaller contribution during all other seasons. In fact, the wintertime HalogLL+VSL x-Loss con- tribution dominates the total ozone loss rate between 1985 and 2020 in the lowermost stratosphere at SH-MLs, with a ClOx–BrOLL+VSL x-Loss contribution to HalogLL+VSL x-Loss reach- ing up to 50%. Due to the expected decrease in the LLCl and LLBr abundances, a transition from ClOx–BrOLL+VSL x-Loss to BrOx-Loss during the course of the 21st century is projected, leading to a less marked seasonal VSLBr impact towards the end of the century. At mid-latitudes, VSLBr introduce a continuous reduction in the TOC during the entire modelled period, with the largest TOC depletion occurring during the period of highest back- ground halogen abundance (1TOC2000 = −8 DU, −2.5 %, for SH-MLs and −5.5 DU, −1.6 %, for NH-MLs). In turn, a signiﬁcant decrease in the impact towards the end of the 21st century at mid-latitudes is projected, with 1TOC trends of 0.15 % and 0.10 % per decade for SH-MLs and NH-MLs, respectively. https://doi.org/10.5194/acp-20-8083-2020 https://doi.org/10.5194/acp-20-8083-2020 References Carpenter, L. J., Wevill, D. J., Hopkins, J. R., Dunk, R. M., Jones, C. E., Hornsby, K. E., and McQuaid, J. B.: Bromoform in trop- ical Atlantic air from 25◦N to 25◦S, Geophys. Res. Lett., 34, L11810, https://doi.org/10.1029/2007GL029893, 2007. Abrahamsson, K., Granfors, A., Ahnoff, M., Cuevas, C. A., and Saiz-Lopez, A.: Organic bromine compounds produced in sea ice in Antarctic winter, Nat. 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https://openalex.org/W2801658393	https://www.nature.com/articles/s41598-018-25816-8.pdf	English	null	Anticancer activity and antibody-dependent cell-mediated cytotoxicity of novel anti-nucleolin antibodies	Scientific reports	2,018	cc-by	10,048	Anticancer activity and antibody- dependent cell-mediated cytotoxicity of novel anti-nucleolin antibodies Received: 28 November 2017 Accepted: 9 April 2018 Published: xx xx xxxx Nucleolin arises as a relevant target for cancer therapy, as it is overexpressed at the surface of cancer and angiogenic endothelial cells thus enabling a dual cellular targeting strategy. Immunotherapeutic strategies, albeit of proven therapeutic relevance, have been scarcely explored against this target. Therefore, this work aimed at engineering an anti-nucleolin VHH-based antibody capable of triggering anticancer immune responses. Herein, anti-nucleolin VHHs have been generated upon grafting F3 peptide-derived nucleolin-binding sequences onto a VHH CDR1 or CDR3. One of these nucleolin-binding CDR3-grafted VHH was subsequently fused to a human IgG1 Fc region, enabling a significant antibody- dependent cell-mediated cytotoxicity (ADCC). The generated anti-nucleolin VHH revealed increased binding and antiproliferative effects against cancer cells, relative to the parental VHH, while the VHH-Fc counterpart presented increased cytotoxicity relative to the corresponding VHH. This VHH-Fc also triggered an ADCC effect, in the nanomolar range, against a nucleolin-overexpressing cancer cell line. This effect was evidenced by a 2 or 1.7-fold increase of cell death, in the presence of PBMCs, relative to the parental VHH-Fc or the VHH counterpart, respectively. Overall, these formats represent the first anti-nucleolin VHHs and the first anti-nucleolin antibody with ADCC activity that have been successfully developed. Nucleolin is a multifunctional protein expressed in the nucleus of exponentially growing eukaryotic cells, where it participates in rRNA synthesis and ribosome biogenesis1. However, in highly proliferating cells, such as cancer cells and angiogenic endothelial cells of the tumour vasculature, nucleolin is translocated to the sur- face2. This translocation makes nucleolin a potential target for anticancer therapy, as it is accessible to drugs administered intravenously, namely the one overexpressed in the tumour vasculature3. In addition, as nucleolin interacts with proteins involved in cell proliferation and migration pathways (such as EGFR4 and CXCR45). As such, nucleolin-based targeting strategies might also disrupt the referred pathways, thus compromising tumour progression6–11. p g Antibodies are nowadays one of the major classes of therapeutics and are currently used against several malig- nancies. These proteins combine a high affinity to their targets through the variable domains (VH and VL) of the antigen binding fragment (Fab), with the capacity to trigger cell death by several mechanisms. These include direct cell death (upon interfering with the signalling pathways in which the target is involved) and immune responses, mediated by the Fc region. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Anticancer activity and antibody- dependent cell-mediated cytotoxicity of novel anti-nucleolin antibodies One of these immune responses is antibody-dependent cell-mediated cyto- toxicity (ADCC)12, which plays a relevant role in the therapeutic outcome of antibodies currently used in the clinic, such as cetuximab, trastuzumab and rituximab13–18. 1CNC – Center for Neuroscience and Cell Biology, Faculty of Medicine (Pólo I), University of Coimbra, Rua Larga, 3004-504, Coimbra, Portugal. 2IIIUC – Institute for Interdisciplinary Research, University of Coimbra, Casa Costa Alemão – Pólo II, Rua Dom Francisco de Lemos, 3030-789, Coimbra, Portugal. 3TREAT U, SA, Parque Industrial de Taveiro, Lote 44, 3045-508, Coimbra, Portugal. 4FFUC – Faculty of Pharmacy, University of Coimbra, Pólo das Ciências da Saúde, Azinhaga de Santa Comba, 3000-548, Coimbra, Portugal. 5iMed.ULisboa - Research Institute for Medicines, Faculty of Pharmacy, University of Lisbon, Avenida Prof. Gama Pinto, 1649-003, Lisbon, Portugal. Correspondence and requests for materials should be addressed to J.N.M. (email: jmoreira@ff.uc.pt) or J.G. (email: jgoncalv@ff.ulisboa.pt) SCIENTIFIC ReportS \| (2018) 8:7450 \| DOI:10.1038/s41598-018-25816-8 1 www.nature.com/scientificreports/ a a b Figure 1. Development of different VHH constructs and binding to human nucleolin. (a) VHHs were developed by grafting a nucleolin-binding peptide sequence, either onto CDR1 or CDR3, with or without flanking linkers, at the end of the grafted CDR. (b) Binding of the generated VHHs, and the corresponding parental VHH, to human nucleolin, following 1 h incubation at 37 °C. Results are from a representative experiment. Figure 1. Development of different VHH constructs and binding to human nucleolin. (a) VHHs were developed by grafting a nucleolin-binding peptide sequence, either onto CDR1 or CDR3, with or without flanking linkers, at the end of the grafted CDR. (b) Binding of the generated VHHs, and the corresponding parental VHH, to human nucleolin, following 1 h incubation at 37 °C. Results are from a representative experiment. Although antibodies have been a breakthrough in cancer therapy, some of their properties constitute a draw- back, as the high molecular weight (around 150 kDa). In this respect, the tumor penetration of smaller antibody variants is expected to take place in a higher extent, while maintaining long circulating time in the blood. The relevance of these features on the overall pharmacodynamics, has led to the development of smaller antibody formats19. In camelids, non-canonical antibodies (HCabs) have been identified, whose antigen binding fragment is solely composed by the heavy chain variable domain, named VHH. Anticancer activity and antibody- dependent cell-mediated cytotoxicity of novel anti-nucleolin antibodies This results in antibodies of approximately 80 kDa20, a molecular size that has enabled higher tumour/blood accumulation ratio, relative to a full-length IgG (150 kD), a scFv (28 kDa) and a diabody (55 kDa), and increased tumour accumulation relative to full-length IgG and a Fab’2 fragment (fusion of two Fab fragments, 110 kDa)19. g g Nucleolin targeting has been widely explored for the delivery of cytotoxic drugs by nanoparticles, using either the nucleolin-binding F3 peptide or the aptamer AS141121. In addition, different nucleolin ligands have shown antiproliferative and/or anti-angiogenic properties, both in vitro and in vivo, including AS14411, pseudopep- tides6,8,10,21 and a rabbit antibody22. Recently, an anti-nucleolin scFv (fused VH and VL domains, ≈35 kDa) has also been generated23 and further fused to a RNase, thus becoming cytotoxic upon internalization24. However, the potential of nucleolin as a target to enable antitumour immune responses remains unexplored. Herein it is hypothesized that generation of anti-nucleolin antibodies, generated upon grafting of the nucleolin binding domain from the F3 peptide, will enable increased cytotoxicity against nucleolin-overexpressing cells and, upon further fusion to a human IgG1 Fc region, will induce an ADCC effect. Results i di Binding of VHHs grafted with a F3 peptide-derived sequence to human nucleolin. Novel VHHs were developed upon grafting a 10-amino acid sequence, derived from the nucleolin-binding F3 peptide, onto either CDR1 or CDR3 of a parental VHH (anti-human TNF-α25), giving rise to two different VHHs (αNCL- CDR1 VHH and αNCL-CDR3 VHH). In addition, a variant of this sequence, flanked by the linker SGGGS at both ends, was also grafted onto each CDR, originating αNCL-CDR1-L VHH and αNCL-CDR3-L VHH (Fig. 1a). The incorporation of these flanking linkers aimed at conferring higher conformational flexibility to SCIENTIFIC ReportS \| (2018) 8:7450 \| DOI:10.1038/s41598-018-25816-8 2 www.nature.com/scientificreports/ Figure 2. Binding of different VHH constructs to nucleolin-overexpressing cells. One hundred thousand of MDA-MB-435S (a,c) or 4T1 (b,c) cells were incubated with parental VHH or VHHs grafted with a nucleolin- binding peptide sequence either onto CDR1 or CDR3, with (αNCL-CDR1-L or αNCL-CDR3-L, respectively) or without (αNCL-CDR1 or αNCL-CDR3, respectively) flanking linkers at the end of the grafted CDR, for 45 min at 4 °C. Binding was assessed with a final incubation with anti-HA-FITC antibody and further analysis by flow cytometry. Competitive inhibition assays were also performed for each cell line (c,d), upon pre- incubation of the cells with 75 µM F3 peptide (gray bars), for 30 min at 4 °C. A control without competitive inhibition was performed (black bars). Data represent the mean ± SD of three independent experiments, performed in duplicate Differences in binding among the VHHs tested were evaluated by one way ANOVA Figure 2. Binding of different VHH constructs to nucleolin-overexpressing cells. One hundred thousand of MDA-MB-435S (a,c) or 4T1 (b,c) cells were incubated with parental VHH or VHHs grafted with a nucleolin- binding peptide sequence either onto CDR1 or CDR3, with (αNCL-CDR1-L or αNCL-CDR3-L, respectively) or without (αNCL-CDR1 or αNCL-CDR3, respectively) flanking linkers at the end of the grafted CDR, for 45 min at 4 °C. Binding was assessed with a final incubation with anti-HA-FITC antibody and further analysis by flow cytometry. Competitive inhibition assays were also performed for each cell line (c,d), upon pre- incubation of the cells with 75 µM F3 peptide (gray bars), for 30 min at 4 °C. A control without competitive inhibition was performed (black bars). Data represent the mean ± SD of three independent experiments, performed in duplicate. Differences in binding among the VHHs tested were evaluated by one-way ANOVA followed by Tukey’s test. Results i di Differences in binding, with or without pre-incubation with F3 peptide, were evaluated by Student’s t-test (p < 0.05, p < 0.01, *p < 0.001). the CDR loop, thus improving antigen binding and recognition26,27. All the new generated anti-nucleolin VHH fragments bound to nucleolin, regardless of the CDR grafted, with the αNCL-CDR3 VHH and αNCL-CDR3-L VHH presenting the highest extent. The nucleolin-binding VHHs grafted onto CDR1 or CDR3 presented a 2- or 3-fold increased binding to nucleolin, respectively, relative to the parental VHH, without engraftment of the F3 peptide-derived sequence (Fig. 1b). This supported the involvement of the F3 peptide-derived sequence on the observed binding of the anti-nucleolin VHH constructs. Binding of anti-nucleolin VHHs to cancer cells. The binding capacity of anti-nucleolin VHHs was assessed with human and mouse cancer cells (MDA-MB-435S and 4T1, respectively)28,29. A concentration-dependent binding of all constructs was observed (Fig. 2a,b). In the case of MDA-MB-435S cells, a significant difference on the binding, at 100 nM, of αNCL-CDR3 (p < 0.01) or αNCL-CDR3-L (p < 0.001) rel- ative to αNCL-CDR1 and αNCL-CDR1-L or to the parental VHH (p < 0.001), was observed. This trend, favour- ing a higher binding extent of the construct grafted onto CDR3, relative to CDR1, was further confirmed at 1000 nM of incubated protein (p < 0.001). At this concentration, a significant difference between the binding SCIENTIFIC ReportS \| (2018) 8:7450 \| DOI:10.1038/s41598-018-25816-8 3 www.nature.com/scientificreports/ Figure 3. Cytotoxicity of anti-nucleolin VHHs against nucleolin-overexpressing cancer cells. Different VHH constructs, grafted with a nucleolin-binding peptide sequence either onto CDR1 or CDR3, with (αNCL- CDR1-L or αNCL-CDR3-L, respectively) or without (αNCL-CDR1 or αNCL-CDR3, respectively) flanking linkers at the end of the grafted CDR, were incubated with (a) MDA-MB-435S or (b) 4T1 cancer cells, at concentrations up to 8 µM, for 72 h at 37 °C. The parental VHH, without the targeting component to nucleolin, was included as control. In the end of the incubation, cytotoxicity was assessed by the MTT assay. Data represent the mean ± SD of at least three independent experiments, performed in duplicate. Differences in cytotoxicity among the tested VHHs were evaluated by one-way ANOVA followed by Tukey’s test (p < 0.05, p < 0.01, p < 0.001). Figure 3. Cytotoxicity of anti-nucleolin VHHs against nucleolin-overexpressing cancer cells. Results i di Different VHH constructs, grafted with a nucleolin-binding peptide sequence either onto CDR1 or CDR3, with (αNCL- CDR1-L or αNCL-CDR3-L, respectively) or without (αNCL-CDR1 or αNCL-CDR3, respectively) flanking linkers at the end of the grafted CDR, were incubated with (a) MDA-MB-435S or (b) 4T1 cancer cells, at concentrations up to 8 µM, for 72 h at 37 °C. The parental VHH, without the targeting component to nucleolin, was included as control. In the end of the incubation, cytotoxicity was assessed by the MTT assay. Data represent the mean ± SD of at least three independent experiments, performed in duplicate. Differences in cytotoxicity among the tested VHHs were evaluated by one-way ANOVA followed by Tukey’s test (p < 0.05, p < 0.01, p < 0.001). of α-NCL-CDR1 VHH or α-NCL-CDR1-L VHH and the parental VHH (p < 0.001) was also observed. At the highest concentration tested, CDR1- and CDR3-grafted VHHs bound to a percentage of cells of approximately 40% and 80%, respectively, whereas the parental VHH bound to less than 10% of the cells (Fig. 2a). The profile of binding of anti-nucleolin VHH constructs to 4T1 cancer cell line, relative to the parental VHH (p < 0.01 for CDR3-grafted VHHs, at 100 nM and p < 0.001 for all anti-nucleolin VHHs, at 1000 nM), was similar to the one reported for MDA-MB-435S (Fig. 2b). The difference in terms of binding arising from peptide grafting either onto CDR1 or CDR3, was not so evident as for MDA-MB-435S, and a decrease on the overall extent of binding of anti-nucleolin VHH was observed, of up to 4-fold, depending on the protein concentration.i p p g p To confirm that nucleolin was involved in the observed binding of the generated VHHs to these cancer cells, a competition assay was performed, by pre-incubating the cells with the F3 peptide. This pre-incubation reduced the extent of binding of the anti-nucleolin VHHs by at least 50% in both cell lines (Fig. 2c,d), suggesting that nucleolin was involved in the binding of the novel VHHs. Cytotoxicity of anti-nucleolin VHHs against cancer cells. All anti-nucleolin VHHs presented oncentration-dependent cytotoxicity against MDA-MB-435S and 4T1 cells, in the micromolar range (Fig. 3).f The parental VHH-Fc antibody, without the targeting component to nucleolin, was included as control. In the end of the incubation, cytotoxicity was assessed by the MTT assay. Data represent the mean ± SD of three independent experiments, performed in duplicate. Differences in cytotoxicity between the VHH-Fc antibodies were evaluated by Student’s t-test (p < 0.01, p < 0.001). Figure 4. Cytotoxicity of an anti-nucleolin VHH-Fc antibody against nucleolin-overexpressing cancer cells. (a) An anti-nucleolin VHH-Fc antibody (αNCL-VHH-Fc) was generated by fusing αNCL-CDR3 to a human IgG1 Fc region and then incubated with (b) MDA-MB-435S or (c) 4T1 nucleolin-overexpressing cancer cells, at concentrations up to 1000 nM, for 72 h at 37 °C. The parental VHH-Fc antibody, without the targeting component to nucleolin, was included as control. In the end of the incubation, cytotoxicity was assessed by the MTT assay. Data represent the mean ± SD of three independent experiments, performed in duplicate. Differences in cytotoxicity between the VHH-Fc antibodies were evaluated by Student’s t-test (p < 0.01, *p < 0.001). Cytotoxicity of an anti-nucleolin VHH-Fc antibody against cancer cells. The αNCL-CDR3 VHH and the parental VHH, without the nucleolin-binding component, were further fused to a human IgG1 Fc region (Fig. 4a). The higher extent of cytotoxicity enabled by the αNCL-CDR3 VHH relative to the CDR1-grafted coun- terpart, and regardless of the presence of the flanking linkers at each end of the grafted CDR, supported the choice of the former to generate the novel αNCL VHH-Fc antibody. Improved cytotoxicity in the nanomolar range was observed with the anti-nucleolin αNCL-VHH-Fc antibody, relative to the VHH counterpart (Fig. 4b,c). A more pronounced decrease of cell viability was observed with αNCL-VHH-Fc, relative to the control antibody (parental VHH-Fc). At the highest concentration tested, αNCL-VHH-Fc led to a 1.7-fold decrease of viability of MDA-MB-435S cells relative to parental VHH-Fc. Antibody-dependent cell-mediated cytotoxicity (ADCC) of anti-nucleolin VHH-Fc antibody against MDA-MB-435S cells. As human IgG1 Fc region enables ADCC responses30, we evaluated cancer cell death upon incubation with anti-nucleolin VHH-Fc antibody and effector cells (PBMCs). In the presence of PBMCs, αNCL-VHH-Fc at 25 nM enabled higher MDA-MB-435S cancer cell death than equimolar concentra- tion of parental VHH-Fc (p < 0.001, Fig. 5a). Important to point out that this difference was completely dissipated in the absence of PBMCs (Fig. 5a). Cytotoxicity of anti-nucleolin VHHs against cancer cells. All anti-nucleolin VHHs presented oncentration-dependent cytotoxicity against MDA-MB-435S and 4T1 cells, in the micromolar range (Fig. 3).f Cytotoxicity of anti-nucleolin VHHs against cancer cells. All anti-nucleolin VHHs presented concentration-dependent cytotoxicity against MDA-MB-435S and 4T1 cells, in the micromolar range (Fig. 3).f In the case of MDA-MB-435S cells, differences relative to the parental VHH became evident at 4 µM, with cell viability reduced to 60% (p < 0.001 for αNCL-CDR1; p < 0.01 for αNCL-CDR1-L) or 30% (p < 0.001) by the proteins grafted onto CDR1 or CDR3, respectively (corresponding to 1.5- or 2.5-fold decrease of cell viability, respectively). CDR3-grafted proteins presented a 2-fold decreased cell viability compared to CDR1-grafted pro- teins (p < 0.001). At 8 µM, differences of activity among anti-nucleolin VHH fragments were dissipated, resulting in less than 20% of viable cells and reaching a 1.5-fold decrease of cell viability relative to the parental VHH (p < 0.001) (Fig. 3a).ht The extent of decrease of cell viability achieved with CDR3-grafted VHHs against 4T1 cancer cells, was similar to the one observed against the MDA-MB-435S cells (at 4 µM, p < 0.05 and p < 0.01 for αNCL-CDR3 and αNCL-CDR3-L, respectively, and at 8 µM, p < 0.001 for all anti-nucleolin VHHs, relative to the parental VHH) (Fig. 3b). Results suggested, once again, that grafting onto CDR3 improved the cytotoxicity efficacy of anti-nucleolin VHHs (relative to grafting onto CDR1). SCIENTIFIC ReportS \| (2018) 8:7450 \| DOI:10.1038/s41598-018-25816-8 4 www.nature.com/scientificreports/ Figure 4. Cytotoxicity of an anti-nucleolin VHH-Fc antibody against nucleolin-overexpressing cancer cells. (a) An anti-nucleolin VHH-Fc antibody (αNCL-VHH-Fc) was generated by fusing αNCL-CDR3 to a human IgG1 Fc region and then incubated with (b) MDA-MB-435S or (c) 4T1 nucleolin-overexpressing cancer cells, at concentrations up to 1000 nM, for 72 h at 37 °C. The parental VHH-Fc antibody, without the targeting component to nucleolin, was included as control. In the end of the incubation, cytotoxicity was assessed by the MTT assay. Data represent the mean ± SD of three independent experiments, performed in duplicate. Differences in cytotoxicity between the VHH-Fc antibodies were evaluated by Student’s t-test (p < 0.01, ***p < 0.001). Figure 4. Cytotoxicity of an anti-nucleolin VHH-Fc antibody against nucleolin-overexpressing cancer cells Figure 4. Cytotoxicity of an anti-nucleolin VHH-Fc antibody against nucleolin-overexpressing cancer cells. (a) An anti-nucleolin VHH-Fc antibody (αNCL-VHH-Fc) was generated by fusing αNCL-CDR3 to a human IgG1 Fc region and then incubated with (b) MDA-MB-435S or (c) 4T1 nucleolin-overexpressing cancer cells, at concentrations up to 1000 nM, for 72 h at 37 °C. Cytotoxicity of anti-nucleolin VHHs against cancer cells. All anti-nucleolin VHHs presented oncentration-dependent cytotoxicity against MDA-MB-435S and 4T1 cells, in the micromolar range (Fig. 3).f Moreover, the effect was partly dependent on the presence of the Fc region, as in the absence of the latter, the αNCL-CDR3 VHH protein enabled a lower level of cancer cell death in the pres- ence of PBMCs (p < 0.01, Fig. 5b). The absence of ADCC activity of the parental VHH-Fc construct was further demonstrated, as it enabled similar levels of cell viability as its VHH counterpart, upon incubation with PBMCs (Fig. 5c). These results supported an overall nucleolin-dependent ADCC effect by the anti-nucleolin VHH-Fc antibody. Increased cytotoxicity of αNCL-CDR3 VHH in the presence of PBMCs, relative to αNCL-CDR3 VHH alone or parental VHH incubated with PBMCs (p < 0.01), was observed.h The extent of cell death for each anti-nucleolin ligand and control proteins as a function of individual PBMCs donors, revealed similar profiles (Fig. 6). Increase in PBCM-dependent cell death ranged from, approximately, 1.3- to 2-fold, relative to the parental VHH-Fc and a 1.3- to 1.7-fold increase relative to the VHH counterpart (p < 0.01). Therefore, and regardless of the PBMC origin, these results supported a Fc-dependent ADCC effect of the anti-nucleolin VHH-Fc against the nucleolin-overexpressing MDA-MB-435S cancer cells. SCIENTIFIC ReportS \| (2018) 8:7450 \| DOI:10.1038/s41598-018-25816-8 5 www.nature.com/scientificreports/ Figure 5. Cytotoxicity of anti-nucleolin VHHs in the presence of PBCMs, against MDA-MB-435S cells. MDA- MB-435S cells, previously cultured in a RTCA plate for 24 h, were incubated with protein, without (solid lines) or with human PBMCs (at a target cells/effector cells ratio of 1:10 or 1:5, dotted lines), for 72 h at 37 °C. Cell index, normalized to the beginning of the incubation, was measured every 15 min, using the xCELLigence system. ADCC effect of the anti-nucleolin VHH-Fc antibody (red) was further supported upon assessing its activity versus (a) 25 nM parental VHH-Fc (blue), (b) 50 nM αNCL-CDR3 VHH (green), (c) 25 nM parental VHH-Fc (blue) versus 50 nM parental VHH (orange). Data are from a representative experiment, performed in duplicate. Figure 5. Cytotoxicity of anti-nucleolin VHHs in the presence of PBCMs, against M Figure 5. Cytotoxicity of anti-nucleolin VHHs in the presence of PBCMs, against MDA-MB-435S cells. MDA- MB-435S cells, previously cultured in a RTCA plate for 24 h, were incubated with protein, without (solid lines) or with human PBMCs (at a target cells/effector cells ratio of 1:10 or 1:5, dotted lines), for 72 h at 37 °C. Cytotoxicity of anti-nucleolin VHHs against cancer cells. All anti-nucleolin VHHs presented oncentration-dependent cytotoxicity against MDA-MB-435S and 4T1 cells, in the micromolar range (Fig. 3).f Cell index, normalized to the beginning of the incubation, was measured every 15 min, using the xCELLigence system. ADCC effect of the anti-nucleolin VHH-Fc antibody (red) was further supported upon assessing its activity versus (a) 25 nM parental VHH-Fc (blue), (b) 50 nM αNCL-CDR3 VHH (green), (c) 25 nM parental VHH-Fc (blue) versus 50 nM parental VHH (orange). Data are from a representative experiment, performed in duplicate. Discussion Differences in cytotoxicity among the tested proteins, upon incubation with PBMCs, were evaluated by repeated measures ANOVA followed by Tukey test. Figure 6. Effect of the PBMCs donor variability on the cytotoxicity of nucleolin-binding proteins against MDA- MB-435S cells. Figures a–d represent the cytotoxicity assays, performed in duplicate, with PBCMs harvested from four donors, using the xCELLigence system. MDA-MB-435S, previously cultured in a RTCA plate for 24 h, were incubated with PBMCs (at a target cells/effector cells ratio of 1:10 or 1:5) and 25 nM anti-nucleolin VHH-Fc antibody (αNCL-VHH-Fc) or the parental VHH-Fc antibody, without the nucleolin-binding component, for 72 h at 37 °C. The VHH counterparts of these antibodies (50 nM αNCL-CDR3 VHH or parental VHH) were also included as controls. Cancer cell death was calculated from the area under the curve (AUC), as described in the Methods. Differences in cytotoxicity among the tested proteins, upon incubation with PBMCs, were evaluated by repeated measures ANOVA followed by Tukey test. ADCC component underlying trastuzumab mechanism of action than the single interference at the level of the HER2-associated intracellular signaling pathway. In fact, in patients with HER2+ metastatic breast tumors, with com- plete or partial remission, upon treatment with preoperative trastuzumab, tumor infiltration of lymphoid cells was iden- tified along with ex-vivo ADCC activity in the presence of PMBCs. In these patients, neither HER2 down-modulation nor changes in proliferation (as evaluated by Ki-67 staining) were observed during therapy18. A recent study also suggested that ADCC contributes to the treatment efficacy of trastuzumab in patients with metastatic gastric cancer. This study reported that the FcγRIIa-131H/H genotype was associated with significantly increased progression-free survival in patients treated with trastuzumab combined with chemotherapy, relative to the arm with chemotherapy alone34. Enhanced ADCC responses were also observed upon incubation of cetuximab with a squamous cell carci- noma of the head and neck (SCCHN) cell line, along with patient-derived NK cells, being also predictive of increased progression-free survival, and further supporting the relevance of ADCC in antibody therapy13.i One of the most common strategies to generate an antibody or an antibody fragment against a specific target encompass: (i) immunization of the animal model used for antibody generation, followed by spleen cell harvesting and antibody recovery, and (ii) generation of a library and selection of binders by display technologies (most fre- quently, phage display). Discussion Several approaches have been developed to target nucleolin-overexpressing tumours, either with targeted drug delivery using nanoparticles21 or nucleolin-binding agents with cytotoxic activity against nucleolin-overexpressing cells31. However, nucleolin targeting with antibodies remains largely unexplored, namely with those enabling an ADCC effect, a mechanism of proven therapeutic relevance for commercially available antibodies, such as cetux- imab, trastuzumab or rituximab13–18.h The importance of ADCC as a mechanism of cell death, enabling a more favorable therapeutic outcome, has been demonstrated in several studies involving polymorphisms in the IgG1 Fc receptors-coding FcγRIIA and FcγRIIIA genes. These receptors present different expression patterns depending on the type of immune cells. FcγRIIa and FcγRIIIa/CD16a are low-affinity activating receptors for IgG1 Fc and are expressed by subpopula- tions of NK cells, macrophages or T cells. Some polymorphisms in these genes augment the affinity of the IgG1 Fc region towards the receptor and correlate with better clinical responses, when compared with the responses in the cohort without polymorphisms. For example, patients with non-Hodgkin lymphoma, presenting a mutation at position 158 of FcγRIIIa (where phenylalanine was replaced by valine), have been associated with complete response to rituximab, subsequent to a stronger binding to the corresponding Fc region16,32. In a cohort of patients with metastatic colorectal cancer, progression free survival was higher in patients with FcγRIIa-131H/H and FcγRIIIa-158V/V genotypes, regardless of the KRAS status15.fi γ g yp g In another setting, treatment efficacy with trastuzumab was increased in patients with metastatic HER2-positive breast cancer presenting V/V or H/H genotype, which correlated with higher ex-vivo ADCC activity of periph- eral blood mononuclear cells (PBMCs)17,33. In this respect, there are experimental evidences suggesting a stronger SCIENTIFIC ReportS \| (2018) 8:7450 \| DOI:10.1038/s41598-018-25816-8 6 www.nature.com/scientificreports/ entificreports/ Figure 6. Effect of the PBMCs donor variability on the cytotoxicity of nucleolin-binding proteins against MDA- MB-435S cells. Figures a–d represent the cytotoxicity assays, performed in duplicate, with PBCMs harvested from four donors, using the xCELLigence system. MDA-MB-435S, previously cultured in a RTCA plate for 24 h, were incubated with PBMCs (at a target cells/effector cells ratio of 1:10 or 1:5) and 25 nM anti-nucleolin VHH-Fc antibody (αNCL-VHH-Fc) or the parental VHH-Fc antibody, without the nucleolin-binding component, for 72 h at 37 °C. The VHH counterparts of these antibodies (50 nM αNCL-CDR3 VHH or parental VHH) were also included as controls. Cancer cell death was calculated from the area under the curve (AUC), as described in the Methods. SCIENTIFIC ReportS \| (2018) 8:7450 \| DOI:10.1038/s41598-018-25816-8 Discussion This is somehow in contrast with the ability of D3 antibody to bind to human nucleolin, but not the one from mouse origin46. Binding to an epitope in a less conserved region of the protein could support the difference in binding, between the two species. The fact that the developed anti-nucleolin VHHs bound to both human and mouse nucleolin, becomes relevant when considering a future characterization of these antibodies in an in vivo setting, as it validates the use of either immunocompromised or immunocompetent mice models, overexpressing human or mouse nucleolin, respectively.hl p y The reported activity of the developed anti-nucleolin VHHs did not depend on loops with increased flexibil- ity, as enabled by SGGGS linkers inserted at the end of CDRs26,27. This is in contrast with the activity of camelid antibodies (HCabs), characterized by the VHH binding domains, instead of the common VH-VL. The longer and more flexible CDR3 loop of Hcabs, favors the access to clefts, characteristic of enzymes’ active site (usually not accessible to conventional antibodies)42,47,48. The results herein presented suggested that the epitope recognized by the developed anti-nucleolin VHHs was easily accessed, not requiring a longer or more flexible loop.h p y q g gl p The anti-nucleolin VHHs antibodies presented cytotoxicity against the cancer cell lines tested, in the micro- molar range. Although anti-nucleolin VHHs presented maximal binding to both cancer cell lines at 1 µM, cyto- toxic effects at this concentration were minimal. To explain this apparent discrepancy, it is important to take into account that in highly proliferative cells, nucleolin is constantly being expressed and translocated to the surface, with a half-life of approximately 1 h49. In the binding experiment (Fig. 2), incubation of the cancer cells with the VHHs was performed at 4 °C for 45 min, whereas the cytotoxicity experiment (Fig. 3) was carried out at 37 °C for 72 h. Differences both in temperature and time scale of the assays, support a higher extent of newly synthesized surface nucleolin in the latter and thus the minimal cytotoxicity at 1 µM of protein, concentration at which max- imal binding was observed.h g The dimeric format of the anti-nucleolin VHHs (VHH-Fc) presented improved cytotoxicity, in the nanomo- lar range. The use of antibodies in this format might enable additional pharmacodynamics features, besides the potential ADCC. Discussion However, the previous demonstration of the binding properties of specific ligands, namely peptide-based, towards a target of interest, enables the grafting of a known binding sequence onto the CDRs of the antibody35,36, thus bypassing the previous described time-consuming steps. In the present work, the development of anti-nucleolin VHHs relied on a grafting strategy based on the nucleolin-binding 31-amino acid F3 peptide, which had previously demonstrated promising in vivo tumour targeting capabilities29,37–39. Grafting was performed with the 10-amino acid sequence responsible for the major component of nucleolin binding by the F3 peptide40. No other studies have reported the use of this small sequence alone for grafting onto antibody formats. SCIENTIFIC ReportS \| (2018) 8:7450 \| DOI:10.1038/s41598-018-25816-8 7 www.nature.com/scientificreports/ Grafting of this 10-amino acid sequence onto CDR1 or CDR3 was justified by the fact that CDR1, and particu- larly CDR3, are the most important domains on antigen binding, especially in VHHs. In this antibody format, the lack of the VL domain is compensated by longer CDR1 and CDR3 domains, as well as a more exposed CDR3. These characteristics favour antigen interaction with these CDRs, mainly the latter41,42. In fact, some antigen interactions with VHH have been reported to occur only through CDR1 and CDR3 (as for an anti-RNase A VHH43) or only through CDR3 (as in an anti-carbonic anhydrase VHH44). Accordingly, grafting either onto CDR1 or CDR3 generated VHHs with the ability to bind nucleolin, as demonstrated with purified nucleolin and cancer cells. Competitive inhibition assays with the F3 peptide further confirmed that nucleolin was involved in the VHH binding to the cancer cell lines. The binding studies, along with the cytotoxicity experiments, supported that the modification onto CDR3 enabled a higher extent of activity relative to the exact same grafting, onto CDR1. This was in agreement with the reports showing that CDR3 usually plays a more relevant role in antigen binding, than other CDRs41,42. The higher binding of the developed anti-nucleolin VHHs to MDA-MB-435S cells, relative to 4T1 cells, is in accordance with the higher density of surface nucleolin of the former relative to the latter (approximately 102000 versus 45000 molecules, respectively; unpublished results).i pp y p y p Binding of the anti-nucleolin VHHs to 4T1 confirmed that these entities also bound to mouse nucleolin, which is line with the 81% homology of the protein between these two species45. Discussion In fact, VHH-Fc are dimeric proteins, thus increasing the binding avidity and, subsequently, the cytotoxic effects, relative to VHHs alone50. This could support the overall increased cytotoxicity enabled by the anti-nucleolin VHH-Fc antibody (in the nanomolar range), relative to its VHH counterpart (in the micromolar range). Although the increased extent of cytotoxicity of the former could be supported by the presence of two VHH monomers50, the fact that even twice the concentration of VHH did not enable an effect as strong as the VHH-Fc counterpart, suggested that other mechanisms are likely to contribute to this effect. In this respect, the possible simultaneous binding of the VHH-Fc to two surface nucleolin molecules could justify the referred dif- ference on cytotoxicity. A similar observation has been reported for cetuximab and its Fab counterpart, with the former presenting higher cytotoxicity than the latter, at concentrations of 66 and 132 nM, respectively. However, a Fab2′ fragment (bivalent) counterpart presented similar cytotoxicity to cetuximab, suggesting that the higher cytotoxicity enabled by the IgG and Fab2′ antibodies, relative to Fab, derived from their ability to simultaneously bind to two EGFR molecules51. The fact that parental VHH-Fc also presented cytotoxicity against MDA-MB-435S cells (although significantly lower than the anti-nucleolin VHH-Fc) could be associated with the anti-(human) TNF-α binding of non-grafted CDRs52,53. In fact, some cancer cell lines (including MDA-MB-435, the parental cell line of MDA-MB-435S) have been reported to express the transmembrane form of TNF-α, tmTNF-α54. The absence of binding to mouse 4T1 cells was in agreement with the lack of cross-reactivity between parental VHH and TNF-α mouse counterpart25.hf The fact that parental VHH-Fc also presented cytotoxicity against MDA-MB-435S cells (although significantly lower than the anti-nucleolin VHH-Fc) could be associated with the anti-(human) TNF-α binding of non-grafted CDRs52,53. In fact, some cancer cell lines (including MDA-MB-435, the parental cell line of MDA-MB-435S) have been reported to express the transmembrane form of TNF-α, tmTNF-α54. The absence of binding to mouse 4T1 cells was in agreement with the lack of cross-reactivity between parental VHH and TNF-α mouse counterpart25. The anti-nucleolin VHH-Fc antibody presented increased effects relative to other agents targeting nucleo- lin, including the AS1411 aptamer and the HB-19 and N6L pseudopeptides, which impacted cancer cell via- bility in the micromolar range6,10,11. Methods Reagents. Human recombinant nucleolin was from Abnova (Taiwan) and F3 peptide (KDEPQRRSARLSAKPAPPKPEPKPKKAPAKK) was custom synthesized by Genecust (Luxemburg). All other chemicals were of analytical grade purity. Cell culture. MDA-MB-435S (ATCC® HTB-129TM, USA) and 4T1 (ATCC® CRL-2539TM, USA) cell lines were cultured in RPMI-1640 (Lonza, Switzerland) and HEK293T cells (ATCC® CRL-3216TM, USA) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Lonza, Switzerland). Both media were supplemented with 10% (v/v) heat-inactivated FBS (HyClone, USA), 2 mM of L-glutamine (Lonza, Switzerland) and 1% (v/v) Pen/Strep/ Fungiezone solution (HyClone, USA). Cells were maintained at 37 °C in a humidified atmosphere of 5% CO2. Production of nucleolin-binding VHH. To generate a nucleolin-binding VHH, CDR1 or CDR3 domains of a parental VHH (anti-human TNF-α25) were grafted with the nucleolin binding F3 peptide-derived 10-aminoacid sequence PQRRSARLSA, giving rise to two different nanobodies (αNCL-CDR1 VHH and αNCL-CDR3 VHH). In addition, a variant of this sequence, flanked by the linker SGGGS at both ends, was also grafted onto each CDR, originating the VHHs αNCL-CDR1-L VHH and αNCL-CDR3-L VHH. Grafting was performed by PCR and, upon digestion of the VHH fragments with the enzymes HindIII and BglII (Thermo Scientific, USA), these were inserted onto a pT7 vector with both HA and His tags. For protein expression, per- formed in E. coli BL21 (DE3) cells, an overnight-grown culture containing the VHH of interest was diluted to 1:100 in SB medium with ampicillin (100 mg/L) and grown at 37 °C, 220 rpm, until reaching an OD between 0.7 and 0.9, at 600 nm. Protein expression was induced with 1 mM of isopropyl β-D-1-thiogalactopyranoside (IPTG, ThermoScientific, USA), for 16 h, 140 rpm. The culture was then centrifuged, resuspended in binding buffer (50 mM phosphate buffer, 300 mM NaCl, 40 mM imidazole, pH 7.0) with EDTA-free protease inhibitor cocktail (Roche, Switzerland) and sonicated. Protein was purified from the soluble fraction using HiTrap Chelating HP columns (GE Healthcare, UK). Production of nucleolin-binding VHH fused to Fc domain. An anti-nucleolin VHH-Fc antibody (αNCL-VHH Fc) was obtained by cloning the VHH sequence of αNCL-CDR3 VHH in the pFuse-hIgG1-Fc2 vector, upon digestion with NcoI and BglII (ThermoScientific, USA). A control antibody (parental VHH-Fc) was also generated, by cloning the parental VHH in the same vector. These were then transfected into HEK293T cells using the calcium phosphate transfection method62. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Importantly, other nucleolin-targeting agents, such as HB-19 and N6L, have been reported to bind not only to nucleolin, but also to nucleophosmin and sulphated glycosaminoglycans8,57. In fact, nucleolin exists in a complex with nucleophosmin58 and interacts with sulphated glycosaminoglycans59, as well as with proteins, including ErbB receptors and Ras60,61. These reports highlighted the complexity of nucleolin interactions and thus of the mechanistic complexity associated with targeting strategies towards this protein. As such, the involvement of other proteins, besides nucleolin, in the activity of the anti-nucleolin VHH/VHH-Fc herein described, should not be ruled out. In summary, herein it has been reported for the first time, and to the authors best knowledge, the development of nanobodies targeting nucleolin, which further fused to a human IgG1 Fc fragment, enabled ADCC activity. The latter (αNCL-VHH-Fc) could in fact emerge as a novel class of therapy against nucleolin-overexpressing tumours. Discussion Although the extent of this effect was lower than the one observed with the anti-nucleolin scFv 4LB5 (IC50 values in the low nanomolar range, 3–58 nM23), the anti-nucleolin antibody herein presented, demonstrated ADCC capacity. g y p p The anti-nucleolin VHH-Fc antibody presented increased effects relative to other agents targeting nucleo- lin, including the AS1411 aptamer and the HB-19 and N6L pseudopeptides, which impacted cancer cell via- bility in the micromolar range6,10,11. Although the extent of this effect was lower than the one observed with the anti-nucleolin scFv 4LB5 (IC50 values in the low nanomolar range, 3–58 nM23), the anti-nucleolin antibody herein presented, demonstrated ADCC capacity. y Several molecules, including EGFR-targeting agents, trigger immune responses even in the absence of a Fc domain (and thus, in an ADCC-independent mechanism)55,56. For this reason, in the ADCC assays, a control using the nanobody counterpart (without Fc domain) was included. The increased MDA-MB-435S cell death associated with the presence of PBMCs enabled by an anti-nucleolin VHH (grafted onto CDR3), relative to the parental VHH, suggested a Fc-independent immune response as an additional component of the mechanism of action of this protein. However, this effect was decreased relative to incubation the VHH-Fc counterpart (in the presence of PBMCs), confirming the ADCC capacity of the anti-nucleolin VHH-Fc. SCIENTIFIC ReportS \| (2018) 8:7450 \| DOI:10.1038/s41598-018-25816-8 8 = + − − × % cell death AUC(protein PBMC) AUC(protein) AUC(PBMC) AUC(untreated cells) 100 (2) (2) Statistical analysis. Analysis of variance (one-way ANOVA) followed by Tukey test was performed to ana- lyse differences among the VHHs, in terms of binding and cytotoxicity. Student’s t-test was performed to analyse differences between binding without and with pre-incubation with F3 peptide and between the cytotoxicity of the nanobody-Fc antibodies. Repeated measures ANOVA followed by Tukey test was performed to analyse dif- ferences in cell death upon incubation with PBMCs and each of the proteins, in the ADCC assay. Analyses were performed with a level of significance of 5%. Data availability. The datasets generated during and/or analysed during the current study are not publicly available due to patent filing but are available from the corresponding author on reasonable request. Methods Five thousan ll culture medium was ex proteins, for a total of 7 etrazolium Bromide (M io-Rad, USA) and perc Absorbance (treated c treated cells) isolated from buffy co t) by a Ficoll-Paque PL 640 medium (Lonza, Sw of L-glutamine (Lonza, S 7 °C in a humidified atm ozen in freezing medium d cultured in the same s 37 °C in a humidified a ADCC assays. of nucleolin-binding against MDA-MB-435S www.nature.com/scientificreports/ cell. Accordingly, assuming a 1:1 nucleolin:antibody binding ratio, the Antibody Binding Capacity defined the cell surface nucleolin density per cell63. cell. Accordingly, assuming a 1:1 nucleolin:antibody binding ratio, the Antibody Binding Capacity defined the cell surface nucleolin density per cell63. Cytotoxicity of nucleolin-binding VHHs against cancer cells. Five thousand MDA-MB-435S or three thousand 4T1 cells were seeded in 96-well plates. After 24 h, cell culture medium was exchanged for fresh one, and cells were incubated with serial dilutions of VHH or VHH-Fc proteins, for a total of 72 h. Cell viability was then determined with 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay64. Absorbance at 595 nm was measured in a microplate reader (Model 680, Bio-Rad, USA) and percentage of cell viability was calculated based on equation (1): = − × % cell viability Absorbance (untreated cells) Absorbance (treated cells) Absorbance (untreated cells) 100 (1 (1) PBMC isolation and culture. Human PBMCs were isolated from buffy coats of healthy volunteers (obtained at Portuguese Institute of Blood and Transplant) by a Ficoll-Paque PLUS density gradient (GE Healthcare, UK). PBMCs were then resuspended in RPMI-1640 medium (Lonza, Switzerland), supplemented with 10% (v/v) heat-inactivated FBS (HyClone, USA), 2 mM of L-glutamine (Lonza, Switzerland), 1% (v/v) Pen/ Strep/Fungiezone solution (HyClone, USA) and placed at 37 °C in a humidified atmosphere of 5% CO2 over- night. After this recovering step, PBMCs were counted and frozen in freezing medium (10% DMSO, v/v, in FBS) and stored at −80 °C. When needed, PBMCs were thawed and cultured in the same supplemented-RPMI-1640 medium as before. PBCMs were maintained overnight at 37 °C in a humidified atmosphere of 5% CO2, to decrease loss of effector capacity65 and only then used in the ADCC assays. Antibody-dependent cell-mediated cytotoxicity of nucleolin-binding VHH-Fc. The ADCC potential of the anti-nucleolin VHH-Fc antibody was tested against MDA-MB-435S cancer cells (7500 cells per well), with the xCelligence Real-Time Cell Analyzer (RTCA; ACEA Biosciences, USA). Effector/target cell ratios of 5:1 or 10:1 and 25 nM of anti-nucleolin VHH-Fc were used. Additional controls comprised cancer cells incu- bated only with effector cells or antibody or the VHH counterparts of the VHH-Fc antibodies. As the VHH-Fc antibodies are dimeric, the VHH fragments were added in a concentration of 50 nM. g Cell index was measured every 15 min for 96 h and the resulting curves were plotted and normalized to 1.0, matching the beginning of the incubation of PBMC and the different tested proteins with the cancer cells. Data were analysed with RTCA Software Package and cancer cell death resulting from incubation with both VHH-Fc antibody and PBMCs was calculated from the area under the curve (AUC) values, approximately between 24 to 72 h, based on equation (2): References References 1. Srivastava, M. & Pollard, H. B. Molecular dissection of nucleolin’s role in growth and cell proliferation: new insights. FASEB J. 13 1911–1922 (1999).h ( ) 2. Hovanessian, A. G. et al. The cell-surface-expressed nucleolin is associated with the actin cytoskeleton. Exp. Cell Res. 261, 31 (2000) 2. Hovanessian, A. G. et al. The cell-surface-expressed nucleolin is associated with the actin cytoskeleton. Exp. Cell Res. 261, 312–28 (2000). ( ) 3. Bergers, G. & Benjamin, L. E. Tumorigenesis and the angiogenic switch. Nat. Rev. Cancer 3, 401–410 (2003). g j g g g 4. Farin, K. et al. Oncogenic synergism between ErbB1, nucleolin, and mutant Ras. Cancer Res. 71, 2140–2151 (201 4. Farin, K. et al. Oncogenic synergism between ErbB1, nucleolin, and mutant Ras. Cancer Res. 71, 2140–2151 (2011). , K g y g , , R R , ( ) 5. Yang, X. et al. Cell surface nucleolin is crucial in the activation of the CXCL12/CXCR4 signaling pathway. Tumor Biol. 35, 333–338 (2014). 6. Destouches, D. et al. A simple approach to cancer therapy afforded by multivalent pseudopeptides that target cell-surface nucleoproteins. Cancer Res. 71, 3296–3305 (2011). 7. Krust, B., El Khoury, D., Soundaramourty, C., Nondier, I. & Hovanessian, A. G. Suppression of tumorigenicity of rhabdoid tumor derived G401 cells by the multivalent HB-19 pseudopeptide that targets surface nucleolin. Biochimie 93, 426–433 (2011). 8. Krust, B., El Khoury, D., Nondier, I., Soundaramourty, C. & Hovanessian, A. G. Targeting surface nucleolin with multivalent HB-19 and related Nucant pseudopeptides results in distinct inhibitory mechanisms depending on the malignant tumor cell type. BMC Cancer 11, 333 (2011). 9. El Khoury, D. et al. Targeting surface nucleolin with a multivalent pseudopeptide delays development of spontaneous melanoma in RET transgenic mice. BMC Cancer 10, 325 (2010).i R g , ( ) 10. Destouches, D. et al. Suppression of tumor growth and angiogenesis by a specific antagonist of the cell-surface expressed nucleolin. PLoS One 3, e2518 (2008).h g , ( ) 10. Destouches, D. et al. Suppression of tumor growth and angiogenesis by a specific antagonist of the cell-surface expressed nucleolin. PLoS One 3, e2518 (2008).h 1. Bates, P. J., Laber, D. A., Miller, D. M., Thomas, S. D. & Trent, J. O. Discovery and development of the G-rich oligonucleotide AS1411 as a novel treatment for cancer. Exp. Mol. Pathol. 86, 151–64 (2009). 11. Bates, P. J., Laber, D. A., Miller, D. Methods Protein purification was performed using Pierce Chromatography Cartridges Protein A columns (Thermo Scientific, USA) and desalting was performed using disposable PD-10 columns (GE Healthcare, UK). The protein was then concentrated (Vivaspin 500, 50 kDa cutoff, GE Healhcare, UK) and stored in 20 mM HEPES, 100 mM NaCl, NaCl, 5% (v/v) glycerol, pH 8.0. Binding assessment of nucleolin-binding VHHs towards nucleolin protein by enzyme-linked immunosorbent assay (ELISA). Ninety-six-well plates (Corning Costar, USA) were coated with 100 ng (19.4 nM) of human nucleolin in 50 mM sodium carbonate buffer, pH 9.6, at 4 °C, overnight, and nonspecific binding sites were blocked with 3% (w/v) BSA in PBS for 1 h, 37 °C. Each nanobody (120 pmol) diluted in 1% (w/v) BSA in PBS) was incubated for 1 h, at 37 °C, and detection was performed with anti-HA-peroxidase anti- body (clone 3F10, Roche, diluted 1:1000 in 1% w/v BSA in PBS), using ABTS substrate (Merck Millipore, USA). Absorbance (405 nm/495 nm) was measured on Model 680 microplate reader (Bio-Rad, USA). Binding assessment of nucleolin-binding VHHs towards cancer cells by flow cytome- try. MDA-MB-435S and 4T1 cancer cell lines, with different levels of surface nucleolin expression (approx- imately 102000 versus 45000 molecules, respectively; unpublished results), were used to evaluate binding of the nucleolin-binding VHHs. One hundred thousand cells, previously treated with dissociation buffer (Merck Millipore, USA), were incubated with VHH proteins, for 45 min at 4 °C, and then with anti-HA-FITC antibody (Y-11 sc-805, Santa Cruz Biotechnology, USA), at room temperature for 30 min. Competitive inhibition assays were performed by pre-incubating the cells with 75 µM F3 peptide, at 4 °C for 30 min, followed by incubation with 1000 nM VHH fragments, at 4 °C for 45 min. Sample acquisition and analysis were performed using Guava easyCyte 5HT and the InCyte software module (Merck Millipore, USA). Determination of the number of nucleolin molecules at the cell surface. Estimation of the density of nucleolin’s cell surface was performed by comparing cell fluorescence (measured by flow cytometry) with stand- ard curves prepared using Quantum™ MESF Alexa488-labeled microsphere kit (Bangs Laboratories, USA) as per manufacturer instructions, upon cell incubation with DSPE-PEG2k-F3 micelles and anti-nucleolin-Alexa®488. This enabled to withdraw the cell Antibody Binding Capacity, i.e. the number of bound antibody molecules per SCIENTIFIC ReportS \| (2018) 8:7450 \| DOI:10.1038/s41598-018-25816-8 9 ratio, the Antibody Bin er cells. References Mol. Biosyst. 6 1186–1194 (2010). 28. Gomes-Da-Silva, L. C. et al. Toward a siRNA-containing nanoparticle targeted to breast cancer cells and the tumor microenvironment. Int. J. Pharm. 434, 9–19 (2012). 9. Moura, V. et al. Targeted and intracellular triggered delivery of therapeutics to cancer cells and the tumor microenvironment: impac on the treatment of breast cancer. Breast Cancer Res. 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Formatted anti–tumor necrosis factor α VHH proteins derived from camelids show superior potency and targeting to inflamed joints in a murine model of collagen-induced arthritis.pdf. Arthritis Rheum. 54, 1856–1866 (2006). 25. Coppieters, K. et al. Formatted anti tumor necrosis factor α VHH proteins derived from camelids show superior potency and targeting to inflamed joints in a murine model of collagen-induced arthritis.pdf. Arthritis Rheum. 54, 1856–1866 (2006). l 26. Fellouse, F. A., Wiesmann, C. & Sidhu, S. S. Synthetic antibodies from a four-amino-acid code: a dominant role for tyrosi antigen recognition. Proc. Natl. Acad. Sci. USA 101, 12467–12472 (2004).h g g 7. Birtalan, S., Fisher, R. D. & Sidhu, S. S. The functional capacity of the natural amino acids for molecular recognition. References M., Thomas, S. D. & Trent, J. O. Discovery and development of the G-rich oligonucleotide AS1411 as a novel treatment for cancer. Exp. Mol. Pathol. 86, 151–64 (2009). p 2. Scott, A. 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A New Technique for the Assay of Infectivity of Adenovirus 5 DNA. Virology 52, 456–467 (1973). Additional Information Competing Interests: V.M. is an employee of TREAT U, SA. The authors are the inventors of a provisional patent application related with the work herein presented (“Anti-nucleolin antibody”, Portuguese provisional patent application number 110214, filed in July 2017). Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre- ative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not per- mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2018 SCIENTIFIC ReportS \| (2018) 8:7450 \| DOI:10.1038/s41598-018-25816-8 12
https://openalex.org/W3211601727	https://www.researchsquare.com/article/rs-999929/latest.pdf	English	null	Bletilla striata Polysaccharide Cryogel Scaffold for Spatial Control of Foreign-body Reaction	Research Square (Research Square)	2,021	cc-by	7,617	Bletilla striata Polysaccharide Cryogel Scaffold for Spatial Control of Foreign-body Reaction Jiaxi Chen University of Macau Huiqun Zhou University of Macau Daping Xie University of Macau Yiming Niu (  yimingniu@um.edu.mo ) University of Macau https://orcid.org/0 Conclusions Collectively, our findings revealed Bletilla striata polysaccharide cryogel scaffold with different pore sizes can spatially control foreign-body reaction. The microstructure of cryogels could differentially guide the distribution of inflammatory cells, affect the formation of blood vessels and fibrous capsules, which eventually influence the material-tissue integration. This work demonstrates a practical strategy to regulate foreign body response and promote the performance of medical devices. Research Article License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at Chinese Medicine on December 1st, 2021. See the published version at https://doi.org/10.1186/s13020-021-00526-y. Page 1/25 Page 1/25 Results Our data demonstrated that cryogels with different pore sizes and modulus can be fabricated by just adjusting the concentration. Besides, the cryogels show well cytocompatibility in the in vitro experiments and exhibited upregulated expression levels of pro-inflammation-related genes (Tnfa and Il1b) with the increase of pore size. In vivo experiments further proved that with the increase of pore size, more immune cells infiltrated into the inner zone of materials. The foreign-body reaction and the distribution of immune- regulatory cells could be modulated by tuning the material microstructure. Methods First, we synthesized methacrylated Bletilla striata Polysaccharide (BSP-MA) and constructed a series of open porous cryogels utilizing this material via the freezing-thawing treatment of solvent-precursors systems. Second, Pore size and rheology were measured to characterize the material properties of cryogels. Live/dead staining of cells and CCK-8 was performed to test the cytocompatibility of the scaffolds. In addition, the Real-Time qPCR experiments were carried for in vitro tests. Finally, the BSP scaffolds were implanted subcutaneously to verify the foreign-body reaction between host tissue and materials. Background Implantation of a biomaterial may induce the foreign-body reaction to the host tissue that determines the outcome of the integration and the biological performance of the implant. The level of foreign-body reaction can be modulated by material properties. Background The rapid development of regenerative medicine has brought much promise to tissue maintenance, repair and host defense.[1–4] As a prominent tool in regenerative medicine, the tissue engineering has been an active field of scientific research for nearly three decades. [5, 6] The key to develop tissue engineering is the design of applicable bioactive materials. With the development materials science and tissue engineering, The range and degree of biomaterial sophistication has also dramatically increase.[7, 8] In addition to the basic characters of biocompatibility, biodegradability, mechanical properties, porosity, Page 2/25 Page 2/25 considering of the further application in clinical, biological activity has been placed in an important position.[9][10] For example: cell adhesion and growth, vascularization, and biological recognition. Therefore, polysaccharides, as a kind of biological polymers, have come to the stage.[11] Chinese medicines, as resources repository, have been widely applied in tissue repair and been proved effective in the past years. [12] Among them, Bletilla striata, has been used as an astringent hemostatic medicinal for thousands of years.[13, 14] The medicinal part is generally regarded to be its pseudobulbs and it has the effects of restraining bleeding, stopping bleeding, reducing swelling, resolving mass and promoting tissue regeneration. The effective component of it has been proved by modern pharmacology to be Bletilla striata polysaccharide (BSP).[15] In general, Chinese medicine derived polysaccharides are barely used in biomaterials. However, natural polysaccharides are demonstrated to be a potential biomaterial and have advanced interaction with tissue in diverse ways. EUP3, as a polysaccharide derived from Eucommia ulmoides, is demonstrated to have an extraordinary interaction with platelet-derived growth factor-BB thus leading to a Growth Factor-affinitive scaffold.[16] Besides, glucomannan (GM) polysaccharide derived from konjac was proved to modulate the action of macrophage[17]. Beyond that, the Bletilla striata polysaccharide we mentioned above has already been designed into scaffold and showed the capacity to promote angiogenesis.[18] Nevertheless, it’s obvious that there are many defects as well. First, it is difficult to extract pure and homogeneous polysaccharide from natural production, thus stable processes and appropriate characterization methods need to be established. Second, since nature derived polysaccharides are not suitable to be used as biomaterials directly, the modification and design strategy of polysaccharides become key issues. Related researches have been done before and discovered that glucomannan can stimulate macrophages to produce pro-regenerative cytokines thus promoting angiogenesis and tissue repairing. Background [19] However, the BSP still has challenges to be a three-dimensional scaffold for tissue engineering, the most common problem is the foreign-body response(FBR), which is a result of the wound healing response altered by the presence of a foreign body.[20–23] The properties of the implantation are provided to effect the degree of reaction. In this study, we modified the polysaccharide with a well-defined structure into a series of three-dimensional scaffolds with only one parameter changing: pore size, and investigated the FBR and its potential application as tissue engineering scaffolds. 1. Materials Bletilla striata (China Pharmaceutical Corporation-Canton, Guangzhou, China); Bletilla striata polysaccharide (BSP) was prepared following our laboratory established and reported protocol; Fetal bovine serum and DMEM medium were obtained from Life Technologies; Calcium AM/PI kit was purchased from shanghaiyisheng (China); CCK8 kit; GoTaq 2-Step RT-qPCR system was purchased from Promega; TRIzol Reagent was obtained from Sigma-Aldrich; Other chemicals and reagents were purchased from Sigma-Aldrich unless otherwise stated. Page 3/25 Page 3/25 The primers of relevant genes were listed as follows： Mouse beta-actin: Forward: 5’-GCTGGTCGTCGACAACGGCTC-3’ Reverse: 5’-CAAACATGATCTGGGTCATCTTTTC-3’； Mouse Nos2: Forward:5’-CCAAGCCCTCACCTACTTCC-3’ Reverse: 5’-CTCTGAGGGCTGACACAAGG-3’; Mouse Il1b: Forward: 5’-GCAACTGTTCCTGAACTCAACT-3’ Reverse: 5’-ATCTTTTGGGGTCCGTCAACT-3’; Mouse Tnfa: Forward:5’-ACGGCATGGATCTCAAAGAC-3’ Reverse: 5’-AGATAGCAAATCGGCTGACG-3’; Mouse Mrc1: Forward:5’- GTGGTCCTCCTGATTGTGATAG-3’ Reverse: 5’- CACTTGTTCCTGGACTCAGATTA -3’; Mouse Tgfb: Forward:5’- TGGAGCAACATGTGGAACTC-3’ Reverse: 5’- TGCCGTACAACTCCAGTGAC -3’; Mouse Arg1: Forward:5’- CAGAAGAATGGAAGAGTCAG -3’ Reverse: 5’- CAGATATGCAGGGAGTCACC -3’; Mouse Osm: Forward:5’- AACTCTTCCTCTCAGCTCCT -3’ Reverse: 5’- TGTGTTCAGGTTTTGGAGGC -3’; Mouse Vegfa: Forward:5’- GTTCAGAGCGGAGAAAGCAT -3’ Reverse: 5’- TCACATCTGCAAGTACGTTCG -3’; 2 Synthesis of methacrylated BSP Reverse: 5’- TCACATCTGCAAGTACGTTCG -3’; 2. Synthesis of methacrylated BSP 2. Synthesis of methacrylated BSP Oxidation of BSP: the C6 primary hydroxyls of Bletilla striata polysaccharide are oxidized to C6 carboxylate groups by TEMPO/NaClO/NaClO2 oxidation system in sodium acetate buffer (0.2 M, pH 5.0). After stirring at 40 °C for 24h, oxidation was quenched by adding excessive ethanol. The precipitate of oxidated BSP was collected by centrifugation. Then, oxidized products were dialyzed (MWCO: 3,500) with milli-Q water, and lyophilized. Page 4/25 4. Methacrylated BSP scaffolds preparation GM scaffolds preparation: methacrylated BSP solution (2%-10%) was synthesized using deionized water as a solvent. Then add tetramethylethylenediamine (TEMED) [0.5% (wt/vol)] and ammonium persulfate (APS) [0.25% (wt/vol)] to the methacrylated BSP solution which was precooled to 4 °C to decrease the rate of polymerization. After a complete incubation in −20 °C refrigerator for one night, the cryogels were put at room temperature to remove ice crystals and washed with milli-Q water. 5. Pore size and rheology measurement of methacrylated BSP scaffolds Page 4/25 Preparation of methacrylated BSP: oxidized BSP was dissolved in a buffer solution (1% w/v, pH 6.5) of 50 mM 2-morpholinoethanesulfonic acid (MES). N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3- dimethylaminopropyl)-carbodiimide hydrochloride (EDC) (molar ratio of NHS:EDC =1:2) were added to the solution to activate the carboxylic acid groups of the oxidized BSP. After activation for 5 min, AEMA (molar ratio of NHS: EDC: AEMA= 1:2:1) was added to the mixture and the reaction was maintained at room temperature for 24 h. The precipitate of methacrylated BSP was collected by centrifugation. Then, oxidized products were dialyzed (MWCO: 3,500) with milli-Q water, and lyophilized. Preparation of methacrylated BSP: oxidized BSP was dissolved in a buffer solution (1% w/v, pH 6.5) of 50 mM 2-morpholinoethanesulfonic acid (MES). N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3- dimethylaminopropyl)-carbodiimide hydrochloride (EDC) (molar ratio of NHS:EDC =1:2) were added to the solution to activate the carboxylic acid groups of the oxidized BSP. After activation for 5 min, AEMA (molar ratio of NHS: EDC: AEMA= 1:2:1) was added to the mixture and the reaction was maintained at room temperature for 24 h. The precipitate of methacrylated BSP was collected by centrifugation. Then, oxidized products were dialyzed (MWCO: 3,500) with milli-Q water, and lyophilized. 3. Characterization of methacrylated BSP 3. Characterization of methacrylated BSP Characterization of oxidized BSP: oxidized BSP was dissolved in deuterated dimethylsulfoxide (DMSO- d6), and the 13C NMR spectra of these glucomannan/DMSO solutions were recorded. Carboxylate content of oxidized BSP was determined by potentimetric titration method. 0.1M HCl was added to methacrylated BSP solution and set pH value in the range of 2.5-3.0, then record HCl volume. 0.1M NaOH solution was added up to pH11 and record NaOH volume. Characterization of methacrylated BSP: methacrylated BSP was characterized by 1H- NMR analysis and the efficiency of BSP methacrylation was determined from 1H-NMR spectra based on the ratio of the integrals for the internal standard protons to the methylene protons of methacrylate. The FTIR spectra of lyophilized pure BSP and BSP-MA were obtained on KBr pellet performed on a FTIR spectrophotometer (MAGNA IR560, Nicolet). All spectra were recorded with the resolution of 4 cm−1 in the range 400-4000 cm−1. 4. Methacrylated BSP scaffolds preparation 8. Cell proliferation assay in scaffolds RAW 264.7 macrophages and HUVECs were seeded in scaffolds as previously mentioned. Scaffolds were placed in a 96-well plate at the density of 2 × 105 cells/scaffold. 6 h after seeding, the cell-laden scaffolds were rinsed with PBS and transferred to another new well to remove the unattached cells. To evaluate cell proliferation, at day 1 and 3, the culture medium was replaced with the cell counting kit-8 (CCK-8) working solution and incubated at 37 °C for 3 h. The CCK-8 solution was collected and the absorbance value was measured with the multi-plate reader at wavelength of 450 nm. 5. Pore size and rheology measurement of methacrylated BSP scaffolds GM scaffolds was stained with fluorescent dyes (FITC). A solution of FITC, 1mM in 20 mM Na-carbonate buffer (pH 9.4) was applied to the stained scaffold for 24 h and thoroughly washed with buffer and water. The stained cryogels were sectioned into slices. Samples were examined by confocal laser scanning microscopy (CLSM) (Leica TCS SP8), using a 20× objective and excitation and emission wavelengths 488 and 530 nm. ImageJ software (http://rsb.info.nih.gov/ij/) was used to analyze images to obtain the pore size and pore size distribution. Page 5/25 Page 5/25 Flow and deformation of materials in response to applied force can be studied by rheology. Elastic modulus and elastic nature of the material is defined as storage modulus (G’). The dissipation (viscous) of the flow is represented by loss modulus (G”). The visco-elasticity behavior or phase angle is the difference between the storage and loss modulus. The cryogels used in experiments had 1.5 cm diameter and 2 mm thickness cylindrical shape. Amplitude sweep (strain sweep) test was applied from 0.01 to 100% at the constant frequency of 1 Hz to determine linear viscoelastic region. Then, frequency sweep measurement was performed from 0.01 to 100 Hz at a controlled strain of 0.2% to investigate the modulus change related to the oscillatory frequency. 6. Cell Culture RAW 264.7, a murine monocyte/macrophage cell line, and human umbilical vein endothelial cells (HUVECs) were purchased from the ATCC (American Type Culture Collection). Cells were cultured in DMEM high glucose medium and RPMI-1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin under 5% CO2 at 37 °C. Cells were passaged after reaching 80% confluence. 7. Live/dead staining of cells in scaffolds RAW 264.7 macrophages and HUVECs were washed by PBS. After cell counting, the cells were centrifuged and re-suspended in culture medium solution at a final concentration of 5×106 cells per milliliter. 3D scaffolds were sterilized in 75% ethanol for one night. Next, the cell solution was added to the 3D scaffolds and cells can be absorbed into the 3D scaffolds. The cell-laden cryogels were then placed in the atmosphere of 37° C with 5% CO2 for 6 h to allow cells attachment inside the scaffold. A live/dead assay was performed to test cell viability in cryogels. Scaffolds loaded with cells in triplicate were incubated with the mixture dye solution containing 1μL of Calcein-AM and 0.5μL propidium iodide (PI) in 1 mL of PBS. After 30min incubation, the cryogels were rinsed with PBS and cells were imaged by confocal laser scanning microscopy (CLSM) (Leica TCS SP8). Green fluorescence represents live cells and red fluorescence was related to dead cells. 8. Cell proliferation assay in scaffolds 11.Implantation of cryogel scaffolds Male C57BL/6J mice (6-8 weeks) were used with cryogel scaffolds subcutaneous implanted in the back for assessing the host response and the biocompatibility of cryogel scaffolds. All procedures were approved by the Animal Ethics Committee, University of Macau. We divided 18 mice into three groups and treated them with hydrogel and BA2 and BA8 respectively embedded. The mice in each group were divided into 2 days and 14 days. Before surgery, the mice were anesthetized with intraperitoneal injection of sodium pentobarbital (70 mg/kg) and then the dorsal hair was shaved. After the sterilization of skin with 75% ethanol, two independent incisions were made on the back, and the scaffolds were embedded in and then the wound were sewn up. 12. Histology Analysis After two days and 2 weeks housing, the mice were sacrificed and the implants along with the 2 cm × 2 cm skin tissue samples were collected and immediately fixed in 4 vol.% formalin and dehydrated by gradient ethanol before embedding in paraffin wax. These samples were cross-sectioned into 6 μm for histological analysis. The sections were deparaffinized and rehydrated for Hematoxylin–eosin (H&E), Masson’s trichrome staining. Besides, the deparaffinized and rehydrated sections were blocked and stained by anti-VEGF, anti-CD31 and anti-CD86 for immunohistochemistry. The images were recorded by a light microscope (BX51; Olympus). ImageJ software was utilized to quantify the fibrous capsule thickness, immunohistochemistry staining (with the assistant of IHC Toolbox plugin), and obtain the cell coordinate datasets followed by calculating the minimum cell distance in R language. 9. Cell infiltration and distribution in scaffolds 9. Cell infiltration and distribution in scaffolds Page 6/25 RAW 264.7 macrophages were seeded in scaffolds as previously mentioned. Calcein-AM solution was added to the sample. After 30 min incubation at 37 °C, the cell-laden cryogels were observed by CLSM. All images were generated by optical sectioning in the z-direction. Optical sections each of 10μm were taken to produce a 250μm z-stack for image processing. images were generated by optical sectioning in the z-direction. Optical sections each of 10μm were taken to produce a 250μm z-stack for image processing. 10. Real-Time qPCR RAW 264.7 macrophages were seeded in scaffolds at a seeding density of 5 × 104 cells/scaffold. Cell- laden scaffolds were rinsed with PBS and transferred to another new well to remove the unattached cells and 1.5 ml of new culture medium was added. Then, samples were incubated in CO2 incubator for 24h. RNA was isolated by kit. RNA was reverse-transcripted into cDNA. Quantitative real time PCR (q-PCR) measurements were performed using a SYBR Green RT-PCR kit. Marker genes including Tnfa，Il1b，Nos, Mrc1, Tgfb，Arg1, Vegfb and Osm were selected for analysis with the primer sequences using the 2−ΔΔct relative quantification method. 11.Implantation of cryogel scaffolds 13. Statistical analysis Statistical differences among samples were studied through t-test or the one-way analysis of variance (ANOVA) with Tukey's multiple comparisons test. The data presented as the mean ± standard deviation Page 7/25 Page 7/25 Page 7/25 was obtained based on at least three independent replicates. Significance was set to p < 0.05. (* p < 0.05, p < 0.01, * p < 0.001, **** p < 0.0001). was obtained based on at least three independent replicates. Significance was set to p < 0.05. (* p < 0.05, p < 0.01, * p < 0.001, **** p < 0.0001). 1. Synthesis and characterization of glucomannan derivates 1. Synthesis and characterization of glucomannan derivates There is no report of methacrylated on BSP up to no. Therefore, we first propose a modification method of BSP to MA. C6-OH groups of BSP are oxidized to C6 carboxylate groups by TEMPO/NaClO/NaClO2 oxidation system (Fig. 1a). [24]The 13C-NMR spectrum with the peak at 180ppm indicated that C6 primary hydroxyls were successfully oxidized to carboxylate groups (Fig. 1b). Determined by potentimetric titration method, the carboxylate content of C6-OH was 2.12 mmol/g and oxidation ratio of C6-OH was 37.06%. (Fig. 1d) Then these carboxylate groups are linked with AEMA to introduce a carbon– carbon double bond, which can be crosslinked via chemical method, TEMED/APS initiation system based on an autocatalytic reaction (Fig. 1a). At last, BSP was successfully methacrylated. The 1H-NMR spectra of methacrylated BSP exhibit peaks of vinyl methylene and methylene protons that were newly formed by the reaction with AEMA, which are located at δ 6.2, 5.7 and 2.9 respectively (Fig. 1b). The FIIR also confirms the successful synthesis of methacrylated BSP (Fig. 1c): the single peak of 1600cm−1 presented the stretching frequency of C=C in the alkene. These results showed that BSP was successfully methacrylated. To calculated the efficiency of BSP methacrylation, the 1H-NMR spectra were recorded on using tetramethylsilane (TMS) as internal standard. The efficiency of BSP methacrylation was calculated to be 20.66% based on the ratio of the integrals for the internal standard protons to the methylene protons of methacrylate (Fig. 1b). 2. Fabrication and characterization of methacrylated BSP scaffolds The process of methacrylation makes the BSP crosslinking to form hydrogel through simply ultraviolet(UV) radiation. [25] The BSP still displays liquid status after 15 seconds UV radiation, however, the BSP-MA with different concentration 0.5%, 1% and 2% were tested to show the different capacity of gelation after the same radiation time. (Fig. 2a) The 2% BSP-MA is shown to change the liquid state to solid state obviously demonstrating the capacity of BSP-MA gelation. However, the pore size in hydrogel is much smaller than cells which is not suitable for cell infiltration and angiogenesis. Page 8/25 Cryogels are gel matrices in which polymerization occurs at subzero temperatures and solvent crystals are defrosted to form interconnected macropores network.[26] BSP cryogels were prepared by freezing and thawing (Fig. 2b). The freezing step causes the ice crystals to form and occupy space, while the subsequent thawing step causes the ice crystals to melt, thus forming large interconnected pores. It shows the macroscopical performance of cryogel BSP-MA, the three colors represent three different concentrations of BSA-MA. (Fig. 2a) It’s obvious that the red one with lowest BSP-MA concentration performs sort of collapse. To explore the concentration effect on material properties, we prepared cryogels with four concentration of BSP-MA and named BA2(2%) BA4(4%), BA6 (6%) and BA8(8%). (Fig. 2c) The concentration can affect the mechanical properties by affecting the pore size[27]. As a result, the cryogels were stained with FITC and pore size and frequency were measured. It’s showed that BSP cryogels contained tunable pore size of 169.49,89.22༌48.29 and 19.51µm (Fig. 2d), The pore size gradually decreases with the increase of concentration. The BA2 and BA4 shows the largest pore size. Next, to observe the inner network and analyze the structure and distribution of the pore in each cryogel group, we measured the swelling ratios and the interconnected porosity. We found that through adjusting the concentration of the initiator and the precursor BSP-MA solution, we developed cryogels with tunable pore size. With the increase of concentration of the precursor BSP-MA solution, porosity was decreased, and some of them were even about 90% (Fig. 2f), while the swelling ratios of cryogels declined from 25 to 13. 2. Fabrication and characterization of methacrylated BSP scaffolds Rheological properties were also investigated to characterized the cryogels to confirm the dimensional stability of the inner work.[28] Rheology test demonstrated that cryogels of different concentration exhibited diverse stiffness at a range from 709.1576 (storage modulus, Pa) to 7803.308 (storage modulus, Pa) (Fig. 2e). As a result, the modulus regulation can be achieved by adjusting pore size, which is a key factor for biomaterial application [29]. 3.Cytocompatibility analysis of the cryogel-based scaffolds To evaluate the cytocompatibility of the cryogels as a biomaterial, we chose BA2 and BA4 cryogels to culture cells due to their relatively large pore size, which is proved to be suitable for cell infiltration and tissue growth. As control, the same concentration of hydrogel was also prepared. As the previous work showed that BSP could regulate macrophages and promote angiogenesis, RAW 264.7 cells and HUVECs were chosen to seed in BA2 and BA4 to evaluate cell distribution and viability. After 6h and 72h of culture, RAW 264.7 cells and HUVECs proliferated well in the BA2 and BA4 scaffolds without any significant difference (Fig. 3a). Compared with cells seeded on hydrogels, OD values of cells increased by around 1.5 times after culturing for 3 days (Fig. 3a). To further compare the cell infiltrating between hydrogel and cryogel, we use live/dead staining. RAW 264.7 cells could infiltrate even up to 450 µm depth in cryogel while the hydrogel can only infiltrate up to 200µm after culturing for 1 day, indicating the construction of interconnected macropores (Fig. 3b). Besides, both BA2 and BA4 cells had high survival rates at 6h and 72h in RAW 264.7 cells and HUVACs. (Fig. 3c) To quantification the ratio of live/dead cells, we used Image J to analyze the fluorescence. (Fig. 3d) For HUVACs, the BA2 shows 93.3% live ratio in 6h and 90.1% in 72h, BA4 shows 71.5% in 6h and 93.3% in 72h. For RAW 264.7 cells, the BA2 shows 95.2% live ratio in 6h and 94.1% in 72h, BA4 shows 87.6% in 6h and 96.5% in 72h. These results demonstrated that BA2 and BA4 cryogels were excellent substrates for cell growth. 4.GM biomaterial based mechanical modulation of macrophages Page 9/25 The above work proves that by only adjusting the concentration, the cryogel can be adjusted to have different mechanical properties, such as pore size, modulus, etc. Besides, cytocompatibility experiments have proved that cryogels are suitable for cells growth. Therefore, in order to investigate the FBR, the expression levels of macrophage related genes were measured by RT-qPCR performing. We evaluated the pro-inflammation related genes (Tnfa,Il1b)༌anti-inflammation related genes ( Mrc1, Tgfb)andrepairrelatedgenes (VegfbandOsm) (Fig.4). in RAW264.7 after 24h culture. It’s exhibited increased expression levels of pro-inflammation related genes including Tnfa and Il1b. 3.Cytocompatibility analysis of the cryogel-based scaffolds (Fig.4a) Specifically, with the increase of pore size, the upregulation of Tnfa and Il1b increases obviously from BA8 to BA2 and BA6 has the similar low level with BA8.It's common that the proinflammatory cytokines increasebecauseofthecryogels intervention. However, it isworth noting that the proinflammatory cytokines gradually decreased as the pore size became smaller, which preliminarily proved that regulating the pore size can modulate inflammatory reaction. The Tgfbas a pleiotropic cytokine were upregulated at the comparable level regardless of the pore size. (Fig.4b) Meanwhile, anti-inflammation related genes such as Mrc1 was downregulated. (Fig.4b) In addition, the expression of angiogenesis-associated gene Vegfb and osteogenesis-related gene Osm genes were increased at the same time (Fig.4c). Therefore, we found that our materials can promote the M1 polarization of macrophage, and the degree of pro-inflammation decrease gradually with the decrease of the pore size of cryogels. Figure 4 Gene expression of RAW 264.7 macrophages response to BSP based cryogels with varying stiffness. qPCR analysis of gene: Tnfa (a),Il1b(a), Mrc1 (b), Tgfb (b), Vegfb (c) and Osm (c) expression of macrophages in response to BA2 – BA8 cryogels. Statistical analysis: Error bars represent standard error (n = 3). One-way analysis of variance (ANOVA) with Tukey's multiple comparisons test, * p < 0.05, p < 0.01, * p < 0.001, **** p < 0.0001. 5. In vivo foreign-body reaction modulated by pore size 5. In vivo foreign-body reaction modulated by pore size To evaluate the biocompatibility and foreign-body reaction of the materials in vivo, we implanted the materials:BA8 and BA2 subcutaneously and hydrogel as control for 2 days and 14 days. What we found is that we can modulate the pore size of materials to regulate the FBR. Firstly, the distribution of immune cells can be regulated by pore size of materials. It can be shown that the materials still have similar morphology as measured in vitro after implantation, which is that BA2 has interconnected macro pores and BA8 has smaller pores, while hydrogel as a whole material has no obvious pores. (Fig. 5) We use Hematoxylin-eosin (H&E) staining and Masson’ trichrome(M&T) to show the distribution of immune cells [30]. It is obvious that host response exists in the implantation site. After 2 days, for BA2, the materials with the largest pore size, the immune cells are distributed in both inner and edge of materials (Fig.5 and 6). The quantitative data further demonstrated immune cells distribution is compatible between inner and edge of the materials (Fig.6b). BA8, with the smaller pore size, immune cells infiltrated both inner and edge as well, but it’s showed that the concentration at the edge of the Page 10/25 Page 10/25 materials (Fig.6c). For hydrogel, with no macro pore, immune cells only exist at the edge of the material, with little inner presence. After 14 days, the immune response was generally diminished and the distribution of both BA2 and BA8 is more even but hydrogel still edge-concentrated. The above results indicated that the pore size can mediate the host response by regulating the distribution of immune cells. At the initial stage of material implantation, the bigger the pore size is, the easier it is for immune cells to infiltrate into the inner of the materials. With the decrease of the pore size, the immune cells are distributed to the edge of the materials. Immunohistochemical staining of CD86 was also performed to identify the location of macrophages in the cryogels (Fig.7a). The distribution of macrophages is similar with it of overall immune cells. Notably, BA2 material shows higher density of macrophages than BA8, which was consistent with the levels of inflammatory cytokines in vitro. Figure 5 Immune cell infiltration in materials. 5. In vivo foreign-body reaction modulated by pore size H&E staining images of (a)BA2 subcutaneously implanted in mice for 2 days and 14 days (b), and BA8 implanted for 2 days (c) and 14 days (d), hydrogel implanted for 2 days (e) and 14 days (f). the yellow triangle shows the location of fibroblasts. The red star shows the angiogenesis. The formation of fibrous capsule can be modulated by pore size of materials.[31] In tissues exposed to the system's immune system, lymphocytes and fibroblasts develop fibrous capsules to fight against biological materials. We evaluate the thickness of the fibrous capsule as an indicator of the FBR. M&T staining (Fig. 6) and the quantification (Fig. 7e and 7f) showed that BA8 has the thinner fibrous capsule than BA2, and hydrogel has the thickest fibrous capsule. This may be due to the larger pore size of BA2, which leads to more macrophages to enter the material, while the macrophages of BA8 and hydrogel concentrating at the edge secrete cytokines to induce collagen deposition by the fibroblasts and form fibrous capsule.[32] Fig. 6 The cell distribution and the formation of fibrous capsule in tissue surrounding the BA2, BA8 and hydrogel. (a) shows the M&T staining of BA2, BA8 andhydrogel after 2 days and 14 days implantation. (b) shows the quantification of the immune cells density. (c) (d)shows the quantification of cell distribution. (e) M&Tstaining of fibrous capsule and the thickness quantification. The green arrows show the immune cells infiltration and the blue crosses show the materials and the purple arrows show the thickness and location of fibrous capsule. For the implantation of biomaterials, the tissue compatibility is also a very important consideration. [33, 34]The pore size can affect the integration of materials and tissues. Immunohistochemical staining of VEGF and CD31 was measured to evaluate the angiogenesis (Fig. 7c-e). It is obvious that the level of VEGF in vivo is consistent with that measured in vitro, which is that BA2 showed the high level contrast with BA8 at both 2 and 14 days. It’s showed that angiogenesis at 14 day which indicated the formation of tissue in BA2 and BA8(Fig. 7e). In addition, BA8 has better tissue compatibility (Fig. 7), BA2, with large pore size, allowed macrophages to infiltrate to inner material thus leading more severe inflammation, which is not conducive to blood vessel growth and tissue integration. The schematic diagram showed of the probable immune cells infiltration.( Fig. Discussion In this study, we have developed a new, natural polysaccharide based cryogel scaffold, which is effective to regulate host response. Notably, this polysaccharide is a Chinese medicine herb derived glucomannan, showing the high biocompatible and stability as a 3D scaffold. Along with the repaid progress of tissue engineering normal biomaterial with biological functions and tunable physical properties are in high demand.[35] On the one hand, natural polysaccharides from ocean have been widely developed. [36–38]On the other hand, polymers from Chinese medicine herbs, despite the evidence bioactivity, still have much room for development, key obstacles include unclear composition and difficulties for material fabrication. [39–41]Here, for the first time, we made macroporous BSP gels suitable for cell culture just by freezing and thawing without adding any pore- forming agents. The in vitro and in vivo date verified our hypothesis, which is the pore size can induce different level of host response. Firstly, our in vitro and in vivo date shows high consistency. The in vivo results: immunohistochemistry staining of CD86 and VEGF is agreed with the in vitro results of RT-PCR. Secondly, perhaps the most remarkable finding is that the material we successfully made can differentially guide to the distribution of immune cells, blood vessels, fibrous capsule. for example, the Fig. 5-7 show the macrophages and other immune cells distribution and amount is associated with the pore size of materials, BA2, with largest pore size, has more macrophages infiltrated and shows more internal distribution, which is also consist with the in vitro data: the upregulation of pro-inflammation related gene. In addition, Many researches have as proved that foreign-body reaction can be modulated by various material properties such as porosity. [42, 43] These exciting findings suggest the future works for the development of BSP cryogel system. First, much work remains to do on the specific types, subtle types of the immune cells into the scaffold at different time points, also interest is that how these differential immune cells profiles lead to tissue remodeling and repairing. Second, as a prove of a consequence study, we implant the gels in health mice, we will evaluate the general potential in specific disease model in future. We believe our exploration will open up a new avenue for the development of Chinese medicine resource for broaden applications. 5. In vivo foreign-body reaction modulated by pore size 8) Page 11/25 Page 11/25 Fig. 7 Inflammatory marker staining and qualification analysis of BA2, BA8 and hydrogel. (a) inflammatory staining and (b)qualification analysis of CD86(c)inflammatory staining of VEGF and (d)qualification analysis (e) inflammatory staining of CD31. The red stars marked the angiogenesis. Fig. 7 Inflammatory marker staining and qualification analysis of BA2, BA8 and hydrogel. (a) inflammatory staining and (b)qualification analysis of CD86(c)inflammatory staining of VEGF and (d)qualification analysis (e) inflammatory staining of CD31. The red stars marked the angiogenesis. Funding This study was funded by the UM Macao Postdoctoral Associateship program and the Science and Technology Development Fund, Macau SAR (File No. 0060/2020/AGJ). Conclusions Taking together, our works revealed that Bletilla striata polysaccharide cryogel scaffold with different pore sizes can spatially control foreign-body reaction. The microstructure of cryogels could differentially guide the distribution of inflammatory cells, affect the formation of blood vessels and fibrous capsules, Page 12/25 which eventually influence the material-tissue integration. This work demonstrates a practical strategy to regulate foreign body response and promote the performance of medical devices. Abbreviations FBR: Foreign-body Reaction; BSP: Bletilla striata polysaccharide; GM: glucomannan; MES: 2- morpholinoethanesulfonic acid；NHS：N-hydroxysuccinimide；EDC：1-ethyl-3-(3-dimethylaminopropyl)- carbodiimide hydrochloride；DMSO: dimethylsulfoxide; TEMED: tetramethylethylenediamine; APS:ammonium persulfate; CLSM: confocal laser scanning microscopy; HUVECs: human umbilical vein endothelial cells; ATCC: American Type Culture Collection; CCK-8: cell counting kit-8; q-PCR: Quantitative real time PCR; H&E: Hematoxylin–eosin; M&T: Masson’ trichrome Declarations Not applicable. Availability of data and materials Not applicable. Acknowledgement Dr. YM Niu acknowledges the UM Postdoctoral Associateship program and the Science and Technology Development Fund, Macau SAR (File No. 0060/2020/AGJ). Author Contributions YMN designed the study. HQZ extracted and purified the natural polysaccharide with the assistant of DPX. HQZ performed major chemical modification and scaffold preparation. JXC contributed to biological and animal experiments. JXC and HQZ were the main drafters of the manuscript. YMN provided funding supports. All authors contributed to data analysis and manuscript drafting. Ethics approval and consent to participate The animal care and experimental procedures used in this study were approved by the Animal Ethics Committee, University of Macau. Page 13/25 Consent for publication Not applicable. Competing interests The authors declare that they have no conflict of interests. The authors declare that they have no conflict of interests. Page 13/25 Page 13/25 References [1] S.J.N.b. Petit-Zeman, Regenerative medicine, 19(3) (2001) 201-206. [1] S.J.N.b. Petit-Zeman, Regenerative medicine, 19(3) (2001) 201-206. [2] A.S. Mao, D.J.J.P.o.t.N.A.o.S. Mooney, Regenerative medicine: current therapies and future directions, 112(47) (2015) 14452-14459. [3] E. Lagasse, J.A. Shizuru, N. Uchida, A. Tsukamoto, I.L.J.I. 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Khademhosseini, DNA-directed self- assembly of shape-controlled hydrogels, 4(1) (2013) 1-10. Figures Page 17/25 Figure 1 Figure 1 Preparation and characterization of methacrylated BSP. (a) The Chinese medicine Bletilla striata and Schematic illustration of methacrylated BSP synthesis procedures and the polymerization under the TEMED/APS initiation system. (b) 13C-NMR and 1H-NMR spectrum analysis of oxidized BSP and methacrylated BSP. (c) The FT-IR of BSP and BSP-MA. (d)The potentiometric titration to measure the degree of substitution. Page 18/25 Page 18/25 Figure 2 Page 19/25 Figure 2 Fabrication and characterization of BSP-based cryogels. (a) Fabrication of hydrogels and cryogels with different concentrations (b) Schematic depiction of cryogels preparation. (c) Confocal laser scanning fluorescence microscopy (CLSM) analysis of BA2 - BA8 cryogels (varying concentration of the precursor solution: 2%, 4%, 6%, 8%, (w/v) %) and average pore diameters and the pore size distributions. Scale bars: 100μm, 50μm, 20μm, 20μm. (d) Rheological analysis of BA2 - BA8 cryogels. Storage modulus (G′) and Figure 2 Page 19/25 Fabrication and characterization of BSP-based cryogels. (a) Fabrication of hydrogels and cryogels with different concentrations (b) Schematic depiction of cryogels preparation. (c) Confocal laser scanning fluorescence microscopy (CLSM) analysis of BA2 - BA8 cryogels (varying concentration of the precursor solution: 2%, 4%, 6%, 8%, (w/v) %) and average pore diameters and the pore size distributions. Scale bars: 100μm, 50μm, 20μm, 20μm. (d) Rheological analysis of BA2 - BA8 cryogels. Storage modulus (G′) and Page 19/25 Page 19/25 loss modulus (G′′) of BA2 - BA8 cryogels on strain sweep measured at 1 Hz of frequency(left), frequency sweep measured at 0.2% of strain (right). (e) Interconnected porosity (n=3) and (f) swelling ratio (n=3) of BA2 - BA8 cryogels. Statistical analysis: Error bars represent standard error (n = 3). One-way analysis of variance (ANOVA) with Tukey's multiple comparisons test, * p < 0.05, p < 0.01, * p < 0.001, **** p < 0.0001. loss modulus (G′′) of BA2 - BA8 cryogels on strain sweep measured at 1 Hz of frequency(left), frequency sweep measured at 0.2% of strain (right). (e) Interconnected porosity (n=3) and (f) swelling ratio (n=3) of BA2 - BA8 cryogels. Statistical analysis: Error bars represent standard error (n = 3). One-way analysis of variance (ANOVA) with Tukey's multiple comparisons test, * p < 0.05, p < 0.01, * p < 0.001, **** p < 0.0001. loss modulus (G′′) of BA2 - BA8 cryogels on strain sweep measured at 1 Hz of frequency(left), frequency sweep measured at 0.2% of strain (right). (e) Interconnected porosity (n=3) and (f) swelling ratio (n=3) of BA2 - BA8 cryogels. Statistical analysis: Error bars represent standard error (n = 3). One-way analysis of variance (ANOVA) with Tukey's multiple comparisons test, * p < 0.05, p < 0.01, * p < 0.001, ** p < 0.0001. Page 20/25 Figure 3 Fi 3 Figure 3 Page 20/25 Page 20/25 Cytocompatibility analysis of cell-laden cryogel: (a) RAW 264.7 cell and HUVECs viability on BA2 hydrogels and cryogels and BA4 hydrogels and cryogels after 24h and 72h culture.(b) Infiltration and distribution images of RAW 264.7 cells seeded on hydrogel and cryogel for 24h. (c-d) Representative images and quantitative analysis of live/dead staining of RAW 264.7 macrophages and HUVECs in BA2 and BA4 cryogels after 6h and 72h of culture. Scale bar: 200μm. Statistical analysis: Error bars represent standard error (n = 3). Figure 2 T-test, * p < 0.001. Cytocompatibility analysis of cell-laden cryogel: (a) RAW 264.7 cell and HUVECs viability on BA2 hydrogels and cryogels and BA4 hydrogels and cryogels after 24h and 72h culture.(b) Infiltration and distribution images of RAW 264.7 cells seeded on hydrogel and cryogel for 24h. (c-d) Representative images and quantitative analysis of live/dead staining of RAW 264.7 macrophages and HUVECs in BA2 and BA4 cryogels after 6h and 72h of culture. Scale bar: 200μm. Statistical analysis: Error bars represent standard error (n = 3). T-test, *** p < 0.001. Figure 4 Figure 4 Page 21/25 Gene expression of RAW 264.7 macrophages response to BSP based cryogels with varying stiffness. qPCR analysis of gene: Tnfa (a)，Il1b(a), Mrc1 (b), Tgfb (b), Vegfb (c) and Osm (c) expression of macrophages in response to BA2 – BA8 cryogels. Statistical analysis: Error bars represent standard error (n = 3). One-way analysis of variance (ANOVA) with Tukey's multiple comparisons test, * p < 0.05, p < 0.01, * p < 0.001, **** p < 0.0001. Gene expression of RAW 264.7 macrophages response to BSP based cryogels with varying stiffness. qPCR analysis of gene: Tnfa (a)，Il1b(a), Mrc1 (b), Tgfb (b), Vegfb (c) and Osm (c) expression of macrophages in response to BA2 – BA8 cryogels. Statistical analysis: Error bars represent standard error (n = 3). One-way analysis of variance (ANOVA) with Tukey's multiple comparisons test, * p < 0.05, p < 0.01, * p < 0.001, **** p < 0.0001. Figure 5 Page 22/25 Immune cell infiltration in materials. H&E staining images of (a)BA2 subcutaneously implanted in mice for 2 days and 14 days (b), and BA8 implanted for 2 days (c) and 14 days (d), hydrogel implanted for 2 days (e) and 14 days (f). the yellow triangle shows the location of fibroblasts. The red star shows the angiogenesis. Figure 6 Page 23/25 The cell distribution and the formation of fibrous capsule in tissue surrounding the BA2, BA8 and hydrogel. (a) shows the M&T staining of BA2, BA8 and hydrogel after 2 days and 14 days implantation. (b) shows the quantification of the immune cells density. (c) (d)shows the quantification of cell distribution. (e) M&Tstaining of fibrous capsule and the thickness quantification. The green arrows show the immune cells infiltration and the blue crosses show the materials and the purple arrows show the thickness and location of fibrous capsule. Figure 2 Figure 7 Page 24/25 Inflammatory marker staining and qualification analysis of BA2, BA8 and hydrogel. (a) inflammatory staining and (b)qualification analysis of CD86（c）inflammatory staining of VEGF and (d)qualification analysis (e) inflammatory staining of CD31. The red stars marked the angiogenesis. Figure 8 Figure 8 The special control of foreign-body reaction by modulation the pore size. The immune cells, including macrophages, monocytes, natural killer cells, dendritic cells, T cells, B cells, fibroblasts diversely distributed in the materials with different pore size and the different level of fibrous capsule formation. Page 25/25 Page 25/25
https://openalex.org/W4245058892	https://www.researchsquare.com/article/rs-30668/v1.pdf?c=1631855857000	English	null	Greater trochanteric bone flap grafting with vascular pedicles for the treatment of femoral head osteonecrosis in patients with systemic lupus erythematosus: A retrospective study	Research Square (Research Square)	2,020	cc-by	4,282	Greater trochanteric bone flap grafting with vascular pedicles for the treatment of femoral head osteonecrosis in patients with systemic lupus erythematosus: A retrospective study Qiang Yang (  yangqiang2020@yeah.net ) Nanyang orthopedic hospital Jixue Zhou Nanyang orthopedic hospital Lei Li Nanyang orthopedic hospital Zhaopeng Guo Nanyang orthopedic hospital Xiaolei Tian Nanyang orthopedic hospital Research article Keywords: Femoral head osteonecrosis, Systemic lupus erythematosus, greater trochanteric bone flap, vascular pedicles Posted Date: May 26th, 2020 DOI: https://doi.org/10.21203/rs.3.rs-30668/v1 DOI: https://doi.org/10.21203/rs.3.rs-30668/v1 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 Page 1/13 Page 1/13 Page 1/13 Abstract Background: Osteonecrosis of the femoral head is a complication of systemic lupus erythematosus,that affect the patient's quality of life seriously.This article reviewed the radiographs of osteonecrosis of the femoral head in patients with systemic lupus erythematosus and assessed the effect of using the greater trochanteric bone flap grafting with vascular pedicles. Methods: We retrospectively reviewed 17 patients (26 hips) with systemic lupus erythematosus who underwent the greater trochanteric bone flap grafting with vascular pedicles for the treatment of osteonecrosis of the femoral head. According to Ficat and Arlet classification, 16 hips were in stage II; 10 hips were in stage III. All patients were followed up for a mean of 32 months (ranging 12~48 months) and were assessed clinically and radiologically according to Harris scoring. Results: No hips were treated with total hip arthroplasty in the follow-up. The mean HHS was improved from preoperative 69.1 points (ranging 52–83 points) to postoperative 89.4 points (ranging 56–100 points). At the latest follow-up, of 26 hips, radiographically 21 hips (80.77%) were improved, 3 hips (11.54%) were unchanged and just 2 hips (7.69%) were worse. Conclusion: The greater trochanteric bone flap grafting with vascular pedicles was successful in maintaining joint function and in delaying the need for joint replacement procedure. Introduction It had reported that vas transplantation was effective for the treatment of ONFH, and the 88.2%(13). At present, there is few studies have reported the treat flap transplantation. At present, few studies have reported vascul of SLE complicated with ONFH. The purpose of this study was to vascularized bone graft for the treatment of secondary ONFH cau Patients Since July 2012 to June 2016, 17 patients (26 hips) with ONFH underwent the procedure of the trochanteric bone flap pedicles grafting. There were 4 males and 13 females. The age of the patients ranged from 18 to 42 years (mean 41.1 years ), cause ranged from 1~6 years and the average duration were 3.4 years. All patients had received corticosteroids threatment. ONFH diagnosis and classification All the patients were corresponding to SLE diagnosis standards established by the 1982 American Rheumatology Association criteria(14). ONFH was identified by one or more of the following imaging techniques: plain X-ray, computsd tomography and MRI. According to the Ficat and Arlet classification(15),16 hips were in stage II (61.5%); 10 hips were in stage III (38.5%). The diagnosis of osteonecrosis was confirmed in all cases by a histological examination of the subchondral bone that was obtained from a core biopsy of the femoral head during the surgery. Materials And Methods All procedures were approved by an Institutional Ethics Review Committee of Nanyang Orthopaedic Hospital. Informed consent was obtained from all individuals who participated in this study. Introduction Systemic lupus erythematosus (SLE) is a diffuse connective tissue disease with multiple system damage mediated by autoimmune reactions. Osteonecrosis of the femoral head (ONFH) is one of the most serious complications of SLE(1). Its morbidity has been reported to be about 4%–30% with an overall average of 10%, which is higher than that of general populatio(1-3).The etiology of this disorder has not been clarified, and no prophylaxis has been established to date. Although the pathogenesis remains unclear, involvement of high-dose corticosteroids therapy(4), immunosuppressive drug therapy(5), hypercoagulability(6), and lipid metabolism abnormality(7) has been suggested. ONFH seriously affects the quality of life of patients , which is an urgent medical problem to be solved. Total hip arthroplasty (THA) is an effective method to treat ONFH(8), which can reduce patients' pain and improve the function of hip joint. Without early surgical intervention, total hip advanced lesions would appear, and eventually THA was unavoidable. However, SLE is more common in younger patients, premature THA will face many problems, such as prosthetic loosening, infection and repeated revision surgery(9, 10). In addition, patients with SLE who take hormones for a long time often have other systemic diseases, which increase the risk of surgery and perioperative complications(9). These will have serious impact on the patient's physiology, psychology and economy, so total hip arthroplasty for younger patient populations need be paid very careful attention. Therefore, on the basis of improvement in symptoms, what kind of surgical methods to delay or even avoid artificial joint replacement has become the focus in theapy research of ONFH. Page 2/13 Page 2/13 Current research methods include core decompression surgery, osteotomy and vascularized bone graft, the therapeutic effect of core decompression and osteotomy has been questioned for the patients whose cartilage damaged and collapsed(11, 12). It had reported that vascularized greater trochanter bone flap transplantation was effective for the treatment of ONFH, and the femoral head preservation rate was 88.2%(13). At present, there is few studies have reported the treatment of ONFH with vascularized bone flap transplantation. At present, few studies have reported vascularized bone flap graft for the treatment of SLE complicated with ONFH. The purpose of this study was to investigate the effect of the vascularized bone graft for the treatment of secondary ONFH caused by SLE. Current research methods include core decompression surgery, os the therapeutic effect of core decompression and osteotomy has cartilage damaged and collapsed(11, 12). Surgical procedure After the patient is anesthetized, they were placed in the supine position with the ilium elevated to 60°. A skin incision was made 4 cm distal to the iliac crest down to the top of greater trochanter, and then extend vertically down along the anterior margin of the trochanter. After we identifing the transverse branch of the lateral femoral circumflex vessel, we separated these vessels to where they entered the greater trochanter. We harvested a vascular graft bone flap about 3x2cm from the anterolateral side of the greater trochanter. Then, we harvested a volume of 1-2 cm3 of cancellous bone from the greater trochanter area.The hip capsule was incised in a T shape to expose the femoral head and neck. We made an approximately 2x2cm bone window at the femoral head-neck junction using an osteotome. According to the imaging evaluation and the judgment of the surgeon,we determined the range of femoral head necrosis.We used the high-speed drill and curette spatula to remove the necrotic bone tissue in the Page 3/13 Page 3/13 femoral head, until the necrotic tissue is removed compeletly and fresh blood flows out. The vascularized bone flap was implanted into the femoral head lesion area after the necrotic bone was removed, and the cancellous bone was filled in insufficient places. The bone flap and the filled cancellous bone were properly tamped to restore the shape of the collapsed femoral head.We required bleeding from the cancellous surface of the greater trochanter graft as an indication of vessel patency. Clinical evaluation Clinical follow-up is performed every 3 months for 1 year and annually thereafter.The mean length of follow-up was 32 months (ranging 12 to 48 months) in our hospital. Before the procedure and at each follow-up, patients were evaluated using the Harris Hip Score (HHS)(16).The HHS is an objective index of hip joint function that quantifies the four categories of (1) pain, (2) walking function and activities of daily living, (3) extent of deformity and (4) joint range of motion, with higher values indicating greater functionality. Harris score no less than 80 points is defined as clinical success. Clinical outcomes were categorised by HHS results at the most recent follow-up as follows: excellent, 90–100; good, 80–89; fair, 70–79; or poor, less than 70. Clinical Results The Surgical procedure took 50 to 90 minutes to complete with the mean of 65 minutes.Mean perioperative blood loss was 320 mL (range 220–510 mL).All patients received perioperative prophylactic antibiotics until after removal of the drains.There were no intraoperative complications. Fat necrosis of incision occurred in one patient postoperatively and the incision healed untill 3 weeks after debridement. There were no other complications, such as infection, hematoma and deep vascular thrombosis, during the period of follow-up. Radiographic evaluation Radiographic progressions of the femoral head collapse (pre-surgery to the most recent follow-up) were evaluated in hips of each classification and staging. According the final radiographs, we are divided into three categories:(1) Improved–those cases in which the osteonecrosis had healed or was being replaced with new bone formation. For the Stage II lesion, the density of cystic lesion had increased with trabecular formation of the greater trochanteric bone flap. For the Stage III lesion, the collapsed lesion healed or became more rounded with trabecular formation of the tip of the greater trochanteric bone flap. (2)Remained unchanged, and the preoperative imaging data showed no significant difference.(3) Worse- staging of disease progression,collapse further aggravated. (2)Remained unchanged, and the preoperative imaging data showed no significant difference.(3) Worse- staging of disease progression,collapse further aggravated. Radiographic outcomes All patients underwented radiological examination at follow-up.Of 26 hips, radiographically 21 hips (80.77%) improved, 3 hips (11.54%) were unchanged and just 2 hips (7.69%) were worse. According to the staging, 14 hips of staging II (67.3%) showed improvement, 2 hips of staging II(12.5%) appeared unchanged and 1 hips of staging II (6.3%) appeared worse.7 hips of staging III (70%) showed improvement (Figs. 3A and 3B),1 hips of staging III (10%) appeared unchanged and 1 hip of staging III (10%) appeared worse. Harris hip scores Page 4/13 At last follow-up,The mean HHS was improved from preoperative 69.1 points (ranging 52–83 points) to postoperative 89.4 points (ranging 56–100 points) (The overall average increase in HHSs is shown in Figure 1).The mean HHS was improved from preoperative 78.4 points (ranging 74–83 points) to postoperative 95.4 points (ranging 79–100 points) for femoral heads with stage II disease and from preoperative 59.8 points (range 52–68 points) to postoperative 83.4 points (range 56–94 points) for femoral heads with stage III disease(HHS is shown in Figure 2)。14 hips (87.5%) with stage II disease achieved excellent and good results and 8 of 10 hips (80%)with stage III disease had excellent and good results (Table 1). Discussion The use of the free fibular flap for the treatment of ONFH was initiated in an effort to enhance revascularization and arrest the progression of the necrosis.Soucacos(26) reported using free vascularized fibular grafting for osteonecrosis of the femoral head in 184 hips, a mean follow-up of 11 years, only 7.6% of patients requiring total hip arthroplasty, and 62% patients without progression.However, the procedure of free fibular flap transfer is technical demanding and time consuming.Moreover, complications associated with the use of fibular flap, such as donor-site pain, peroneal nerve neuropathy and fracture of the femoral neck, have also been reported(27, 28). The greater trochanter bony structure with cancellous bone, peripheral rich blood supply, transplantation can effectively improve the blood supply, reduce blood supply reconstruction process.The thin cortical bone is very similar to head of femur, is ideal selected area to treat ONFH; and is able to improve the mechanical properties of necrosis of the femoral head effectively. After removing the necrotic bone completely, the transferred greater trochanter and the remaining part of femoral metastases fit closely to promote fracture healing process; greater trochanter and femoral trabecular bone trabecular have same nature, which restored the continuity of cancellous bone trabecular, rebuilding the supporting role of trabecular bone of the femoral head, to recovey femoral neck bearing bracket, can be adapted to load normal joint activities; meanwhile as the cystic degeneration repair and new bone reconstruct, the femoral head restoring the force area, so that the stress beared by unit trabecular bone area reduced, the mechanical properties of the femoral head strengthened, preventing the collapse effectively. In addition, the vascularized bone flap can reconstruct a new blood supply system, accelerate the repair and rebuilding of bone necrosis, thereby restoring the biological characteristics of the femoral head necrosis. And by mechanics, we proved the cut of the greater trochanter does not damage the mechanical properties and structure of the proximal femur trochanter major stress conductive zones, thus from a mechanical point of view, the selection of bone flap from the greater trochanter is safe and feasible. The bone flap transplantation located in front of the femoral neck, this area is a region of low stress distribution, can reduce the impact on the mechanical characteristics of the proximal femur. Discussion A satisfactory treatment for osteonecrosis of the femoral head in patients with SLE remains elusive.Without operative intervention ONFH could result in collapse and deterioration of the joint. Some scholars reported(17) a 53% rate of progression to collapse of the femoral head articular surface in 79 hips treated nonoperatively.Current treatment options include core decompression, various osteotomy techniques and vascularized bone graft(18-21). Core decompression provides pain relief,especially for patients with earlier stages of avascular necrosis(22).There is evidence that core decompression is an effective intervention in lupus patients with Ficat stage I and II.Several studies report , failure of core decompression and progression of osteonecrosis in lupus patients, especially in patients with Ficat stage III(23). Maniwa(24) have evaluated the results of core decompression for osteonecrosis of the hip in patients with SLE. The report suggest that patients with avascular necrosis of the femoral head and lupus do not respond as well as nolupus patiens treated with core decompression. Page 5/13 In 1978, Sugioka(25) first introduced rotary osteotomy through greater trochanter of femur to treat ONFH， which was intertrochanteric osteotomy of the femoral head necrosis rotation. The principle is the upper part of the front of the femoral head necrosis is transferred to the non-load-bearing parts, in order to prevent progressive collapse of the articular surface of the femoral head, and correct head and acetabulum mismatch caused by subluxation occured after the collapse of the articular surface of the femoral head. Then a variety of improved surgical procedures used in clinical, there is a big difference in the reported results. Although it is considered this kind of surgical mothod can prevent the collapse of the new weight-bearing area, but increased the incidence of instability and the corresponding joint osteoarthritis. Due to the high incidence of this surgical complication and followed by deformity of the proximal femur, the clinical application is limited. Greater trochanter of femur mainly comprised by spongy bone, have rich blood supply, bone flap have large range blood supply, the blood supply in the affected areas can be improved effectively after transplantation, the rebuilding process of the blood supply can be shortened. Conclusion The effects of the greater trochanteric bone flap in patients with lupus on plain x-ray changes and HHSs demonstrate the benefits of this technique. The greater trochanteric graft flap with pedicles are easy to perform and could be indicated in young patients with Ficat and Arlet stage II to III disease. Its could provide the necrotic femoral head with sufficient blood supply and prevention of femoral head collapse. Ethics approval and consent to participate We declar that all procedures were approved by an Institutional Ethics Review Committee of Nanyang Orthopaedic Hospital, and obtained the consent of the participants. Consent for publication All the contents obtained the consent of the patients for publication. Discussion This provides bone flap entering the channel and select specific operative incision from femur head and neck in greater trochanter vascularized bone grafting a strong theoretical basis, but also proves the safety of this surgical technique, and fewer complications. On the use of vascularized the greater trochanteric bone flap graft treating 26 hips with secondary ONFH caused by SLE, by an average of 32 months of follow-up,medium-term clinical results were satisfactory,the excellent and good rate was 88.5% , this surgery can slow down or even stop the progress of ONFH. During follow-up, femoral head necrosis increasing with the progression of stage in two patients, DSA angiography showed: blood filling poorly in vascularized bone or no contrast agent filling the femoral head, maybe caused by the twist or spasm of the vascular pedicle, or constriction from Page 6/13 surrounding tissue swelling, until to the last follow-up, both patients have no other further treatment requirements in addition to functional rehabilitation treatment. surrounding tissue swelling, until to the last follow-up, both patients have no other further treatment requirements in addition to functional rehabilitation treatment. But we are also aware of some limitations of our study. First, we had no control group treated with alternative joint-preserving procedures. Second, the length of follow-up is only 32 monthes. Longer term outcome analysis will be necessary to prove the longevity of the procedure. Third, the current study has only 26 hips; therefore, significant statistical conclusions are more difficult to make. Availability of data and materials All data generated or analysed during this study are included in this publis Abbreviations SLE, systemic lupus erythematosus; ONFH ,Osteonecrosis of the femoral head; THA ,Total hip arthroplasty Competing interests The authors declare that they have no competing interests. Fundings Authors' contributions Page 7/13 Page 7/13 Page 7/13 Conception and design of the research:Qiang Yang; Acquisition, analysis and interpretation of data: Jixue Zhou and Lei Li; Statistical analysis: Zhaopeng Guo and Xiaolei Tian; Drafting the manuscript: Qiang Yang; Manuscript revision for important intellectual content: Qiang Yang. All authors have read and approved the manuscript. Acknowledgement Not applicable. References 1. Gladman D, Dhillon N, Su J, Urowitz M. Osteonecrosis in SLE: prevalence, patterns, outcomes and predictors. Lupus. 2018;27(1):76-81. 2. Mertelsmann-Voss C, Lyman S, Pan TJ, Goodman S, Mandl LA. Arthroplasty Rates Are Increased Among US Patients with Systemic Lupus Erythematosus: 1991-2005. Journal of Rheumatology. 2014;41(5):867-74. 3. Kasturi S, Goodman S. Current Perspectives on Arthroplasty in Systemic Lupus Erythematosus: Rates, Outcomes, and Adverse Events. Current Rheumatology Reports. 2016;18(9):59. 3. Kasturi S, Goodman S. Current Perspectives on Arthroplasty in Systemic Lupus Erythematosus: Rates, Outcomes, and Adverse Events. Current Rheumatology Reports. 2016;18(9):59. 4. Nevskaya T, Gamble MP, Pope JE. A meta-analysis of avascular necrosis in systemic lupus erythematosus: prevalence and risk factors. Clinical and Experimental Rheumatology. 2017;35(4):700. 4. Nevskaya T, Gamble MP, Pope JE. A meta-analysis of avascular necrosis in systemic lupus erythematosus: prevalence and risk factors. Clinical and Experimental Rheumatology. 2017;35(4):700. 5. Systemic lupus erythematosus in a multiethnic US cohort (LUMINA): XXIV. Cytotoxic treatment is an additional risk factor for the development of symptomatic osteonecrosis in lupus patients: results of a nested matched case–control study. Annals of the Rheumatic Diseases. 2006. 6. Oinuma K, Harada Y, Nawata Y, Takabayashi K, Abe I, Kamikawa K, et al. Sustained hemostatic abnormality in patients with steroid-induced osteonecrosis in the early period after high-dose corticosteroid therapy. Journal of Orthopaedic Science Official Journal of the Japanese Orthopaedic Association. 2000;5(4):374-9. 7. Belmont H, Lydon E. Avascular necrosis prevention with lipitor in lupus erythematosus. Lupus. 2005;14(10):869-70. 8. Li Z, Du Y, Xiang S, Feng B, Weng X. Risk factors of perioperative complications and transfusion following total hip arthroplasty in systemic lupus erythematosus patients. Lupus. 2019;28(9):096120331986260. 9. Kennedy JW, Wasim K. Total Hip Arthroplasty in Systemic Lupus Erythematosus: A Systematic Review. International Journal of Rheumatology. 2015;2015:1-8. 9. Kennedy JW, Wasim K. Total Hip Arthroplasty in Systemic Lupus Erythematosus: A Systematic Review. International Journal of Rheumatology. 2015;2015:1-8. 10. Zangger P, Gladman DD, Urowitz MB, Bogoch ER. Outcome of total hip replacement for avascular necrosis in systemic lupus erythematosus. Journal of Rheumatology. 2000;27(4):919. 11. Scully SP, Aaron RK, Urbaniak JR. Survival Analysis of Hips Treated with Core Decompression or Vascularized Fibular Grafting Because of Avascular Necrosis. Journal of Bone and Joint Surgery, Page 8/13 Page 8/13 American Volume. 1998;80(9):1270-5. 12. Halland AM, Klemp P, Botes D, Van Heerden B, Loxton A, Scher AT. AVASCULAR NECROSIS OF THE HIP IN SYSTEMIC LUPUS ERYTHEMATOSUS: THE ROLE OF MAGNETIC RESONANCE IMAGING. Rheumatology. 1993;32(11):972-6. 13. References Zhao D, Wang B, Guo L, Yang L, Tian F. Will a Vascularized Greater Trochanter Graft Preserve the Necrotic Femoral Head? Clinical Orthopaedics & Related Research. 2010;468(5):1316-24. 14. Tan EM, Cohen AS, Fries JF, Masi AT, Winchester RJ. The 1982 revised criteria for the classification of systemic lupus erythematosus. . Arthritis & Rheumatology. 1982;25(11):1271-7. 15. J FRA. Necrosis of the femoral head. In: Hungerford DS,editor Ischemia and Necrosis of Bone Baltimore, MD: Williams &Wilkins;. 1980:55-74. 16. Harris WH. Traumatic arthritis of the hip after dislocation and acetabular fractures: treatment by mold arthroplasty. An end-result study using a new method of result evaluation. The Journal of bone and joint surgery American volume. 1969;51(4):737-55. 17. OHZONO K, SAITO M, SUGANO N, TAKAOKA K, ONO K. The Fate of Nontraumatic Avascular Necrosis of the Femoral Head: A Radiologic Classification to Formulate Prognosis. Clin Orthop Relat Res. 1992;277(277):73-8. 18. Pedesen DR, Brown TD, Poggie RA. Finite element characterization of a porous tantalum material for treatment of avascular necrosis. Trans Orthop Res Soc. 1997;22:598. 18. Pedesen DR, Brown TD, Poggie RA. Finite element characterization of a porous tantalum material for treatment of avascular necrosis. Trans Orthop Res Soc. 1997;22:598. 19. Shuler MS, Rooks MD, Roberson JR. Porous Tantalum Implant in Early Osteonecrosis of the Hip: Preliminary Report on Operative, Survival, and Outcomes Results. Journal of Arthroplasty. 2007;22(1):0-31. 20. Tsao, A. K. Biomechanical and Clinical Evaluations of a Porous Tantalum Implant for the Treatment of Early-Stage Osteonecrosis. Journal of Bone & Joint Surgery American Volume. 2005;87(suppl_2):22. 21. Veillette, J.H. C. Survivorship Analysis and Radiographic Outcome Following Tantalum Rod Insertion for Osteonecrosis of the Femoral Head. Journal of Bone & Joint Surgery. 2006;88(suppl_3):48. 22. Miyahara HdS, Rosa BB, Hirata FY, Gurgel HdMC, Ejnisman L, Vicente JRN. What is the role of core decompression in the early stages of osteonecrosis of the femoral head? Evaluation of the surgical result by functional score and radiological follow-up. Revista Brasileira De Ortopedia. 2018;53(5):537-42. 23. MONT, Michael A, FAIRBANK, Adrian C, MICHELLE, HUNGERFORD, et al. Core Decompression for Osteonecrosis of the Femoral Head in Systemic Lupus Erythematosus. Clinical Orthopaedics & Related Research. 1997;334(334):91-7. 24. Maniwa S, Nishikori T, Furukawa S, Kajitani K, Iwata A, Nishikawa U, et al. Evaluation of core decompression for early osteonecrosis of the femoral head. Archives of Orthopaedic & Trauma Surgery. 2000;120(5-6):241-4. 24. Maniwa S, Nishikori T, Furukawa S, Kajitani K, Iwata A, Nishikawa U, et al. osteotomy operation. Clin Orthop. 1978;130. osteotomy operation. Clin Orthop. 1978;130. 26. Soucacos PN, Beris AE, Malizos K, Koropilias A, Zalavras H, Dailiana Z. Treatment of Avascular Necrosis of the Femoral Head With Vascularized Fibular Transplant. Clinorthoprelatres. 2001;386:120-30. 27. URBANIAK JR. Treatment of osteonecrosis of the femoral head with free vascularized fibular grafting. : A long-term follow-up study of one hundred and three hips. J Bone Joint Surg. 1995;77. 27. URBANIAK JR. Treatment of osteonecrosis of the femoral head with free vascularized fibular grafting. : A long-term follow-up study of one hundred and three hips. J Bone Joint Surg. 1995;77. grafting. : A long term follow up study of one hundred and three hips. J Bone Joint Surg. 1995;77. 28. Urbaniak JR, Harvey EJ. Revascularization of the Femoral Head in Osteonecrosis. J Am Acad Orthop Surg. 1998;6(1):44-54. 28. Urbaniak JR, Harvey EJ. Revascularization of the Femoral Head in Osteonecrosis. J Am Acad Orthop Surg. 1998;6(1):44-54. 28. Urbaniak JR, Harvey EJ. Revascularization of the Femoral Head in Osteonecrosis. J Am Acad Orthop Surg. 1998;6(1):44-54. Table Table 1. Clinical Outcome of HSS in osteonecrosis of the femoral head. Outcome Case Ficat and Alert Stages Stage II Stage III Excellent 19 13 6 Good 3 1 2 Fair 3 2 1 Poor 1 0 1 Total 26 16 10 Outcome Case Ficat and Alert Stages Stage II Stage III Excellent 19 13 6 Good 3 1 2 Fair 3 2 1 Poor 1 0 1 Total 26 16 10 Figures References Evaluation of core decompression for early osteonecrosis of the femoral head. Archives of Orthopaedic & Trauma Surgery. 2000;120(5-6):241-4. Page 9/13 25. Sugioka Y. Transtrochanteric anterior rotational osteotomy of the femoral head in the treatment of osteonecrosis of the femoral head in the treatment of osteonecrosis affecting the hips ; a new Page 9/13 25. Sugioka Y. Transtrochanteric anterior rotational osteotomy of the femoral head in the treatment of osteonecrosis of the femoral head in the treatment of osteonecrosis affecting the hips ; a new osteotomy operation. Clin Orthop. 1978;130. osteotomy operation. Clin Orthop. 1978;130. Figures Page 10/13 Page 10/13 Figure 1 The effect of the greater trochanteric bone flap with vascular pedicles grafting on HHS is shown. Figure 1 The effect of the greater trochanteric bone flap with vascular pedicles grafting on HHS is shown. The effect of the greater trochanteric bone flap with vascular pedicles grafting on HHS is shown. Page 11/13 Page 11/13 Figure 2 The effect of the greater trochanteric bone flap with vascular pedicles grafting on Harris hip score (HHS) in stage II and III is shown. g Figure 3 Figure 3 Page 12/13 A 32-year-old woman with lupus and stage III osteonecrosis of the left femoral head with collapse in the preoperative X-ray examination. Anteroposterior radiographs were taken preoperatively (3A) and 36 months postoperatively (3B), at which time the greater trochanter bone flap graft was well incorporated and the forma of the femoral head did not show progressive collapse. A 32-year-old woman with lupus and stage III osteonecrosis of the left femoral head with collapse in the preoperative X-ray examination. Anteroposterior radiographs were taken preoperatively (3A) and 36 months postoperatively (3B), at which time the greater trochanter bone flap graft was well incorporated and the forma of the femoral head did not show progressive collapse. Page 13/13
https://openalex.org/W2922864234	https://bib-pubdb1.desy.de/record/419101/files/s41598-018-36216-3.pdf	English	null	Femtosecond phase-transition in hard x-ray excited bismuth	Scientific reports	2,019	cc-by	5,935	Femtosecond phase-transition in hard x-ray excited bismuth Received: 23 August 2018 Accepted: 14 November 2018 Published: xx xx xxxx Received: 23 August 2018 Accepted: 14 November 2018 Published: xx xx xxxx The evolution of bismuth crystal structure upon excitation of its A1g phonon has been intensely studied with short pulse optical lasers. Here we present the first-time observation of a hard x-ray induced ultrafast phase transition in a bismuth single crystal at high intensities (~1014 W/cm2). The lattice evolution was followed using a recently demonstrated x-ray single-shot probing setup. The time evolution of the (111) Bragg peak intensity showed strong dependence on the excitation fluence. After exposure to a sufficiently intense x-ray pulse, the peak intensity dropped to zero within 300 fs, i.e. faster than one oscillation period of the A1g mode at room temperature. Our analysis indicates a nonthermal origin of a lattice disordering process, and excludes interpretations based on electron-ion equilibration process, or on thermodynamic heating process leading to plasma formation. The typical response time of the internal microscopic degrees of freedom in a solid, such as the arrangement of the electrons and atoms, ranges between few fs to few ps. Ultrashort laser pulses can excite materials on time scales faster than those response times, often revealing unique behaviour1–5 and furthering the understanding of the interactions between electrons and atomic lattice6–8. In the case of ultrafast melting, photoexcitation drives the material in a highly non-equilibrium state where the electrons are excited while the lattice is still cold. As the electrons thermalize, the lattice disorders in hundreds of femtoseconds due to a significant shift in the atomic potential energy surface (PES) driven by the excited electrons. Bismuth (Bi), well known for its Peierls distorted lattice structure, is an important example of such a phase transition.h p p p The crystal lattice of Bi can be readily excited by ultrashort laser pulses promoting electronic excitations that typically trigger a coherent oscillation of the optical Γ-point A1g phonon mode1–4,9. The characteristic parameters of this oscillation, for example its frequency (~2.9 THz10,11), are strongly dependent on the excitation intensity and are directly correlated with the out-of-equilibrium potential energy surface12 (PES). Results from a pioneer- ing infrared-pump experiment13 and subsequently from theoretical models8, suggested presence of a non-thermal melting process in Bi at an absorbed dose above its thermal melting threshold. Received: 23 August 2018 Accepted: 14 November 2018 Published: xx xx xxxx www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports OPEN Femtosecond phase-transition in hard x-ray excited bismuth M. Makita1, I. Vartiainen1, I. Mohacsi1,2, C. Caleman3,4, A. Diaz 1, H. O. Jönsson4,5, P. Juranić1, N. Medvedev6,7, A. Meents 3, A. Mozzanica1, N. L. Opara1,8, C. Padeste 1, V. Panneels 1, V. Saxena3,9, M. Sikorski10, S. Song 10, L. Vera1, P. R. Willmott1, P. Beaud 1, C. J. Milne 1, B. Ziaja-Motyka3,11 & C. David1 Femtosecond phase-transition in hard x-ray excited bismuth To date, however, to the best of our knowledge, neither experiments nor calculations have been able to describe the dynamics in a range above an absorbed dose of 1.2 eV/atom13, which is still well below a strong fast ionisation regime. In the interest of time-resolved studies, several experiments explored excitation regimes up to the regime of nonthermal melting in Bi3,9,14 and in other materials with state-of-the-art techniques15,16. Up to now, structural studies at high temporal resolution (<100 fs) sensitive to the excitation of phonon modes in Bi have never been carried out neither in the non-thermal melting regime or above it. It is therefore of a fundamental interest to investigate the time-resolved dynamical lattice response of Bi to an intense electronic excitations, with high temporal resolution. y p g p To address this question, we performed a study of the ultrafast lattice dynamics of a bismuth bulk crystal, excited with a femtosecond hard x-ray pulse. The advantages of using hard x-rays, instead of optical lasers, are: (i) x-rays allow to highly excite the material with negligible non-linear effects from electric and magnetic fields of the 1Paul Scherrer Institut, CH-5232, Villigen PSI, Switzerland. 2Synchrotron SOLEIL, L’Orme des Merisiers, 91190, Saint-Aubin, France. 3CFEL, Deutsches Elektronen-Synchrotron DESY, 22607, Hamburg, Germany. 4Department of Physics and Astronomy, Uppsala University, SE-751 24, Uppsala, Sweden. 5Department of Applied physics, KTH Royal Institute of Technology, SE-106 91, Stockholm, Sweden. 6Institute of Physics, Czech Academy of Sciences, 182 21, Prague 8, Czech Republic. 7Institute of Plasma Physics, Czech Academy of Sciences, 182 00, Prague 8, Czech Republic. 8C-CINA Biozentrum, University of Basel, CH-4058, Basel, Switzerland. 9Institute for Plasma Research, Bhat, Gandhinagar, 382428, India. 10Linac Coherent Light Source, SLAC National Accelerator Laboratory, Menlo Park, California, 94025, USA. 11Institute of Nuclear Physics, Polish Academy of Sciences, 31-342, Krakow, Poland. Correspondence and requests for materials should be addressed to M.M. (email: mikako.makita@xfel.eu) Scientific Reports \| (2019) 9:602 \| https://doi.org/10.1038/s41598-018-36216-3 1 www.nature.com/scientificreports/ Figure 1. (a) Y-Z plane view of the setup. The label ‘G1 gratings’ denotes a stack of 10 different diamond gratings. Only three are shown here for simplicity. Each of these gratings diffracts a small portion of the incoming x-ray pulse in the y-z plane, at varying angles defined by their pitch. The diffracted pulses are then re- diffracted back by the G2-gratings, to overlap with the transmitted primary pulse within ±3 μm precision. (b) X-Z plane view of the setup. Results Th The experiment was performed at the XCS station20 of the Linac Coherent Light Source (LCLS)21, using an x-ray photon energy of 5 keV, a nominal pulse energy of 2 mJ and a nominal pulse duration of 35 fs FWHM. The sche- matic arrangement of the transmission gratings is illustrated in Fig. 1a. The transmitted part of the x-ray pulse through the gratings acted as a ‘pump’, whilst those diffracted by the gratings ‘probed’ the sample at precise time delays determined by their extended optical paths relative to the transmitted pulse. On the sample, half of the probe beams are spatially overlapped with the pump pulse, while the other half was separated by 70 μm from the pumped region. The uniqueness of the setup has been demonstrated previously19. The two major differences from the pre- vious work in this experiment are: finer time steps of 20–50 fs for an overall coverage of 300 fs, allowing higher probing time resolution, and, 2–5 times higher grating diffraction efficiency resulting in better signal-to-noise ratio. Further details about the setup can be found in the Methods and in refs.19,22. To ensure the spatial over- lap, spot sizes of the pump and the probe pulses were focused to a FWHM of 35 ± 5 μm and 12.5 ± 2.5 μm, respectively. The sample was oriented so as to direct the Bragg reflections of all the pulses onto a 2D detector.l hl Figure 2a,b show typical single-shot images of the Bi (111) Bragg peak reflections which is sensitive to the A1g phonon mode, as recorded on the detector. Diffraction patterns with and without the pump pulse are com- pared (Fig. 2a,b). For both images, the group of signals on the left-hand side, marked as “reference”, probed the unpumped region of the sample, while that marked as “probe” on the right probed the excited area of the sample. The delay time increases as the signal distance increase from the (blocked) pump pulse at the centre of the image. The flare caused by the pump beam was fitted with a 1D Gaussian profile for background removal (Fig. 2c).lfl hl yii g g Figure 3 shows the time evolution of the (111) reflection recorded for different x-ray pump fluences, with the relative pump incidence at time 0 (centre of the pump pulse). Femtosecond phase-transition in hard x-ray excited bismuth All the transmitted and diffracted pulses satisfy the Bragg condition of the sample in the x-z plane. The pulses that diffract from the sample are separated along the y-axis and aligned along the x-axis. They are then recorded on a 2D detector (JUNGFRAU). Figure 1. (a) Y-Z plane view of the setup. The label ‘G1 gratings’ denotes a stack of 10 different diamond gratings. Only three are shown here for simplicity. Each of these gratings diffracts a small portion of the incoming x-ray pulse in the y-z plane, at varying angles defined by their pitch. The diffracted pulses are then re- diffracted back by the G2-gratings, to overlap with the transmitted primary pulse within ±3 μm precision. (b) X-Z plane view of the setup. All the transmitted and diffracted pulses satisfy the Bragg condition of the sample in the x-z plane. The pulses that diffract from the sample are separated along the y-axis and aligned along the x-axis. They are then recorded on a 2D detector (JUNGFRAU). focused laser17. In this way a purely electronic response of the material can be studied, and (ii) x-ray irradiation creates a low photoelectron density gradient within the pump pulse penetration depth under an incidence close to normal, yielding a relatively homogeneous secondary electron density therein. The technical difficulties typically associated with an x-ray pump and x-ray probe experiment using SASE pulses18 were overcome by using a novel x-ray splitting setup19. As a result, an extremely high temporal resolution was achieved, limited only by the XFEL pulse duration. Importantly, this technique allows for combined characteristics which: (1) is free of any timing and spatial jitter between the pump and the probe, or between the probe beams, (2) provides consecutive prob- ing (8 in this work) from a single x-ray pulse, thus eliminating stochastic effects, and (3) allows for independent focusing between pump and the probe pulses. Scientific Reports \| (2019) 9:602 \| https://doi.org/10.1038/s41598-018-36216-3 Results Th The fluence levels are 2.46 J/cm2, 1.48 J/cm2, and 0.91 J/cm2, corresponding to the average absorbed dose values of 3.5 eV/atom, 2.1 eV/atom, and 1.3 eV/atom, respectively. (The chosen x-ray energy does not induce a resonant absorption by inner-shell electrons). Prior to the pump events, probe pulse intensities were measured and confirmed low enough not to damage or affect the Scientific Reports \| (2019) 9:602 \| https://doi.org/10.1038/s41598-018-36216-3 2 www.nature.com/scientificreports/ Figure 2. (a,b) Typical raw images showing (a) unpumped, and (b) pumped events of Bragg reflection signals. The pump pulse is blocked in front of the detector. The integrated plots of these images are shown in (c), red (pumped) and blue (unpumped) lines. The time delay increases symmetrically left and right from the pump- pulse at the centre. A dashed grey line indicates the 1D Gaussian background level in the pumped case. After background removal, the pumped signals are then integrated and normalised by their counterpart from unpumped signals. The normalised signals are plotted as red open circles. Figure 2. (a,b) Typical raw images showing (a) unpumped, and (b) pumped events of Bragg reflection signals. The pump pulse is blocked in front of the detector. The integrated plots of these images are shown in (c), red (pumped) and blue (unpumped) lines. The time delay increases symmetrically left and right from the pump- pulse at the centre. A dashed grey line indicates the 1D Gaussian background level in the pumped case. After background removal, the pumped signals are then integrated and normalised by their counterpart from unpumped signals. The normalised signals are plotted as red open circles. crystal. On average, 50 ± 10 selected shots for each fluence level were used. We emphasize here that the signal dynamics seen in Fig. 3 cannot be due to x-ray intensity and spectral fluctuations, or due to any crystal imper- fections, since the time sequences were taken from the same pulse in single shot and from the same region of the crystal.h The dynamics at the lowest absorption dose of 1.3 eV/atom (at 0.91 J/cm2) in our case could be considered sim- ilar to the highest dose cases presented in ref.13 (1.2 eV/atom for Bi) and in ref.23 (0.35 eV/atom for Sb) – implying that the PES map at this x-ray dose should be highly anharmonic and described by a single-minimum-well. Results Th For such a case, it is expected that the disordering of the lattice follows a relatively direct path, possibly by a direct anharmonic coupling of the initially excited A1g motion coordinate to that of the degenerate Eg optical mode8,13. F th b b d d f 2 1 V/ t d 3 5 V/ t th i t it d t ithi 300f f t th p g y 1g g g p For the absorbed doses of 2.1 eV/atom and 3.5 eV/atom, the intensity drops to zero within ~300 fs, faster than one oscillation period of the unperturbed Bi A1g phonon mode (342 fs, 2.92 THz)10–12. This is also highlighted in the inset graph in Fig. 3, which compares our observation to previous Bi (111) diffraction experiments in which bond softening or lattice damage at later times were observed2,3,9. Our signal decays in less than half a picosecond ruling out the possibility of a thermal melting process. We also note that the observed femtosecond transition triggered by 1013~1014 W/cm2 pump intensity excludes the possibility of fast collisional heating through photoex- cited electrons24 as a dominant heating process. More details about these analyses will be given in the discussion below.l An intermediate signal plateau present in all fluence cases shown in Fig. 3 (shaded green area) has been observed at a comparable timescale in x-ray irradiated diamond within its transient transmission of optical pulses25. An exponential fitting to extrapolate the decay time constant is possible for reflections from lattice insensitive to optical phonon-modes5,13. In our case, however, due to the sensitivity of (111) reflection to the A1g phonon mode, we cannot uniquely exclude the possible origin of the plateau in Fig. 3 from a damped oscillatory motion along the distortion coordinate26,27. In such a case, the major initial atomic potential shift would be along the (111) direction, leading to minima of diffracted intensity when Bi atomic potential passes through or near the lower symmetry equilibrium position of the parent cubic lattice. A possibility of such a process could be validated in the future by performing dedicated experiments with a longer time delay window. Discussion In the thermal melting regime, dramatic changes in the crystal structure can only be observed after significant atomic heating due to the electron-ion energy exchange. This process takes time typically of the order of ps28,29 at the pump pulse fluences considered here. Therefore, the complete signal drop within 300 fs, seen in Fig. 3, casts out the lattice disorder through its thermalisation as a possible cause.fi If, on the other hand, the diffraction signal drop is due to strong ionisation only, one would expect a significant change of the Bi atomic scattering factor due to ionisation of core-electrons. However an ionisation degree esti- mated with the non-local thermodynamic-equilibrium (non-LTE) radiation transfer code, CRETIN30, points to less than 1 electron per atom being excited in the first 100 fs after the pump, at our highest intensity of 1014 W/cm2 Scientific Reports \| (2019) 9:602 \| https://doi.org/10.1038/s41598-018-36216-3 3 www.nature.com/scientificreports/ Figure 3. Temporal evolution of the normalised signals from x-ray irradiated Bi crystal at three different absorbed doses: 3.5 eV/atom (red circles), 2.1 eV/atom (magenta triangles) and 1.3 eV/atom (blue squares). Error bars are taken from standard deviation. Dashed lines are guides for the eye. The green shaded area marks roughly the plateau region discussed in the text. The inset graph shows the comparison of the case with [A] 3.5 eV/atom absorbed dose from 5 keV x-ray pulse, with the extrapolated data from previous reports on optical excitation of Bi at various absorbed doses: [B] Harmand et al. ([9]), [C] Fritz et al. ([2]), and [D] Johnson et al. ([3]). Figure 3. Temporal evolution of the normalised signals from x-ray irradiated Bi crystal at three different absorbed doses: 3.5 eV/atom (red circles), 2.1 eV/atom (magenta triangles) and 1.3 eV/atom (blue squares). Error bars are taken from standard deviation. Dashed lines are guides for the eye. The green shaded area marks roughly the plateau region discussed in the text. The inset graph shows the comparison of the case with [A] 3.5 eV/atom absorbed dose from 5 keV x-ray pulse, with the extrapolated data from previous reports on optical excitation of Bi at various absorbed doses: [B] Harmand et al. ([9]), [C] Fritz et al. ([2]), and [D] Johnson et al. ([3]). (equivalent to 3.5 eV/atom) (see Supplementary Fig. (ii)). Discussion Indeed, an estimate of the scattered intensity based on XATOM code31 suggest that, to realise the observed signal drop, the normalised scattering factors of Bi should be at least ~0.8. This corresponds to an average ionisation degree of more than ~10 per atom in the initial state (see Supplementary Fig. (i)). To reach such a degree of ionisation would require orders of magnitude higher x-ray intensity than we have in this experiment, which would push the sample into a plasma state. y p p p p This leaves a non-thermal melting process as the most likely interpretation of the observed transition, where a modification of interatomic potential due to high electronic excitation induces a lattice instability13,32. Given the average absorbed pump doses (1.3~3.5 eV/atom) in our cases, we conclude that 0.3~1.4 atoms for every 1000 atoms underwent photoabsorption. Therefore, ~0.1% of the atoms have one inner-shell hole due to photo-excitation, which is immediately filled through an Auger process occurring in a sub-fs timescale31. Immediately after photo-excitation, this process creates only ~0.1% holes in the valence or conduction bands, and the core holes are promptly filled. We can then assume that in the first instants after photoexcitation the inter- atomic potential is not significantly perturbed. Meanwhile the free energy of the sample increases by the amount of the absorbed energy, which is stored in high energy (>keV) photoelectrons. In a conventional non-thermal melting processes, the following dynamics has been discussed, especially for the case of semiconductors5,32,33. A fast electron distributes its energy through collisional interactions repetitively until it settles down into bound states. At the same time the electron cascading processes further excite electrons from the valence to the conduc- tion band, altering the electron density distribution and thus the interatomic potential, and consequently leading to lattice disorder.f Given that x-ray diffraction is mainly sensitive to the changes in core electron density, it should be noted that Bragg diffraction signal is, in principle, not directly sensitive to the PES. Indeed, for hard x-ray excited Si with similar absorbed doses to our case5, the start of the Bragg diffraction signal decay was observed after ~110 fs from the incidence of the pump. The similar excitation condition in Bi in our case also implies a significantly low ionisation degree, thus low photoelectron population, despite the absorbed dose is up to a few times higher than its damage threshold13. Scientific Reports \| (2019) 9:602 \| https://doi.org/10.1038/s41598-018-36216-3 Methods i Experimental setup. The schematic arrangement of the transmission gratings is illustrated in Fig. 1a. A set of 10 linear gratings (G1 gratings), made of diamond, was placed 0.3 m downstream from the beamline exit window. A set of 20 linear gratings (G2 gratings), made of Ir and SiO2, was positioned 3.3 m downstream of the G1 gratings to receive the diffracted beams from the G1 gratings. All the gratings were fabricated by means of electron-lithography and reactive ion etching, at the Laboratory for Micro- and Nanotechnology, Paul Scherrer Institut. The sample was positioned 6.6 m downstream of the G1 gratings, accepting both the transmitted (pump) beam and 20 diffracted (probe) beams from the G2 gratings. On the sample, half of the probe beams spatially overlap with the pump pulse, while the other half is separated by 70 μm to the unpumped region. In this way, the whole sequence of pumped and unpumped signals are obtained simultaneously, from a single x-ray pulse. In this experiment, the grating efficiencies are enhanced by factors of 2~5 compared to those reported earlier. The grating pitches were optimized to sequentially increase the delay timings with precisely defined intervals of 20 fs to 50 fs. This small time steps were continued up to ~300 fs delay, with the probe focus on early-onset of the damage to the crystal.hlh g y The Bragg angle for the (111) reflection in Bi at 5 keV is 18° with a measured rocking curve width of 0.2°. The varying incident angles in y-z plane of the pump and probe beams do not affect the Bragg condition, because the diffraction angle from the sample is only sensitive to the angle in the x-z plane, which is perpendicular to the gratings diffraction plane (Fig. 1b). Each pulse are then incident at the detector at spatially separated locations due to the angular separations in the y-z planes created by the diffraction gratings, thus retaining the delay-time information. Fresh sample surface was used for every shot. p y The Bragg reflected probe beam signals were recorded using the JUNGFRAU detector, an integrating two-dimensional pixel detector comprised of arrays of 75 × 75 μm2 pixels, with single photon sensitivity over a dynamic range of 2.5 × 104 for 5 keV photons. More details about the JUNGFRAU detector can be found in34. Discussion For reflections insensitive to optical modes, similarly to the Si or diamond cases5,25,33, one could expect a short period of delay until the onset of signal drop can be observed. However, from Bi (111) the onset of the signal decay is almost immediate, especially for the highest excitation case. It indicates an atomic dislocation process occurring in parallel to non-thermal melting process.hl p g p g p The fluence dependence of our measurements (as seen in Fig. 3), carries additional information on the possible phenomena occurring upon photoexcitation. Scenarios such as thermal excitation of the lattice (Debye-Waller) would exhibit a fluence dependence only in the amplitude of the exponentially decreasing diffracted intensity. In contrast, here both the amplitude and the gradient leading to the normalised intensity of ~0.5 in Fig. 3 show fluence dependence – an observation compatible with the displacive excitation of coherent phonons (DECP)12. Theoretical modelling of the DECP process is primarily dependent on the pump pulse duration (shorter than the phonon oscillation period) and on the absorbed power density. Presence of energetic excited electrons leads to Scientific Reports \| (2019) 9:602 \| https://doi.org/10.1038/s41598-018-36216-3 4 www.nature.com/scientificreports/ excitation of many outer electrons, thus a coherent motion of the lattice is induced as a result of changes of PES, likely contributing to the building up of a non-thermal melting process. Assuming that DECP process is initiated, the potential implication following here is a strong anharmonic coupling between the A1g and the Eg optical modes leading to a phase transition on the timescale less than one period of the A1g mode, as discussed also in ref.8. We therefore speculate that this coherent lattice motion could be the precursor to the observed non-thermal melting process on such a rapid timescale. These stages of transition dynamics initiated by hard x-rays are thus most likely relevant for materials with A1 optical modes in general.i y y y y 1 p g In conclusion, we have performed the first-time observation of x-ray induced lattice disordering in Bi occur- ring within less than 300 fs. The (111) Bragg peak dynamics indicates that the phase transition proceeds as a com- plex multistep process. Our data set an important benchmark for future experiments and modelling of the hard x-ray induced ultrafast phase-transition in Bi, which should reproduce the observed fluence dependence. Methods i The intensity of each diffracted peak on the detector was determined by integrating the counts up to half the distance to the neighbouring peaks. The integrated peak intensities were then normalised against the unpumped counter- parts, which were then rescaled using the normalised and averaged “reference” signals.h p g g g The size of the pump beam spot was determined by observing the attenuated x-ray beam on a YAG screen at the sample position, using a microscope lens and a Charge Coupled Device (CCD) detector. The different spot sizes of the pump and the probe pulses were achieved by using Be focusing lenses positioned at two places. The first set was placed upstream of the G1 gratings and therefore affected the focusing of the direct pump beam and the probe beams. The other set was placed on the optical axis at the G2 grating position (not shown in the figure) and only affected the focusing of the pump pulse. This arrangement allowed us to choose the size of the pump and the probe beams independently.ht p p y The unattenuated pump pulse energy at the sample position (after passing through the gratings and the focus- ing lenses) was measured with a calorimeter to be ~100 μJ, which is ~5% of the nominal total emitted pulse energy. In order to vary the fluence level, but not the probe intensities, the pump pulse was attenuated using 10 μm and 20 μm Al foils placed on the optical axis of the transmitted beam, close to the G2 grating (Fig. 1a). With these conditions, the pump beam fluence varied from 0.9 to 2.5 J/cm2. The energy of each probe beam was 0.01 to 0.2% of the pump beam at the sample position. In the absence of the pump beam, the crystal structure remains intact, confirming that the probe pulses are sufficiently weak to not damage the crystal. Discussion For that purpose, there is a strong need for a development of dedicated theoretical models capable of following such complex non-equilibrium dynamics in time. Promising candidates currently being developed are time-dependent Density Functional Theory or Hartree-Fock based schemes. References 1. Sokolowski-Tinten, K. et al. Femtosecond X-ray measurement of coherent lattice vibrations near the Lindemann stability limit. Nature 422, 287–289 (2003). 2. Fritz, D. M. et al. Ultrafast Bond Softening in Bismuth. Science. 315, 633–636 (2007). 3. Johnson, S. L. et al. Directly Observing Squeezed Phonon States with Femtosecond X-ray Diffraction. Phys. Rev. Lett. 102, 175503 (2009). 4. Johnson, S. L. et al. Nanoscale Depth-Resolved Coherent Femtosecond Motion in Laser-Excited Bismuth. Phys. Rev. Lett. 100, 155501 (2008). 5. Pardini, T. et al. Delayed Onset of Nonthermal Melting in Single-Crystal Silicon Pumped with Hard X Rays. Phys. Rev. Lett. 120, 265701 (2018). 6. Murray, É. D. & Fahy, S. First-principles calculation of femtosecond symmetry-breaking atomic forces in photoexcited bismuth. Phys. Rev. Lett. 114, 055502 (2015). 7. Fahy, S., Murray, É. D. & Reis, D. A. Resonant squeezing and the anharmonic decay of coherent phonons. Phys. Rev. B 93, 134308 (2016). References et al. Soft x-ray induced femtosecond solid-to-solid p 25. Tavella, F. et al. Soft x ray induced femtosecond solid to solid phase transition. High Energy Density Phys. 24, 22 27 (2017). 26. Beaud, P. et al. A time-dependent order parameter for ultrafast photoinduced phase transitions. Nat. Mater. 13, 923–7 (2014). p p p p ( ) 27. Huber, T. et al. Coherent structural dynamics of a prototypical charge-density-wave-to-metal transition. Phys. Rev. Lett. 113, 02640 (2014).hi 8. Sokolowski-Tinten, K. et al. Thickness-dependent electron – lattice equilibration in laser-excited thin bismuth films. New J. Phys. 1 113047 (2015).f 29. Arnaud, B. & Giret, Y. Electron cooling and Debye-Waller effect in photoexcited bismuth. Phys. Rev. Lett. 110, 016405 (2013). 29. Arnaud, B. & Giret, Y. Electron cooling and Debye Waller effect in photoexcited bismuth. Phys. Rev. Lett. 110, 016405 (2013). 30. Scott, H. A. Cretin—a radiative transfer capability for laboratory plasmas. J. Quant. Spectrosc. Radiat. Transf. 71, 689–701 (200 30. Scott, H. A. Cretin a radiative transfer capability for laboratory plasmas. J. Quant. Spectrosc. Radiat. Transf. 71, 689 701 (2001). 31. Son, S.-K., Young, L. & Santra, R. Impact of hollow-atom formation on coherent x-ray scattering at high intensity. Phys. Rev. A 83, 033402 (2011). 2. Medvedev, N., Jeschke, H. O. & Ziaja, B. Nonthermal phase transitions in semiconductors induced by a femtosecond extrem ultraviolet laser pulse. New J. Phys. 15, 015016 (2013).h p y 33. Medvedev, N., Li, Z. & Ziaja, B. Thermal and nonthermal melting of silicon under femtosecond x-ray irradiation. Phys. Rev. B 91, 054113 (2015). 4. Mozzanica, A. et al. Characterization results of the JUNGFRAU full scale readout ASIC. J. Instrum. 11, C02047–C02047 (2016). Acknowledgements We thank Dr. Steve Johnson, Dr. Stephen Fahy, and Dr. Eamonn Murray for insightful advices and discussions. The research leading to these results has received funding from the European Community’s Seventh Framework Programme (FP7/2007–2013) under grant agreement no. 290605 (PSI-FELLOW/COFUND). The diamond membranes used for the gratings were provided by the Diamond Materials GmbH. Silicon-Nitride membranes used for the gratings were fabricated by N.P. Opara and J. Lehmann. Partial financial support from the Czech Ministry of Education (Grants LTT17015 and LM2015083) is acknowledged by N. Medvedev. C. Caleman thanks Swedish Research Council (2013–3940) and Helmholtz Association, through Center for Free-Electron Laser Science at DESY. Support from Swiss Nanoscience Institute via project P1305 is acknowledged by N. L. Opara. Preliminary experiments were conducted at the cSAXS beamline at the Swiss Light Source, Paul Scherrer Institut, Villigen, Switzerland, as preparation for the experiment at the FEL. Use of the Linac Coherent Light Source (LCLS), SLAC National Accelerator Laboratory, is supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences under Contract No. DE-AC02–76SF00515. Author Contributions C.D. conceived and planned the project together with M.M. The time-resolved sequential-probing with single- shot setup was built by C.D., M.M., I.V., I.M., and A.Mo. C.D. treated the Bi samples. M.M. fabricated the gratings used for the experiment. M.M., I.V., I.M., A.D., P.J., A.Me., A.Mo., N.L.O., C.P., V.P., L.V., P.R.W., C.J.M., and C.D. carried out the experiment. M.S. and S.S. supported the use of the XCS beamline at the LCLS facility. A. Mo operated the JUNGFRAU detector. C.C. and H.O.J. operated the CRETIN code. B.Z.-M., and V.S. operated the XATOM code. M.M. analysed the data together with B.Z.-M., N.M., C.M., and P.B. The manuscript was written by M.M. with inputs from all the co-authors. References References 1. Sokolowski-Tinten, K. et al. Femtosecond X-ray measurement of coherent lattice vibrations near the Lindemann stability limit. Nature 422, 287–289 (2003). 2. Fritz, D. M. et al. Ultrafast Bond Softening in Bismuth. Science. 315, 633–636 (2007). 3. Johnson, S. L. et al. Directly Observing Squeezed Phonon States with Femtosecond X-ray Diffraction. Phys. Rev. Lett. 102, 175503 (2009). 4. Johnson, S. L. et al. Nanoscale Depth-Resolved Coherent Femtosecond Motion in Laser-Excited Bismuth. Phys. Rev. Lett. 100, 155501 (2008). 5. Pardini, T. et al. Delayed Onset of Nonthermal Melting in Single-Crystal Silicon Pumped with Hard X Rays. Phys. Rev. Lett. 120, 265701 (2018). 6. Murray, É. D. & Fahy, S. First-principles calculation of femtosecond symmetry-breaking atomic forces in photoexcited bismuth. Phys. Rev. Lett. 114, 055502 (2015). 7. Fahy, S., Murray, É. D. & Reis, D. A. Resonant squeezing and the anharmonic decay of coherent phonons. Phys. Rev. B 93, 134308 (2016). 2. Fritz, D. M. et al. Ultrafast Bond Softening in Bismuth. Science. 315, 633–636 (2007). 155501 (2008). 5. Pardini, T. et al. Delayed Onset of Nonthermal Melting in Single-Crystal Silicon Pumped with Hard X Rays. Phys. Rev. Lett. 120, 265701 (2018). É & h S l l l f f d b k f h d b h 6. Murray, É. D. & Fahy, S. First-principles calculation of femtosecond symmetry-breaking atomic forces in photoexcited bism Phys. Rev. Lett. 114, 055502 (2015). y ( ) 7. Fahy, S., Murray, É. D. & Reis, D. A. 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Short-pulse laser - plasma interactions. Plasma Phys. Control. Fusion 38, 769–793 (1996). l d d bf d l f lf l fi d b 18. Emma, P. et al. Femtosecond and subfemtosecond x-ray pulses from a self-amplified spontaneous-emission-based free-electron laser. Phys. Rev. Lett. 92, 074801 (2004). 9. David, C. et al. Following the dynamics of matter with femtosecond precision using the X-ray streaking method. Sci. Rep. 5, 7644 (2015).h 0. Alonso-Mori, R. et al. The X-ray Correlation Spectroscopy instrument at the Linac Coherent Light Source. J. Synchrotron Radiat. 22 508–513 (2015). 21. Emma, P. et al. First lasing and operation of an ångstrom-wavelength free-electron laser. Nat. Photonics 4, 641–647 (2010).f Emma, P. et al. First lasing and operation of an ångstrom-wavelengt 22. Opara, N. L. et al. Demonstration of femtosecond X-ray pump X-ray probe diffraction on protein crystals. Struct. Dyn. 5, 05 (2018). 23. Bauerhenne, B., Zijlstra, E. S. & Garcia, M. E. Molecular dynamics simulations of a femtosecond-laser-induced solid-to- transition in antimony. Appl. Phys. A 123, 608 (2017). y pp y 24. Vinko, S. M. et al. Creation and diagnosis of a solid-density plasma with an X-ray free-electron laser. Nature 482, 59–62 (2012 g y p y ( ) 25. Tavella, F. et al. Soft x-ray induced femtosecond solid-to-solid phase transition. High Energy Density Phys. 24, 22–27 (2017). 25. Tavella, F. Additional Information Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-36216-3 Competing Interests: The authors declare no competing interests. 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https://openalex.org/W4281611539	https://zenodo.org/record/7246592/files/Decker_Feigel_Foster__Kepl_2022.pdf	English	null	“Press On, Continue On”: Rural Parents’ Experiences of Transitions Within Early Intervention	Rural special education quarterly	2,022	cc-by	12,865	7080 RSQXXX10.1177/87568705221097080Rural Special Education QuarterlyDecker et al. 7080 RSQXXX10.1177/87568705221097080Rural Special Education QuarterlyDecker et al. Research Reports https://doi.org/10.1177/87568705221097080 Rural Special Education Quarterly 1–14 © Hammill Institute on Disabilities 2022 Article reuse guidelines: sagepub.com/journals-permissions DOI: 10.1177/87568705221097080 journals.sagepub.com/home/rsq Keywords l y early intervention, transition, family involvement, parents, rural values/concerns Transitional periods are an important part of each family’s experience since all families of young children experience change over time (Britto & Pérez-Escamilla, 2013; Bush & Price, 2020; McGoldrick et al., 2011). Yet, for families of children with exceptionalities, they may experience even more transitions throughout their child’s earliest years (Hebbeler & Spiker, 2016; Rous & Hallam, 2012), such as those related to a variety of early intervention services for their children (e.g., Early Head Start; Maternal, Infant, and Early Childhood Home Visiting Program; Nurse–Family Partnership). Early intervention services may include those provided by Part C of the Individuals with Disabilities Education Act (IDEA, 2004), which supports children less than the age of 3 with an identified delay and/or disability. After the age of 3, some children with exceptionalities will transition out of Part C services and into Part B services of IDEA, commonly known as special education. Other mean- ingful transitions may also take place for these families and their children during this time period, such as changes in providers, beginning or ending additional services based on the needs of the child or family, and changes in day-to-day needs (Gothberg et al., 2017; Hebbeler & Spiker, 2016; Pang, 2010; Rous & Hallam, 2012). Part C services and/or transitions over time (Decker et al., 2020, 2021; Elpers et al., 2016; Hallam et al., 2009; Murphy et al., 2013; Singh et al., 2019). The U.S. Census Bureau defines rurality as areas that have populations of 50,000 residents or less (Ratcliffe et al., 2016). Although young children in rural areas are more likely to be eligible for Part C services (Roberts et al., 2014) and receive spe- cial education services at a higher rate than children in urban areas (Grace et al., 2011), these children are fre- quently underrepresented in rural education research (Capizzano & Fiorillo, 2004; Grace et al., 2011; Thier et al., 2021). This is important given that parents in rural communities have identified barriers that include travel time or a lack of access to services, providers, or early intervention information (Cummings et al., 2017; Decker et al., 2020, 2021; Elpers et al., 2016; Hallam et al., 2009; Mann & Williams, 2011; Singh et al., 2019). 1Montana State University, Bozeman, USA 2Eastern Michigan University, Ypsilanti, USA Abstract Although all families experience a variety of transitions over time, families of children with exceptionalities tend to encounter more transitions during their children’s earliest years. Transitions can be stressful and often include changes as part of early intervention or special education services. It is important to understand these transitions because the support families receive can influence child and family well-being. Therefore, we interviewed parents (N = 28) across a rural state about their experiences of transition over time. Using thematic analysis, three common themes emerged: (a) change is constant, (b) positive relationships support changing needs and priorities, and (c) parents need more support, information, or access to services or providers. Parents reported relationships and collaboration with providers to be important, yet insufficient, in supporting transitions. Rurality added some challenges to parents’ experiences with transition. Recommendations include empowering families, providing more access and/or removing barriers to services, and building family efficacy through family-focused services. Corresponding Author: Kalli B. Decker, Montana State University, 115 Herrick Hall, Bozeman, MT 59717, USA. Email: kalli.decker@montana.edu “Press On, Continue On”: Rural Parents’ Experiences of Transitions Within Early Intervention Kalli B. Decker, KD, PhD1 , Alexandra J. Feigel, AF, MS1, Tricia D. Foster, TD, PhD2, and Rachel L. Kepl, RK, BS1 Keywords l The experi- ences that rural families face may exacerbate the stress The location in which families live, such as how urban or rural an area is, may also influence their experiences of Rural Special Education Quarterly 00(0) 2 and difficulties that are often associated with times of transition. Studies focused on the educational experiences of young children and their families in rural areas are rare, and more scholarship is needed (Thier et al., 2021). This study aims to fill this gap in the literature related to how families living in rural areas are supported by early inter- vention and the educational system during times of transition. ending additional services (e.g., therapies), and entering or exiting special education services (Douglas et al., 2022; Gothberg et al., 2017; Hebbeler & Spiker, 2016; Pang, 2010; Rous & Hallam, 2012; Walsh & Taylor, 2010). The intersection of these typical family transitions with the experiences of living in a rural area and accompanying challenges, such as limited access to services or providers (Cummings et al., 2017; Decker et al., 2020, 2021; Elpers et al., 2016; Hallam et al., 2009; Murphy et al., 2013; Singh et al., 2019), is not well understood, especially in the realm of early intervention. While a number of studies have focused specifically on families’ experiences of transition into Part B services (Douglas et al., 2022; Malone & Gallagher, 2009; Podvey et al., 2013), few have focused more broadly on the multi- tude of transitions faced by families who have children with exceptionalities (Hebbeler & Spiker, 2016; Rous & Hallam, 2012) and even fewer have focused on transitions within early childhood for families living in rural areas (Murphy et al., 2013). Therefore, little is known about families’ broader experiences with transitions over time or the extent to which they have felt supported during these periods of change. Better understanding families’ experi- ences of transition, including but not limited to their child’s exit from Part C and/or entry into Part B services, could fill gaps in the current literature and guide practices of how families are supported during times of shifting needs or priorities. Families and Children With Exceptionalities The transitions that families of children with exceptionali- ties face are more extensive than those of families without children with exceptionalities, and these transitions can cre- ate high levels of stress for families (Douglas et al., 2022; Pang, 2010; Waters & Friesen, 2019). These transitions include meaningful changes related to the family (e.g., rela- tionships), environment (e.g., living situations, schools, or facilities for medical care), or amount of support received throughout these interactions (Pang, 2010; Waters & Friesen, 2019). Other major changes are also meaningful, such as formal transitions that occur related to Part C and/or Part B since this frequently leads to changes in the location, providers, and/or goals of services (Pang, 2010; Rous & Hallam, 2012; Walsh & Taylor, 2010). According to the Council for Exceptional Children’s Division of Early Childhood’s (DEC) recommended practices (2014), transi- tion is defined as “events, activities, and processes associ- ated with key changes . . . during the early childhood years” (p. 16). Therefore, transitions could include any number of changes all families may experience, as well as those related to changes that occur from hospital to home, entering and exiting Part C services, or entering preschool special educa- tion services (Part B). Keywords l Therefore, this study aims to examine parents’ perceptions of transitions when they have a child with exceptionalities, such as during formal transitions (e.g., exiting Part C services and entering Part B services) and other meaningful life transitions (e.g., child- or fam- ily-level changes such as those related to development, providers, or family structure). Families and Children With Exceptionalities Transitions During the Early Years All families of young children experience change and tran- sitions over time (Britto & Pérez-Escamilla, 2013; Bush & Price, 2020; McGoldrick et al., 2011). Family responses to transition depend on contextual influences and internal qualities of the family system (Bush & Price, 2020; McGoldrick et al., 2011). Adjustments to change for one family may be different for another (Bush & Price, 2020; McGoldrick et al., 2011). The ways families respond to transitions are indicative of child and family well-being and include making necessary shifts to support family members experiencing change (Bush & Price, 2020; McGoldrick et al., 2011). In addition, families with young children typi- cally experience common transitions including the birth of a child, changes in child development, parental role changes, increasing or decreasing social support networks, and parental occupation changes (Britto & Pérez-Escamilla, 2013; Bush & Price, 2020; McGoldrick et al., 2011). However, families of children with exceptionalities may face additional types of hardships including finding access to medical services, changes in providers, beginning or The transition from Part C to Part B services marks an important change for children and their families—as this typically means that there is a major shift from home-based services that are geared toward the family to center- or school-based services where the family is not present (Douglas et al., 2022; Pang, 2010; Walsh & Taylor, 2010). Historically, parents’ experiences with transitions into Part B services are fraught with challenges (Douglas et al., 2022; Podvey et al., 2010; Rous et al., 2007). Parents have reported wanting more clarification of their role(s) once entering Part B services (Podvey et al., 2013; Rous et al., 2009), with open and honest communication between practitioners and the family being a main priority for parents (Doudna et al., 2015; Douglas et al., 2022; Malone & Gallagher, 2009; Waters & Friesen, 2019). Additional barriers parents report facing include socioeconomic status, availability of resources, and rurality (Malone & Gallagher, 2009). In a Decker et al. 3 types of supports these children and families may need dur- ing transitions. small qualitative study, parents reported feeling like “out- siders” within the context of Part B services; parents felt as if they were more involved in their child’s learning and care in Part C services, as compared with their level of involve- ment in Part B (Podvey et al., 2013). Transitions During the Early Years There is a clear need for high-quality support during transitions, since this can affect both child- and family-level outcomes (Douglas et al., 2021; Murphy et al., 2013; Rous et al., 2007). Current Study Supporting parents of children with exceptionalities through the many transitions they may experience in the early years of their children’s lives is an important component of early intervention services (DEC, 2014; IDEA, 2004). However, parents whose children receive Part C services do not always receive supportive or consistent transition services (Rous & Hallam, 2012). Existing research on transitions in Part C has traditionally focused on outcomes related to for- mal transitions within and between intervention systems (Douglas et al., 2021; Gothberg et al., 2017; Malone & Gallagher, 2009; Podvey et al., 2010; Rous et al., 2007), rarely also exploring the informal transitions and everyday changes that family experience during their children’s early years (Hebbeler & Spiker, 2016). Furthermore, since fami- lies living in rural areas frequently face barriers to accessing services (Cummings et al., 2017; Decker et al., 2020, 2021; Elpers et al., 2016; Hallam et al., 2009; Mann & Williams, 2011; Murphy et al., 2013; Singh et al., 2019), these fami- lies may also face additional barriers during times of transi- tions. Therefore, this study asks: For families in a Western rural state who have young children with exceptionalities, what are parents’ experiences of transitions over time? Supporting Families Through Transitions Given the frequency with which families of children with additional support needs undergo transitions in their chil- dren’s earliest years, and the stress that often accompanies these transitions, it is important to consider what types of practices best support families and children. Transitions can be a nerve-wracking experience for families, so including families in the planning and execution process is important in addressing their needs and/or concerns (Doudna et al., 2015; Douglas et al., 2022; Gothberg et al., 2017; Pang, 2010). Parents who feel supported during transitions by their providers are more likely to be involved and engage in better decision-making, resulting in better overall positive outcomes for their child and family (Doudna et al., 2015; Gothberg et al., 2017; Pang, 2010). Successful transitions are characterized by collabora- tion among all members of the early intervention team (Doudna et al., 2015; Douglas et al., 2022; Gothberg et al., 2017; Walsh & Taylor, 2010). For example, indicators for a successful transition out of Part C services or from Part C to Part B services include the following: (a) the creation of transition outcomes within the child’s Part C Individualized Family Service Plan (IFSP) developed by the family, service coordinator, and any other providers; (b) notification to the school district of potentially eligible children for Part B special education services; and (c) holding a transition conference between the family, the lead education agency or school district, and any other providers within the child’s life (IDEA, 2004). The pro- cess of preparing for formal transition into Part B special education proves valuable in supporting families and increasing collaboration between educators, providers, and family members (DEC, 2014; Doudna et al., 2015; Douglas et al., 2022; Walsh & Taylor, 2010). Setting This study took place in a rural state in the Western United States (Ratcliffe et al., 2016), also regarded as frontier (Rural Health Information Hub, 2018) as many individuals must travel far distances to receive health care and other services. The state in which these data were collected is considered a “research desert” related to rural education (Thier et al., 2021, p. 9). Many counties in the state are con- sidered medically underserved (Health Resources & Services Administration, 2018), which has implications for Part C services (Decker et al., 2020, 2021) There are regional programs in the state in which the data were col- lected that employ service coordinators for children’s Part C services and Individualized Family Service Plans (IFSPs). The Part C team frequently includes occupational therapists (OTs), physical therapists (PTs), and speech language pathologists (SLPs), in addition to the dedicated service coordinator. In the state in which these data were collected, nearly all of these therapists work for independent agencies, clinics, or hospitals outside of the Part C system. Therefore, the child’s service coordinator partners with therapists or other providers to coordinate the child’s IFSP team. Once a child reaches age 3, if they are eligible, they can enter Regardless of the type of changes families experience, approaching transitions using family-centered practice, a focus on understanding, supporting, and addressing fami- lies’ changing needs via collaborative relationships is seen as the most supportive route (Doudna et al., 2015; Douglas et al., 2021; Pang, 2010; Rous & Hallam, 2012). Although families in rural areas may face more difficulties in access- ing and receiving early intervention services (Cummings et al., 2017; Decker et al., 2020, 2021; Elpers et al., 2016; Hallam et al., 2009), little is known about what distinct Rural Special Education Quarterly 00(0) 4 preschool special education (Part B) services and have an Individualized Education Plan (IEP) and/or additional fam- ily-based services and continue to have an IFSP. exceptionalities that had occurred since participating in Wave 1. Participants from Wave 1 were contacted and invited to participate in Wave 2 interviews. Twenty-eight of the original 30 parents from Wave 1 participated in Wave 2. The PI worked with interested families to set up a date, time, and location for the data collection visit. Sample Data used for this study are from Wave 2 of a longitudinal, qualitative study of parents (N = 28) whose children had received Part C services during Wave 1 of the study. Each of the state’s 5 regions was represented in Wave 2 (3 partici- pants from Region 1; 6 from Region 2; 12 from Region 3; 3 from Region 4; and 4 from Region 5). All participants iden- tified as the child’s parent and primary caregiver, and all identified as female. One participant had three children whom they discussed as part of this study, another partici- pant had two children, and the remaining participants each had one child; this led to a total of 31 children discussed (12 female, 19 male) in Wave 2. Twenty-three participants iden- tified as Caucasian, 3 as American Indian and Caucasian, 1 as American Indian, and 1 as Hispanic/Latino and Caucasian. The majority of the children represented were Caucasian (n = 23), one child was American Indian, six children identified with more than one race/ethnicity, American Indian and Caucasian (n = 4); American Indian and Hispanic/Latino (n = 1); American Indian, Asian, and Caucasian (n = 1), and for one race/ethnicity information was not provided for the child; this race/ethnicity data gen- erally reflect the demographics of the state in which these data were collected. Participants reported their information about the child/children’s reason for originally receiving Part C services, including one or more of the following: developmental delays in one or more areas; diagnosed con- ditions (e.g., autism spectrum disorder, cerebral palsy, hydrocephalus); and other factors such as prematurity and heart or brain disorders. Children ranged between 15.8 and 49.8 months of age at the time of Wave 2 (M = 34.8, SD =11.1 months). See Table 1 for additional parent and child demographic information. The data for this study are based on the in-depth inter- view conducted in Wave 2. The first part of the Wave 2 interview was focused on reviewing information gathered in Wave 1 of data collection. The interview then focused on updates and/or changes to parents’ goals, priorities, or needs, or changes related to their child’s development or services. Parents were asked to reflect on past and current services and providers, levels of support from providers based on any changes that had occurred, and past and pres- ent transitions out of or into new services. Sample The primary interview questions used for the current study included: Do you feel that anything has changed over the last year related to your child’s development or your family’s needs or pri- orities? Do you feel that services have met your family’s and your child’s needs over the last year? What are your feelings about the professionals you and your child have worked with over the last year? Looking back, what could have made these services better? Setting Most of the interviews were led by two research assistants (RAs) trained in the interview protocol; one RA led the interview, while another RA was primarily responsible for attending to the child(ren) so that the parent could participate with fewer distractions. The interviews began with the RA outlining the purpose of the study, answering questions, and obtain- ing written consent from the parent. Data collection took approximately 1 to 2 h; this included reviewing information gathered at the last interview, providing an option for par- ents to update their demographic information collected dur- ing Wave 1 of the study, and a semi-structured interview to gather additional information regarding parents’ Part C experiences and changes in the child and families’ lives since the Wave 1 interview. All interviews were conducted in English, with video and audio recordings taken to be able to transcribe the data. After completion of the interview, families were given a $50 gift card and a children’s book. Procedures The following procedures were approved by a university Institutional Review Board. This study was not an official evaluation of the state’s Part C services, but the principal investigator (PI) did receive support from the state’s Part C agencies to help recruit participants for Wave 1. Regional Part C agencies sent fliers about the study to families who had one or more child receiving Part C services. Families who were interested in participating then contacted the PI. See Decker et al. (2020, 2021) for more information about Wave 1 of the study. Analysis aNumbers sum to more than 100% because children could have received more than one type of therapy and/or received therapies in different % (n) or M (range, SD) Marital status Married Single Divorced Employment status Stay-at-home parent Working part-time Working full-time Education level High school or General Education Diploma Some college or associate’s degree Bachelor’s degree Master’s degree Household income before taxes Less than $12,000 $20,000–39,999 $40,000–59,9999 $60,000–79,999 $80,000 or more Information about children (N = 31) Age of initial diagnosis of delay or disability (in months) Age of entry into Part C services (in months) Age at time of Wave 2 data collection (in months Age at time of Wave 2 data collection (in months) Status of children’s services Still receiving Part C services Exited Part C, did not transition into Part B or additional family-based services after Part C Exited Part C, transitioned into Part B services Services provided by a service coordinator Location of services provided by service coordinator Home Services provided by PT, OT, and/or SLP Location of services provided by PT, OT, and/or SLPa Services provided by PT, OT, and/or SLP Location of services provided by PT, OT, and/or SLPa Home 38.9% (7) Clinic 61.1% (11) School 55.6% (10) Home Clinic School Home Clinic School Note. Eleven of the 28 families chose not to fill out a new demographic form during Wave 2, so information in this table comes from Wave 1 demographic forms for those families. PT = physical therapist; OT = occupational therapist; and SLP = speech language pathologist. aNumbers sum to more than 100% because children could have received more than one type of therapy and/or received therapies in different locations. Note. Eleven of the 28 families chose not to fill out a new demographic form during Wave 2, so information in this table comes from Wave 1 demographic forms for those families. PT = physical therapist; OT = occupational therapist; and SLP = speech language pathologist. aNumbers sum to more than 100% because children could have received more than one type of therapy and/or received therapies in different locations. concise and specific, and giving each code a description and examples. Next, the fourth author coded all remain- ing transcripts, and the first author reviewed each tran- script. The authors met to discuss and resolve any differences, and finally, they reviewed the coded data to identify overarching themes. Analysis We used inductive thematic analysis to analyze the data, which is appropriate when investigating an under-studied research question (Braun & Clarke, 2006). The inductive thematic process includes familiarizing oneself with the data, generating initial codes, searching for trends and patterns, reviewing codes and themes, defining and nam- ing themes, and producing a final report (Braun & Clarke, 2006). Therefore, data analysis began with transcribing all interviews. The first and fourth authors then completed each of the next steps. The authors began data immersion The design of Wave 2 of this study was intended to gather information from parents about changes in their needs, priorities, or services related to their child with 5 Decker et al. Demographic information % (n) or M (range, SD) Parent demographics (N = 28) Marital status Married 78.6% (22) Single 14.3% (4) Divorced 7.1% (2) Employment status Stay-at-home parent 50.0% (14) Working part-time 25.0% (7) Working full-time 25.0% (7) Education level High school or General Education Diploma 21.4% (6) Some college or associate’s degree 28.6% (8) Bachelor’s degree 42.9% (12) Master’s degree 7.1% (2) Household income before taxes Less than $12,000 14.3% (4) $20,000–39,999 25.0% (7) $40,000–59,9999 17.9% (5) $60,000–79,999 21.4% (6) $80,000 or more 21.4% (6) Information about children (N = 31) Age of initial diagnosis of delay or disability (in months) 10.1 (0.0-30.0, 10.3) Age of entry into Part C services (in months) 13.8 (1.0-30.0, 9.9) Age at time of Wave 2 data collection (in months) 34.8 (15.8-49.8, 11.1) Status of children’s services Still receiving Part C services 25.8% (8) Exited Part C, did not transition into Part B or additional family-based services after Part C 38.7% (12) Exited Part C, transitioned into Part B services 35.5% (11) Services provided by a service coordinator 100% (31) Location of services provided by service coordinator Home 100% (31) Services provided by PT, OT, and/or SLP 58.1% (18) Location of services provided by PT, OT, and/or SLPa Home 38.9% (7) Clinic 61.1% (11) School 55.6% (10) Note. Eleven of the 28 families chose not to fill out a new demographic form during Wave 2, so information in this table comes from Wave 1 demographic forms for those families. PT = physical therapist; OT = occupational therapist; and SLP = speech language pathologist. Analysis We then determined the number and percentage of participants whose interviews included information relevant to each theme. Each par- ticipant was only counted once per theme even if their transcript included multiple passages coded within that theme. Codes and themes can be found in Table 2. separately by reading each transcript and making notes about which portions pertained to the research question and which did not. Transcripts were then discussed, parts of the transcripts to be coded were agreed upon, and ini- tial codes were generated based on participants’ words and phrases. The authors coded four transcripts separately to refine and edit the coding scheme and demonstrate consistency. This resulted in a kappa score of .75, which indicates substantial agreement (Landis & Koch, 1977). The authors moved on to defining each code which included changing the names of some codes to be more Rural Special Education Quarterly 00(0) 6 Table 2. Parents’ (N = 28) Reports of Their Experiences With Transitions. Theme and code Example Theme 1: Change is Constant (N = 28, 100%) Anticipation or uncertainty of future changes or needs “[They] told me that could indicate a cognitive delay. So, that’s kind of on our radar. . . . We know he had prenatal drug and alcohol exposure, and ADHD is definitely a possibility, behaviora issues, learning delays, cognitive delays. I mean there are a lot of things that are possibilities.” Changes in child development “[Good news] would be the fact that [my child] can do this. I mean she is not always in her bed. That killed me for months. I was like ‘Gosh, I want to take her outside. I want to take her to the store. How do I let her sit in a cart when her head is so not stable and her body flops everywhere?’ But she is getting better and she is not completely immobile anymore. . . . It is getting easier to hold her.” Changes in services or providers “I’m not sure how much [our new provider] is going to see us, she doesn’t know. Which kind of sucks because [our old provider] had been working with [my child] for a year and a half now.” Family-level changes “Our priorities have changed a lot . . . as far as our needs, we want a bigger place. Example That was a little bit of a stressor.” “A little bit better communication [would have helped]. . . . There wasn’t as much volunteered information as there could have been as far as what resources were available and stuff.” “I was [my child’s] advocate and I was really the one that was calling the shots as far as, ‘This is what’s going on; this is what needs to happen.’ But at the end of the day, the resources weren’t provided to me to continue doing that to the best of my ability.” “[Part B services are] not as personalized as the [Part C] services were. . . . You don’t have the kind of the centralized support. . . . It would have been nice to have like the same level that she received before.” and providers. Importantly, parents described these changes as a continuous process which required them to “press on, continue on.” Parents were keenly aware of the fact that transitions and change over time would remain a continu- ous part of supporting their child. Example ment “[Good news] would be the fact that [my child] can do this. I mean she is not always in her bed. That killed me for months. I was like ‘Gosh, I want to take her outside. I want to take her to the store. How do I let her sit in a cart when her head is so not stable and her body flops everywhere?’ But she is getting better and she is not completely immobile anymore. . . . It is getting easier to hold her.” g g “I’m not sure how much [our new provider] is going to see us, she doesn’t know. Which kind of sucks because [our old provider] had been working with [my child] for a year and a half now.” “Our priorities have changed a lot . . . as far as our needs, we want a bigger place. That’s part of why I am going back to work, so that we can put some money back and get into a bigger place because our family has outgrown our apartment.” “These people wanted me to succeed as a parent and wanted my kids to succeed. I always felt that way with everybody I worked with.” “Everybody has really come together and pulled through for [my child].” “Everybody has really come together and pulled through for [my child].” “[Our provider] has done a really good job of seeking out the kind of services that we would need, and suggesting things that I wouldn’t even have thought of that we need.” “[Our provider] has done a really good job of seeking out the kind of services that we would need, and suggesting things that I wouldn’t even have thought of that we need.” “[The Part B transition meeting] was good. It was informative and I felt like they really listened to me” ) “There was a lack of [providers] to be able to come out and help, and we were supposed to get that going in September, and it took all the way until January to start services. That was a little bit of a stressor.” ) “There was a lack of [providers] to be able to come out and help, and we were supposed to get that going in September, and it took all the way until January to start services. Results Although each parent’s experience of transition over time was unique, our analysis resulted in three common themes: (a) change is constant, (b) positive relationships support changing needs and priorities, and (c) needing more. See Table 2 for information about each of the themes. Unless otherwise noted, the term “provider” refers to any early intervention professional families discussed. Quotes from transcripts are shared below to describe the findings. Brackets are used to protect parent, child, and/or provider identity or to provide contextual information. Transitions experienced over time. Parents had much to say about their child’s developmental transitions over time (e.g., “He progressed” and “I can see growth.”), even if it only felt like “subtle differences.” Parents attributed changes in their children’s development to aging and the services they had received. These developmental transitions were meaningful to parents: Note. ADHD = attention-deficit/hyperactivity disorder. Analysis That’s part of why I am going back to work, so that we can put some money back and get into a bigger place because our family has outgrown our apartment.” Theme 2: Positive Relationships Support Changing Needs and Priorities (N = 28, 100%) Appreciation of providers “These people wanted me to succeed as a parent and wanted my kids to succeed. I always felt that way with everybody I worked with.” Collaboration among providers “Everybody has really come together and pulled through for [my child].” Responsiveness to child’s or family’s needs and priorities “[Our provider] has done a really good job of seeking out the kind of services that we would need, and suggesting things that I wouldn’t even have thought of that we need.” Support during transition from Part C to Part B “[The Part B transition meeting] was good. It was informative and I felt like they really listened to me” Theme 3: Parents Need More (n = 27, 96.4%) Lack of adequate access to resources, services, or providers “There was a lack of [providers] to be able to come out and help, and we were supposed to get that going in September, and it took all the way until January to start services. That was a little bit of a stressor.” Lack of communication between providers and parent “A little bit better communication [would have helped]. . . . There wasn’t as much volunteered information as there could have been as far as what resources were available and stuff.” Parent did not feel adequately prepared by provider for transition “I was [my child’s] advocate and I was really the one that was calling the shots as far as, ‘This is what’s going on; this is what needs to happen.’ But at the end of the day, the resources weren’t provided to me to continue doing that to the best of my ability.” Support reduced once Part B or additional family-based services began after Part C “[Part B services are] not as personalized as the [Part C] services were. . . . You don’t have the kind of the centralized support. . . . It would have been nice to have like the same level that she received before.” Theme 2: Positive Relationships Support Changing Needs and Priorities All parents (N = 28, 100%) described ways in which they felt supported over time. Many parents shared their deep appreciation for their services overall (e.g., “It was a really positive experience.”) and their providers, in particular. Providers were described as collaborative and responsive as children’s or parents’ needs and priorities transitioned over time. Many parents also experienced transitions between providers, sometimes due to retirements, which services a child was receiving (e.g., ending private therapy services and beginning school-based therapy when transitioning from Part C to Part B), or because parents were not happy with a provider (e.g., “We’re currently in transition for physical therapy [PT]. I was butting heads with our PT.”). However, changes in providers most often occurred due to staff turnover. Some parents talked about having multiple providers within a few months that was unrelated to a child starting or ending services. Parents said, “[The clinic] went through three different therapists during the short time we were there,” and “One of the things that I found was a hard transition, was when [our first service coordinator] was replaced. They did n’t go directly to [our current service coordinator]. They had [a different service coordinator] come for a month.” Providers’ support and expertise. Parents described many positive aspects of providers’ interpersonal skills, saying things like, “They’re never gonna judge. . . . I really appre- ciate having people that are real, down to earth, that I can be honest with,” and that providers were “really kind, warm people, who are easy to welcome into your home.” More specifically, parents’ comments about appreciation focused on feeling genuinely cared for by their providers. Parents summarized this by saying: “They truly did care,” and “It felt like they were interested in her, like specifically as an individual versus just a kid with [a specific need].” Parents perceived that their providers were deeply committed to helping (e.g., “Everybody has been flexible and open, and never rigid in their thoughts. . . always willing to do some- thing different for her because it’s probably going to take something different for her.”). Parents discussed feeling cared for by providers who they viewed as responsive, which included being listened to, validated, and supported during times of shifting needs or priorities for the child, par- ent, or family. One parent said, “People on the team have always been very good about listening to our concerns . Theme 1: Change Is Constant She has seriously learned so much in this amount of time. And that makes me happy because I have had doctor after doctor tell me, ‘She’s not gonna walk, she’s not gonna talk, she’s not All parents (N = 28, 100%) described transitions they expe- rienced in their child’s skills, family life, or with services 7 Decker et al. “[My child] shouldn’t have long term issues”), others described this anticipation in more uncertain terms (e.g., “It could never be an issue or it could be a huge issue, but we won’t know until he starts talking.”). When discussing the future, another parent said, gonna eat, she’s not gonna be doing this.’ . . . It can’t be working out any better—you want to prove the doctors wrong, and that’s what she’s doing. Not only did parents discuss the ways in which their child had changed, but they also described many transitions their families had experienced. For example, parents described divorce, moving, and mental health crises. Some parents shared multiple changes that had affected their family sys- tem, which had in turn influenced their child. One parent shared, “His biological dad. . . moved away and it caused a lot more behavioral shifts [for my child].” Some of the changes parents mentioned were positive or supportive of the child or family (e.g., “I adopted [my child],” and “I don’t have to work anymore, so I can stay with him. That’s helped a lot.”). There is a good possibility that my daughter won’t ever be able to go to school. I mean that is real shit. And it is not fun. It’s scary and it makes you want to cry all the time, but if you did that then you wouldn’t be present and you wouldn’t be able to enjoy what she’s doing now. She is definitely the light of my life. That smile just slays me. Theme 3: Needing More Despite feeling supported in a variety of ways, most parents (n = 27, 96.4%) also described feeling like they needed more from services or providers. Parents most often described needing additional support, information, or access to services or providers as their child’s or family’s needs and priorities transitioned over time. The need for more information. Many parents reported feeling shocked, confused, or unprepared when their child transi- tioned out of or between programs (i.e., Part C, Part B, and/or family-based services after Part C) because proper informa- tion had not been provided to them. One parent described feeling like they had been “dropped off” by their provider when Part C services ended. Other families described similar feelings when their child no longer qualified for Part C. One parent shared, “I didn’t really know what I was supposed to be doing. [Our provider] just told us he should go to Head Start and I had to figure it out from there.” Some parents whose children exited Part C services and were not eligible for other services felt that their child was still in need of ser- vices, but only one child continued to receive therapy ser- vices after exiting Part C. Parents shared the following about transition out of Part C: “He no longer qualified even though he did really need it,” and “It was a little bit depressing because then he was not eligible to go to the [Part B] pre- school and he missed the cutoff for Head Start by two days.” Collaboration among providers. Parents also described col- laboration among their providers as an important part of how they were supported via positive relationships. Some aspects of collaboration overlapped with areas of apprecia- tion parents had mentioned, such as valuing providers who demonstrated expertise (e.g., “We all work really closely together. [They] are people I feel comfortable with, people that I trust, and I’ll listen to.”) or commitment to helping (e.g., “They’re learning from each other, they’re willing to try unconventional methods. They’re willing to work out- side the box, and that means a lot.”). Parents also described that collaboration included providers communicating and cooperating in ways that then led to better support for their child. Theme 3: Needing More Examples of this type of collaboration included pro- viders working as “a team,” frequently across disciplines (e.g., “Our PT is working really hard at some sensory stuff to be able to get [my child] to calm himself down before he gets to speech that day.”). Some parents felt that there was not enough information provided about various programs or services available, including information about eligibility criteria, processes, or differences between programs. For parents whose chil- dren had entered Part B services, some described feeling most in need of additional support by their providers during the qualification and transition process. Some parents reported receiving little to no information about this qualifi- cation and transition process. One parent described the ini- tial process of being told about Part B services as: “It was kind of like, ‘Here’s some brochures. Here’s some pam- phlets.’” Another family felt that they had been given false information about the Part B starting age: Cross-discipline collaboration was mentioned most often by parents who felt supported when their child had transitioned from Part C to Part B services. This type of teamwork helped parents begin to form positive relation- ships with new providers and feel reassured that everyone who would be supporting their child was involved; this included many individuals, such as their service coordina- tor, therapists, and transportation professionals. Parents felt most supported through this type of transition when they were informed and involved with the transition process (e.g., “It was a really easy transition. They knew what we were working on, what [my child] needed to work on, and so we had all that documented [in the IEP].”). A positive transition experience frequently included providers giving parents an ample amount of information regarding the pro- cess, paperwork, and changes to come (e.g., “It was very informational. . . . They went over everything”). When par- ents had a positive Part C to Part B transition, they regularly mentioned their child’s service coordinator. One parent said, “It was pretty seamless. We had all the meetings set up The superintendent led the [IEP] meeting. . . and explained that [my child] was not ready for preschool. Theme 2: Positive Relationships Support Changing Needs and Priorities . . and that’s very nice because then you feel like your con- cerns are validated, they’re working with the team, and they’re really listening to you.” Expected transitions in the future. As part of parents’ discus- sions of the transitions and changes they had already expe- rienced, each parent also discussed the future. Parents described their anticipation or uncertainty for what the future might hold for their child. For instance, parents regu- larly described some aspect of developmental progress their child had recently made and then quickly followed this by talking about the next skills their child was working toward (e.g., “I think she’s a lot more confident. But she’s still, I think, behind on developmental skills,” and “She’s defi- nitely making progress. Compared with peers the same age, I don’t think she’s caught up.”). Parents described anticipa- tion for these changes to continue as part of their child’s progression over time. Parents shared: “Goals might change within six months, because kids develop and grow,” and “We haven’t achieved all of our goals yet, but they take time. You know, development doesn’t just happen overnight because of these services. . . . But I think everything’s headed in the right direction.” While some parents seemed more certain of what they anticipated in the future (e.g., Parents viewed their providers as experts whose advice they valued, and this provided them with reassurance when transitions caused uncertainty. Most of parents’ comments about providers’ expertise focused on therapists. Parents shared: “I need their expertise, I need their. . . specialized training to meet his needs,” and “We’ve made huge strides Rural Special Education Quarterly 00(0) 8 00(0) a month before. . . . [Our service coordinator] kind of han- dled everything and she had everybody there.” in the last year and I am a firm believer that if we didn’t have these therapies none of this would be happening.” Parents described feeling reassured by their providers that they “were on the right path with everything.” One parent said, “[Our provider] made me feel like I wasn’t crazy! That there was reason to believe what I was saying and that it wasn’t all in my head.” Many parents were grateful when their providers had specialized expertise specific to their child’s needs, such as resources, referrals to other providers, and advice. Theme 2: Positive Relationships Support Changing Needs and Priorities Parents described receiving information about “helpful tips and tricks and stuff to do,” as well as opportu- nities to build their skills or understanding (e.g., “There are all kinds of classes and stuff for me to do. They are phenom- enal at giving me the opportunity to gain the knowledge I need.”). Theme 3: Needing More Well, “Oh, I’m on my way.” [I thought,] “Well, you could’ve sent that to me when our appointment actually was!” Parents whose children had transitioned into Part B shared that they wanted more communication and transparency about what was occurring during services. For example, “What happens at school I have really no idea about, because that’s not really communicated. . . . They’ll discuss how she’s doing toward meeting her goals or whatever,” and “This is her school notebook. This is the communica- tion from the school. It doesn’t really give me any informa- tion. . . . From this I don’t really know what she did. I don’t know what they’re working [on].” Parents whose children had transitioned into Part B shared that they wanted more communication and transparency about what was occurring during services. For example, “What happens at school I have really no idea about, because that’s not really communicated. . . . They’ll discuss how she’s doing toward meeting her goals or whatever,” and “This is her school notebook. This is the communica- tion from the school. It doesn’t really give me any informa- tion. . . . From this I don’t really know what she did. I don’t know what they’re working [on].” Many parents also commented on their need for better access to resources, services, or providers after frequently experiencing waitlists or a general lack of services avail- able. Some parents made broad statements about the lack of resources: “There’s just not the resources here that we need,” and “There isn’t really much for support around here. . . . There weren’t resources really.” Other parents talked about how they felt there was a lack of funding for programs and that led to a lack of resources being available as part of their services (e.g., “Funding! . . . The things we couldn’t get was because of funding.”). Parents mentioned having to start “paying for speech services” when their Part C agency stopped doing so, needing “adaptive equipment” for their child but being told it was not necessary, and not being able to receive respite care. Related to the reduction of support provided to parents via Part B and/or family-based services after Part C, some parents commented on how the goals outlined for these pro- grams were not in line with realistic needs of their child. Theme 3: Needing More One parent said, “I really don’t understand what the difference between this program and that program is. . . . Other parents also experienced confusion about their child exiting Part C and entering into the state’s family-based ser- vices that can continue past Part C, when children are eligi- ble. One parent said, “I really don’t understand what the difference between this program and that program is. . . . They didn’t really go into depth.” This lack of communica- tion about programs and services also was evident when one parent did not know any additional programs existed to serve children over the age of 3 years after they exited Part C. They didn’t really go into depth.” This lack of communica- tion about programs and services also was evident when one parent did not know any additional programs existed to serve children over the age of 3 years after they exited Part C. For parents whose children had exited Part C services and begun Part B and/or additional family-based services, many of them felt that the support they received had drasti- cally reduced. In general, parents’ comments indicated that their expectations for Part B and/or additional family-based services were not being met. Some parents stated these ser- vices were not as personalized as the services they received when in Part C. More specifically, parents stated that their providers made less frequent visits, they felt less knowl- edgeable about their child’s progress, and that services were less comprehensive. Parent said, “I really liked [Part C ser- vices]. They were way more involved,” and “[The services] are there if I need it, but it isn’t as interactive. . . . When she was younger it was way more involved. Now it’s just kind of, ‘We’re here if you need us.’” Some parents felt that the additional family-based services provided after Part C, in particular, were generally less supportive than Part C ser- vices had been. One parent said that these services were “definitely a step backwards more than helping,” while another receiving family-based services after Part C said, “Now that she turned three. . . they are not really helpful.” Well the biggest [challenge] was the communication. . . . Appointment changes, being late. Like the point in time would come and I hadn’t heard from her and then got worried that something had legitimately happened. Theme 3: Needing More The entire team said he was not ready for preschool, and I said, “Well, what do you mean he’s not ready for preschool?’ They said, ‘He’s not [age] four,” and I said, “But state and federal law says that the age is three and that’s why we are doing this meeting.” And they said, “Well, no. It’s [age] four.” Similarly, for parents whose children transitioned out of Part C and into the state’s additional family-based services, 9 Decker et al. some found the process to be unclear. One parent shared that their child was initially not considered for family-based services because they had entered into Part B services, though children can receive both services at the same time: maybe cookie cutter,” while another shared “[My child] is not anywhere near ready to work on [the goals set]. . . . [The provider did not ask], ‘Are you okay with it?’ But like kind of said, ‘[Your child] will be ready.’” I got like a letter after he transitioned [into Part B] saying, “Well, since you’ve indicated you don’t want services anymore, we’re taking you off our list.” So, I called and was like, “Whoa whoa whoa whoa! I never indicated I didn’t want services anymore.” And they were like, “Well, when you transition to Part B, we take you out.” The need for better communication. Parents also described a need for better communication. Some parents indicated that they experienced tension, misunderstandings, or felt unin- formed about their child’s services in general. One parent summed this up by saying, “You don’t know what you don’t know.” Another parent said, “There’s been surprises along the way like, ‘Oh, you should do this.’ ‘Really? I would have liked to know that five years ago!’” Parents felt that there should have been more regular information provided about the services that their children were receiving, espe- cially related to services that the child received while the parent was not present (e.g., therapy provided in clinics for Part C services, therapy in the school for Part B services, or Part B services more broadly). Some parents’ concerns about the lack of communication were broader, such as wanting better communication with their provider: Other parents also experienced confusion about their child exiting Part C and entering into the state’s family-based ser- vices that can continue past Part C, when children are eligi- ble. Theme 3: Needing More Some parents discussed how the process of creating goals, for Part B IEPs or IFSPs for the additional family-based services after exiting Part C, became less meaningful. Many of the parents felt that the providers created goals that were unrealistic for the child or the school, or that were not in line with parents’ priorities. One parent said, “The goals need to be rewritten. . . they were unattainable at the [local] school district level. . . . So I think some of those goals were 10 Rural Special Education Quarterly 00(0) Some parents also commented on how they felt the general lack of providers available led to their Part C service coor- dinators being overworked or underqualified. So while many parents commented on valuing the expertise of their providers—primarily therapists—they also felt that they were sometimes missing out on specialized knowledge, information, or advice, specifically from their Part C ser- vice coordinators. Parents said, “I think they are just very understaffed and too much is expected of one person,” and Some parents also commented on how they felt the general lack of providers available led to their Part C service coor- dinators being overworked or underqualified. So while many parents commented on valuing the expertise of their providers—primarily therapists—they also felt that they were sometimes missing out on specialized knowledge, information, or advice, specifically from their Part C ser- vice coordinators. Parents said, “I think they are just very understaffed and too much is expected of one person,” and Many parents commented on the lack of adequate access to providers and how this influenced the services they received. Parents perceived there were not as many provid- ers available as were needed, which led to challenges with availably and waitlists; this was true for families living in some of the most densely populated areas of the state and was even more challenging for those living in more rural areas. Parents shared that it was “hard to get an appoint- ment” with various types of providers, mostly pediatric therapists. Sometimes rurality led to challenges with find- ing pediatric therapists (e.g., “We’re in [a] rural [state]. . . . We have a doctor in [a town ~250 miles away], so they have no idea what we do or don’t have up here.”), especially ones who have specialized areas of expertise (e.g., “There isn’t another speech therapist that does feeding and swallowing. . Theme 3: Needing More . . It’s a problem in [our area]. . . . At this point we don’t have anyone”). One parent said, “They need to hire a speech therapist. . . . I just don’t think they can get anyone to come to [our small, rural town]. . . . I think the closest OT is [approximately ~115 miles away] and they’re booked.” Many parents also talked about very long waitlists, saying: “It took a while to get into [SLP services] because they have such huge caseloads and they didn’t have enough availabil- ity.” This challenge of waitlists was exacerbated for parents who would have had to travel long distances to receive those services. Parents said: “It was over a six month wait to get into [a clinic ~350 miles away for] pediatric speech and OT services,” and “I called in October and they said, ‘Well, the first thing we have is in January, but we could put you on our cancel list.’ Well that’s fine and dandy but we’re [~220 miles away].” [Our service coordinator] is definitely spread very thin. She’s getting done what’s needed to get done and what is expected to get done. . . . Their job is to bring different ways and ideas and things, and I don’t think she has time to think of different ways and ideas. Parents described their concerns regarding not always get- ting the personalized support and help they needed. One parent said, “I think I expected them to be more therapeutic than they are. So, more of the technical training piece, like helping parents learn new strategies with their kids and I personally haven’t felt like I’ve received that.” Some par- ents commented on the lack of specialized education or training of their service coordinators, and how this may contribute to feeling that they needed more support than they were receiving. One parent summarized this: I think that’s probably my main concern with early intervention in [our state], is that we’re not hiring professionals who have degrees in early intervention, early education, or special ed, because we’re paying them poorly. . . . So, there isn’t a huge depth to the services that they’re providing. Given the challenges with the lack of availability of pro- viders, parents felt that this also influenced the frequency and location of services provided. Parents described receiv- ing some services weekly and other services monthly. Theme 3: Needing More Some parents commented on how they felt that there was a need for their child to receive “more frequent” services, and that their children were not “getting worked with as much as [they] should.” One parent said, “My expectations were too high. I thought they’d be here every week, but they’re here every month.” While some of these services were provided in their homes, many parents were required to travel long distances for their child’s therapy services (e.g., “We some- times have to travel four and a half hours or three hours.”). A few parents commented on their desire to have in-home therapy services, but that it was not realistic given the lack of availability of providers more generally. One parent said, Discussion The purpose of this study was to gain an understanding of rural parents’ experiences of transitions over time when they have a young child with exceptionalities. The findings of this study included three common themes in parents’ interviews. First, parents reported many transitions they experienced related to their child, their family, and/or aspects of their services; they viewed these transitions as a constant part of parenting their child, which included antici- pation or uncertainty about future changes or needs their child may experience. Second, parents described how posi- tive relationships, including those with their providers or among their providers, helped provide necessary support during periods of transition. Finally, though parents described many ways in which they had been supported, they also mentioned many ways in which they needed more support. Parents reported needing more communication and support from their providers to feel informed and prepared for transitions, especially transitions out of Part C services that had felt more personalized and inclusive of the family. I want [our state] to do better. I don’t know what the answer is as far as recruiting more therapists or paying them better, but I wish we could get in home services. . . because it’s the most natural environment for my baby to learn, and it’s easiest for our family to have a therapist come here but there aren’t any. 11 Decker et al. In addition, parents needed overall better access to services and highly trained providers. which is recommended to increase positive outcomes in times of transition (DEC, 2014; Doudna et al., 2015; Malone & Gallagher, 2009). It is encouraging that parents in this study identified numerous examples of these types of sup- portive practices. Parents described feeling supported not only as they transitioned through everyday informal changes, such as moving or welcoming a new sibling, but also as they experienced through more formal changes, such as getting a new provider or entering special education services from home-based services. Many parents described providers who listened, validated their experiences, and really took an interest in helping, which supported parents during times of uncertainty and/or change. Feeling that their provider cared for, and really knew, their child and family was important to parents. Cultivating caring relationships is an evidence-based recommended practice (Murphy et al., 2013). Based on parents’ reports, this was a strength of pro- viders in this study. Discussion One of the most salient messages communicated from parents in this study was that change is ubiquitous as they move across the timeframe of their child’s early years, which is consistent with and extends existing family sys- tems research on early developmental changes (Britto & Pérez-Escamilla, 2013; Pang, 2010). In this study, we inter- acted with parents during a period of many and significant transitions—such as becoming parents for the first time, expanding their family, and learning about relevant systems and support networks. Many of these transitions are experi- enced by all families, including uncertainty or joy experi- enced in their child’s early years (Bush & Price, 2020; McGoldrick et al., 2011). However, families of children with exceptionalities, especially those in early intervention services, face far more transitions and changes in early development years; in sum, the challenges and uncertainty of transitions are amplified for these families (Douglas et al., 2022; Gothberg et al., 2017; Hebbeler & Spiker, 2016; Malone & Gallagher, 2009; Podvey et al., 2010; Rous & Hallam, 2012; Waters & Friesen, 2019). In this study, par- ents described a variety of experiences similar to all fami- lies of young children, but they also described unique experiences that were intensified given their child’s addi- tional needs. It is not that parents identified change as bad per se, but rather, the uncertainty and disempowerment that often came along with some transitions were difficult for some families to weather. Strong relationships are a necessary foundation, but insufficient on their own for providing high-quality early intervention services (Foster et al., 2020). The current study demonstrated that regardless of strong provider–family relationships, parents continued to identify significant bar- riers to supportive services that ultimately undermined chil- dren’s, parents’, and/or families’ well-being. Similar to others’ findings (Douglas et al., 2021; Podvey et al., 2013; Rous et al., 2009; Waters & Friesen, 2019), some parents needed more from their providers, including more commu- nication, expertise, access to services, and information related to transition into or out of services. Most notable was parents feeling disempowered and uninformed during times of formal transitions between services. It is notable that much of what parents identified, both in the realm of change and in what they identified as “good” practice, was operating at the child level. Discussion For instance, while parents described change as constant, much of the change they referenced happened as part of everyday rhythms of family life (e.g., getting a new job and moving), but most change they attributed to the early intervention process itself happened to the child (e.g., the child learning new skills and being able to talk more). It is significant that parents did not talk about changes within themselves, their parenting, or family processes or well-being as a result of early interven- tion services, especially as this contrasts with the tenets of family-centered practice and parent empowerment in early intervention (DEC, 2014). It also may contribute to the sense of loss some families described as their child moved into Part B services out of the home setting—these families may have internalized that it is the provider working with the child, rather than the parents themselves, who are the most important drivers of development for their children. The importance of the context of rurality for these fami- lies cannot be overstated—the large, rural nature of a state may have direct implications for the types of providers available for children and their families, how many or how often providers are available, and the locations or distance required to travel for services. Research on rural parents’ perceptions of early intervention services is limited yet, as demonstrated in this and other studies, families in rural areas may face more difficulties in accessing and receiving services (Cummings et al., 2017; Decker et al., 2020, 2021; Elpers et al., 2016; Hallam et al., 2009; Mann & Williams, 2011; Singh et al., 2019). In this study, parents validated the challenges of living in a rural state and were willing to travel long distances to accept any services available, pri- oritizing their child’s needs over the stresses caused by these barriers. While certain approaches like telehealth might mitigate the need for travel in rural states and thus increase availability of services (Rooks-Ellis et al., 2020), these approaches may introduce new challenges or may not be feasible for all families. For instance, some families, especially those in the most rural areas, may not be able to afford or have access to high-speed Internet. Regardless of how parents in this study viewed their own role in early intervention services, it was clear they valued their relationships with providers. Limitations and Future Directions study also indicated that these supportive relationships are a necessary but insufficient ingredient as they navigate through important life transitions. Based on recommended practices that are intended to guide services for children with exceptionalities, the work of others, and our findings, there should be concerted efforts to: This is a qualitative study based on a small sample from one state; this limits the ability to generalize the findings to other rural areas or states. A further limitation of this study is that most parents and children were Caucasian. While this is not reflective of a more diverse national population, it does reflect the demographics of the state in which these data were collected and other large, rural states. Moreover, although this study utilized a sample from each of the regional Part C agencies in the state, the experiences of the parents included may not accurately represent all parents’ experiences of transition across the state. These findings may not represent parents’ experiences in other rural areas or in other states and regions. Furthermore, this study relied on parent’s reports of their perception on their child’s ser- vices and the family’s experiences, and as such, the data may not fully represent the complete nature of the early intervention services, such as quantitative child or family outcomes or providers’ perspectives. However, understand- ing parents’ experiences is critical to informing family- centered practice and, as a broader concept of transitions over time, it is not well studied in the current literature. One important strength of this study is that families were able to comment on the changing nature of their services, as many families had experienced months and years of services, rather than provide data relative to one point in time. In addition, while the qualitative nature of this study leads to natural limitations in generalizations, it did allow for a deep understanding of parents’ experiences, informed by the voices of parents themselves. Building on this study, future research should investigate how providers view transitions with children and families, how they are trained to adminis- ter transition support, and the barriers they may face. The importance of rurality in these parents’ experiences should also be explored further, especially as the rural context relates to access, communication, and transitions within families and between providers and systems. Discussion Similar to the findings of other studies, parents felt as if communication and collabo- ration were essential in bridging the gap between important transitions (Douglas et al., 2022; Waters & Friesen, 2019), Rural Special Education Quarterly 00(0) 12 Limitations and Future Directions •• Address the specific needs related to the context of rurality, specifically regarding access to providers, reducing waitlists, and enhancing collaboration and communication between all members of the early intervention team, especially as services change and evolve (Decker et al., 2020, 2021; Mann & Williams, 2011; Singh et al., 2019). •• Recruit, hire, and retain providers with specialized training (and/or provide that training), specifically aimed at how to work with families throughout tran- sitions (Douglas et al., 2022; Murphy et al., 2013; Podvey et al., 2013). DEC (2014) identifies numer- ous recommended practices to this end (e.g., see rec- ommended practices TR1 and TR2). •• Provide training and education of providers, with the specific aim of bolstering the use of family-centered practices and family capacity building (e.g., see DEC, 2014 recommended practices F1-F10; Doudna et al., 2015; Pang, 2010; Rous & Hallam, 2012). •• Empower families and reduce the uncertainty that accompanies transitions, by better preparing them for the qualification process/entry into Part B and other services, and/or the end of Part C or other ser- vices (Douglas et al., 2022; Murphy et al., 2013; Podvey et al., 2013). Although change is an expected part of life, providers should expect that families are not always prepared for these changes. Providers have a special role to play in mitigating stress and uncertainty, and maximizing success and empower- ment, during these times. This study offers insight regarding early intervention practices that may be working well as services unfold over time—communication, collaboration, and relationships— and also gives us direction into what may need to be tackled head-on—empowerment, family-centered services, and access—to best support families and children. Conclusions and Practical Implications This study adds to the limited existing research related to parents’ experiences with transitions over time, which includes but is not limited to exiting Part C early interven- tion services and/or entering into Part B special education services. Change is a unifying aspect of parenthood, but this study adds to our understanding of some of the possible unique needs of parents of children with exceptionalities as they experience transitions over time. This study also pro- vides insight into the additional needs that families living in rural areas may experience. Parents’ relationships with pro- viders, a relative strength in our sample, are critical for par- ents to feel supported throughout the many changes they and their children experience. However, parents in this Acknowledgment We wish to thank the parents who participated in this study, as well as Montana’s Part C agencies that helped with recruitment for this study. References Braun, V., & Clarke, V. (2006). Using thematic analysis in psy- chology. Qualitative Research in Psychology, 3(2), 77–101. https://doi.org/10.1191/1478088706qp063oa Britto, P. R., & Pérez-Escamilla, R. (2013). No second chances? Early critical periods in human development. Social Science & Medicine, 97, 238–240. https://doi.org/10.1016/j.socs- cimed.2013.09.001 Hallam, R. A., Rous, B., Grove, J., & LoBianco, T. (2009). Level and intensity of early intervention services for infants and tod- dlers with disabilities. Journal of Early Intervention, 31(2), 179–196. https://doi.org/10.1177/1053815109331914 Bush, K. 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Declaration of Conflicting Interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. 13 Decker et al. Douglas, S. N., Meadan, H., & Schultheiss, H. (2022). A meta- synthesis of caregivers’ experiences transitioning from early intervention to early childhood special education. Early Childhood Education Journal, 50, 371–383. https://doi. org/10.1007/s10643-021-01165-6 Funding The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: Research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number P20GM103474. This research was also supported by funding from Montana’s Department of Public Health & Human Services, and Montana State University including (a) the College of Education, Health and Human Development; (b) an Outreach & Engagement grant; and (c) a Scholarship & Creativity grant. The content is solely the responsibility of the authors and does not necessarily repre- sent the official views of the National Institutes of Health or any other funders. Elpers, J., Lester, C., Shinn, J. B., & Bush, M. L. (2016). Rural family perspectives and experiences with early infant hear- ing detection and intervention: A qualitative study. Journal of Community Health, 41(2), 226–233. https://doi.org/10.1007/ s10900-015-0086-1 Foster, T. D., Decker, K. B., Vaterlaus, J. M., & Belleville, A. (2020). How early intervention providers describe family- centered practice: A collective broadening of the definition. 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https://openalex.org/W2361718120	https://jelectrochem.xmu.edu.cn/cgi/viewcontent.cgi?article=1557&context=journal	Chinese	null	Electrochemical Lithium Insertion of Nanosized TiO_2	Deleted Journal	2,004	cc-by	1,319	Journal of Electrochemistry Journal of Electrochemistry Journal of Electrochemistry Journal of Electrochemistry Ying LAN Jian-wen LIU Xue-ping GAO Xing-di ZHOU Jin-qiu QU Feng WU De-ying SONG Recommended Citation Recommended Citation Journal of Electrochemistry Journal of Electrochemistry Volume 10 Issue 2 Ying LAN, Jian-wen LIU, Xue-ping GAO, Xing-di ZHOU, Jin-qiu QU, Feng WU, De-ying SONG. Electrochemical Lithium Insertion of Nanosized TiO_2[J]. Journal of Electrochemistry, 2004 , 10(2): 133 136 Electrochemical Lithium Insertion of Nanosized TiO_2 Electrochemical Lithium Insertion of Nanosized TiO_2 Ying LAN Jian-wen LIU Xue-ping GAO Xing-di ZHOU Jin-qiu QU Feng WU De-ying SONG Recommended Citation Recommended Citation Ying LAN, Jian-wen LIU, Xue-ping GAO, Xing-di ZHOU, Jin-qiu QU, Feng WU, De-ying SONG. Electrochemical Lithium Insertion of Nanosized TiO_2[J]. Journal of Electrochemistry, 2004 , 10(2): 133-136. DOI: 10.61558/2993-074X.1557 Available at: https://jelectrochem.xmu.edu.cn/journal/vol10/iss2/3 Available at: https://jelectrochem.xmu.edu.cn/journal/vol10/iss2/3 This Article is brought to you for free and open access by Journal of Electrochemistry. It has been accepted for inclusion in Journal of Electrochemistry by an authorized editor of Journal of Electrochemistry. 电化学第10 卷　第2 期 2004 年5 月 Vol. 10 　No. 2 May 2004 Vol. 10 　No. 2 May 2004 电化学 EL ECTROCHEMISTRY 收稿日期:2003212226 ,修订日期:2004201209 3 通讯联系人, Tel : (86222) 23502604 ,E2mail : xpgao @nankai. edu. cn 国家重点基础研究发展规划项目(2002CB211800) ,国家自然科学基金(90206043) ,教育部科学技术研究重点项目(03047) ,高等学校博士学科点专项科研基金(20020055007) 资助 2. 1 　样品表征图1 为质子钛酸盐纳米管(a) 和500 ℃下煅烧后得到的纳米柱TEM 照片. 如图1 (a) 所示,水热制备的产物具有纳米管结构,该纳米管分散性好、管径(约10～15 nm) 均匀. 另经 TGA 证明,该纳米管含有大量水分,文献[7 ]称此为质子钛酸盐纳米管. 而煅烧后,该纳米管逐渐转变为纳米柱(图1b) ,直径约15～20 nm ,长度200～400 nm ,但无大量团聚颗粒形成. 图2 示出经500 ℃下煅烧后的纳米柱样品的X - 射线衍射图谱. 对照J CPDS 标准数据可知,该样品具有标准的锐钛矿相四方结构. 图1 　纳米管(a) 和纳米柱(b) 的TEM 图 Fig. 1 　TEM images of the nanotubes (a) and nanorods (b) 　图2 　TiO2纳米柱的X2射线衍射图谱　Fig. 2 　X2ray diffraction pattern of the TiO2 nanorods 2. 2 　样品的充放电性能图3 给出TiO2纳米柱电极的第1 周充放电曲线,其充放电电位限定在2. 5～1. 0V 之间,充放电电流为36mA/ g. 如图所示,该TiO2纳米柱电极在1. 75 V 左右开始出现一段较长的放电平台, 与文献[8 ]报道的大体相近,相应的初始放电容量可达到232 mAh/ g. 图3 中放电后期平台出现倾斜,这与一维纳米柱的表面拥有大量缺陷、电化学嵌锂过程存在多种嵌入位置相关. 图3 中,充电过程于1. 90 V 左右处出现一段较宽的充电平台,首次充电容量达182 mAh/ g ,充放电效率达78 %. 实验表明,第2 周放电容量可达198 mAh/ g(相当图1 　纳米管(a) 和纳米柱(b) 的TEM 图 Fig. 1 　TEM images of the nanotubes (a) and nanorods (b) 图1 　纳米管(a) 和纳米柱(b) 的TEM 图 Fig. 1 　TEM images of the nanotubes (a) and nanorods (b) 纳米TiO2的电化学嵌锂研究兰　英,刘建文,高学平3 ,周杏第,曲金秋,吴　锋,宋德瑛 (南开大学新能源材料化学研究所,天津300071) 摘要: 　应用苛性钠水热法制备粒度均匀、分散性好、质子钛酸盐纳米管(直径约10～15 nm) . 经加热烧结脱水后,该纳米管逐渐转变成具有锐钛矿相结构的纳米柱(直径约15～20 nm) . 初步研究表明, 这种具有锐钛矿相结构的纳米柱,其电化学可逆嵌/ 脱锂容量较高,但循环稳定性还有待改进提高. 二氧化钛可广泛应用于光催化、工业催化、染料、光敏化电池以及锂离子二次电池材料等, 是目前的研究热点. 一维二氧化钛纳米管于1998 年由Kasuga 等率先制备[1 ] ,近年来采用水热反应法、模板法和电化学沉积法等已经制备出各种不同形态的TiO2纳米管、纳米柱和纳米丝[2～4 ] ,并在光催化方面表现出优越的性能[5 ]. 另一方面,由于一维纳米材料的特殊结构,而以TiO2作为锂离子电极材料也表现出许多优势,例如,TiO2在锂离子嵌入/ 脱出过程中的体积膨胀小(3 %) [6 ] ,嵌入/ 脱出深度小、行程短,加之循环稳定性好,放电平台电位高(约为1. 7 V) ,因而是用于锂离子电池的一种理想基质材料. 但目前有关一维纳米结构二氧化钛的电化学嵌锂性能的研究尚未见报道. 本文应用苛性钠水热法制备了质子钛酸盐的纳米管,经过加热烧结脱水后,该纳米管逐渐转变成具有锐钛矿相结构的纳米柱. 初步研究了这种具有锐钛矿相结构纳米柱的电化学嵌锂性能. 实　验主要原料为金红石相二氧化钛(天津试剂三厂) 和氢氧化钠(天津医药公司) . 二氧化钛纳米管制备按文献[1 ]报道方法:将TiO2与NaOH 混合于150 ℃恒温水热反应2 d ,生成的白色沉淀物经洗涤至p H = 8～9 ,再于100 ℃下烘干, 500 ℃下充分脱水. 采用X2射线衍射 (Rigaku , D/ max - 2500) 和透射电镜(FEI , Tecnai 20) 分析表征纳米管和纳米柱的微结构. 工作电极是由活性物质、导电材料(乙炔黑) 和粘接剂(聚四氟乙烯) 按85∶10∶5 混合制收稿日期:2003212226 ,修订日期:2004201209 3 通讯联系人, Tel : (86222) 23502604 ,E2mail : xpgao @nankai. edu. cn 国家重点基础研究发展规划项目(2002CB211800) ,国家自然科学基金(90206043) ,教育部科学技术研究重点项目(03047) ,高等学校博士学科点专项科研基金(20020055007) 资助收稿日期:2003212226 ,修订日期:2004201209 电　化　学 · 4 3 1 · 2004 年 2004 年成,对电极和参比电极均为锂金属电极. 电解液为1 mol· L - 1LiPF6 (溶剂由60 %的碳酸乙烯酯、30 %碳酸丙烯酯和10 %的碳酸二甲酯混合组成) . 模拟电池制备及处理均在手套箱中进行. 电池容量和循环寿命测试采用Land2CT 2001A 电极测试仪于常温下进行,充放电电流36 mA/ g ,起止电位2. 5～1. 0 V (vs. Li + / Li) . 2 　结果与讨论 2. 1 　样品表征 2 　结果与讨论 2 1 　样品表征 2. 2 　样品的充放电性能图3 给出TiO2纳米柱电极的第1 周充放电曲线,其充放电电位限定在2. 5～1. 0V 之间,充放电电流为36mA/ g. 如图所示,该TiO2纳米柱电极在1. 75 V 左右开始出现一段较长的放电平台, 与文献[8 ]报道的大体相近,相应的初始放电容量可达到232 mAh/ g. 图3 中放电后期平台出现倾斜,这与一维纳米柱的表面拥有大量缺陷、电化学嵌锂过程存在多种嵌入位置相关. 图3 中,充电过程于1. 90 V 左右处出现一段较宽的充电平台,首次充电容量达182 mAh/ g ,充放电效率达78 %. 实验表明,第2 周放电容量可达198 mAh/ g(相当图2 　TiO2纳米柱的X2射线衍射图谱　Fig. 2 　X2ray diffraction pattern of the TiO2 nanorods 兰　英等:纳米TiO2的电化学嵌锂研究第2 期第2 期 · 5 3 1 · · 5 3 1 · 于嵌锂系数0. 62) ,这比此前报道的微米级的锐钛矿和金红石相二氧化钛颗粒的容量高(161 mAh/ g ,相当于嵌锂系数0. 5) [9 ]. 于嵌锂系数0. 62) ,这比此前报道的微米级的锐钛矿和金红石相二氧化钛颗粒的容量高(161 mAh/ g ,相当于嵌锂系数0. 5) [9 ]. 图4 给出TiO2纳米柱电极的充放电循环性能. 如图可见,首次放电存在不可逆容量(50 mAh/ g) ,与微米级的锐钛矿TiO2的嵌锂特征不同[8 ]. 随着充放电循环次数的增加,相应的充放电效率也随之提高,最终可接近于100 %. 但与微米级的锐钛矿TiO2相比,纳米柱电极循环衰减仍然较快. 至第20 周,其放电容量仅保持152 mAh/ g ,较之第2 周,约下降23 %. 总之,尽管该TiO2纳米材料的电化学嵌锂容量较高,但循环稳定性还有待改进提高. 放电效率也随之提高,最终可接近于100 %. 但与微米级的锐钛矿TiO2相比,纳米柱电极循环衰减仍然较快. 至第20 周,其放电容量仅保持152 mAh/ g ,较之第2 周,约下降23 %. 总之,尽管该TiO2纳米材料的电化学嵌锂容量较高,但循环稳定性还有待改进提高. 3 　结　论应用水热法制备了粒度均匀、分散性好、质子钛酸盐纳米管. 该纳米管经加热烧结脱水后, 逐渐转变成具有锐钛矿相结构的纳米柱. 与微米级颗粒相比,这种锐钛矿相纳米柱的电化学可逆嵌/ 脱锂容量高,但其循环稳定性还有待改进提高. Electrochemical Lithium Insertion of Nanosized TiO2 LAN Ying , L IU Jian2wen , GAO Xue2ping 3 , ZHOU Xing2di , QU Jin2qiu , WU Feng , SON G De2ying ( Institute of New Energy M aterial Chemistry , N ankai U niversity , Tianjin 300071 , China) Abstract : The protonic titanate nanotubes with the diameter of 10～15 nm were synthesized via hydrothermal method in NaOH solution and the calcined nanotubes at 500 ℃were transferred 管该纳米材料的电化学嵌锂容量较高但循环稳定性还有待改进提高 3 　结　论应用水热法制备了粒度均匀、分散性好、质子钛酸盐纳米管. 该纳米管经加热烧结脱水后逐渐转变成具有锐钛矿相结构的纳米柱. 与微米级颗粒相比,这种锐钛矿相纳米柱的电化学可逆嵌/ 脱锂容量高,但其循环稳定性还有待改进提高. 管该纳米材料的电化学嵌锂容量较高但循结　论应用水热法制备了粒度均匀、分散性好、质子钛酸盐纳米管. 该纳米管经加热烧结脱水后, 逐渐转变成具有锐钛矿相结构的纳米柱. 与微米级颗粒相比,这种锐钛矿相纳米柱的电化学可逆嵌/ 脱锂容量高,但其循环稳定性还有待改进提高. Electrochemical Lithium Insertion of Nanosized TiO2 LAN Ying , L IU Jian2wen , GAO Xue2ping 3 , ZHOU Xing2di , QU Jin2qiu , WU Feng , SON G De2ying ( Institute of New Energy M aterial Chemistry , N ankai U niversity , Tianjin 300071 , China) Abstract : The protonic titanate nanotubes with the diameter of 10～15 nm were synthesized via hydrothermal method in NaOH solution and the calcined nanotubes at 500 ℃were transferred 电　化　学 2004 年 · 6 3 1 2004 年 to anatase nanorods with the diameter of 12～20 nm due to dehydration. The electrochemical be2 havior of the anatase nanorods as lithium insertion materials was examined. The results showed that the nanorod electrode had the high initial discharge capacity of 232 mAh/ g and second re2 versible capacity of 198 mAh/ g. The capacity of nanorod electrode maintained 152mAh/ g after 20 cycles. Key words : TiO2 , Nanotubes , Nanorods , Lithium2ion battery Key words : TiO2 , Nanotubes , Nanorods , Lithium2ion battery References : [1 ] 　Kasuga T , Hiramatsu M , Hoson A , et al. Formation of titanium oxide nanotube[J ]. Langmuir , 1998 , 14 : 3160. [2 ] 　Wang Y Q , Hu G Q , Duan X F , et al. Microstructure and formation mechanism of titanium dioxide nan2 otubes[J ]. Chem. Phys. Lett. , 2002 , 365 : 427. [2 ] 　Wang Y Q , Hu G Q , Duan X F , et al. Microstructure and formation mechanism of titanium dioxide nan2 otubes[J ]. Chem. Phys. Lett. , 2002 , 365 : 427. [3 ] 　Yuan Z Y, Colomer J F , Su B L. Titanium oxide nanoribbons[J ]. Chem. Phys. Lett. ,2002 , 363 : 362. [3 ] 　Yuan Z Y, Colomer J F , Su B L. Titanium oxide nanoribbons[J ]. Chem. Phys. Lett. ,2002 , 363 : 362. [4 ] 　Zhang X Y, Yao B D , Zhao L X , et al. Electrochemical fabrication of single2crystalline anatase TiO2 nanowire arrays[J ]. J . Electrochem. Soc. , 2001 , 148(7) : G398. [4 ] 　Zhang X Y, Yao B D , Zhao L X , et al. Electrochemical fabrication of single2crystalline anatase TiO2 nanowire arrays[J ]. J . Electrochem. Soc. , 2001 , 148(7) : G398. [5 ] 　Zhang S L , Zhou J F , Zhang ZJ , et al. Morphological structure and physicochemical properties of TiO2nan2 otube[J ]. Chin. Sci. Bull. , 2000 , 45 :1 533. [5 ] 　Zhang S L , Zhou J F , Zhang ZJ , et al. Morphological structure and physicochemical properties of TiO2nan2 otube[J ]. Chin. Sci. Bull. , 2000 , 45 :1 533. [6 ] 　Wagemaker M , Kearley G J ,Well A A van , et al. Multiple Li positions inside oxygen octahedra in lithiated TiO2 anatase[J ]. J . Am. Chem. Soc. ,2003 , 125 : 840. [7 ] 　Chen Q , Du G H , Zhang S , Peng L M. The structure of trititanate nanotubes[J ]. Acta Cryst. B ,2002 , 58 : 587. [8 ] 　Huang S Y, Kavan L ,Exnar I , et al. Rocking chair lithium battery based on nanocrystalline TiO2 (anatase) [J ]. J . Electrochem. Soc. , 1995 , 142(9) : L142. [9 ] 　Gover R K B , Tolchard J R , Tukamoto H , et al. References : Investigation of ramsdellite titanates as possible new nega2 tive electrode materials for Li batteries[J ]. J . Electrochem. Soc. , 1999 , 146(12) : 4 348.
https://openalex.org/W1974822865	https://zenodo.org/records/2413964/files/article.pdf	English	null	SUPERHEATERS. (INCLUDING APPENDIX).	Minutes of proceedings	1,908	public-domain	6,559	(Paper No. 3 71 9.) (‘ Superheaters.” (Paper No. 3 71 9.) (‘ Superheaters.” (Paper No. 3 71 9.) (‘ Superheaters.” (Paper No. 3 71 9.) 365 365 Papers.] PREECE ON RUPBI1IIEATEI1S. By FRANK HUGH PREECE, Assoc. M. Inst. C.E. IT is only within late years that the use of superheated steam has been recognized as commercially advantageous, although its properties were known many years ago to those interested in the development of steam-engines. Its application was restricted for various reasons, the chief one being the bad effect of the high temperature on the lubricants then in use ; these difficulties have now, to a large extent, been overcome, and a superheater in one form or another is to be found in most modern installations. The factors which are adverse to xte:tm-economy are variollh, but chief among them are initial condensation and leakage past the valves, and of these there is much doubt as to which causes the greater loss ; it is impossible to deny, however, that the use of superheated steam has diminished both to a large extent. Prom early days the effect of condensation was considered to be the greatest factor of the so-called “missing quantity,” and various methods have been brought into use to prevent the cooling of the cylinder-walls, notably steam-jacketing m c l the use of non- conducting compositions. These improvements were combined with the use of higher initial pressures, a better adjustment of the number of expansions-thus obtaining more useful work from each pound of steam,-and later m-ith the use of superheated steam, all these having reduced the steam required by first-class engines to an extent which would formerly have been deemed impossible. p First with regard to the view that the “missing quantity” is @wed by the initial condensation in the cylinders, asld i s due to the Downloaded by [ University of Sussex] on [17/09/16]. Copyright © ICE Publishing, all rights reserved. 266 PREECE ON SUPERHEATERS. [Selected cooling of the cylinder-walls below the temperature of the entering steam : during steam admission this cooling will cause a con- densation of a portion of the steam, and owing to re-evaporation the pressure will rise above that of the theoretical curve of expan- sion. The use of superheat will raise the temperature of the walls so that the maximum and minimum variation will be very slight, and therefore will cause the expansion of the steam more closely to follow the theoretical law. By the adoption of a suitable degree of superheat the cylinder is always at a temperature exceeding that at which steam will condense, and this will account for the important reduction in the amount of loss by condensation. This is very clearly shown by comparing the din.grams obtained from an engine, first with saturated steam, and then with superheated steam, when the improvement in the ‘‘ dryness fraction ” and the consequent gain in economy is at once apparent. Next, taking the view that the greater part of the “ missing quantity ’’ is caused by leakage past the valve from the steam-inlet to the exhaust without entry to the cylinder ; its advocates point to experiments of Messrs. Callendar and Nicolson-which show that when the valve-faces were heated by artificial means the losses were greatly diminished-and contend that the same effect occurs with superheated steam. In the discussion on a Paper at the Institution of Mechanical Engineers in July, 1904,l Mr. Longridge stated that in his opinion superheating played an important part in reducing leakage. He went on to say that the velocity of flow through a small orifice, such as might be supposed to exist between a valve and face, depended partly on the viscosity of the fluid and partly on the square root of the difference in pressure on the two sides of the orifice. With fluids of small viscosity, such as steam and water at high pressure, the velocity would depend almost entirely on the difference of the pressure, and would be therefore practically equal under a given pressure, whether the fluid were steam or water. Downloaded by [ University of Sussex] on [17/09/16]. Copyright © ICE Publishing, all rights reserved. ’ Proceedings of the Institution of Mechanical Engineers, 1904, p. 1018. Downloaded by [ University of Sussex] on [17/09/16]. Copyright © ICE Publishing, all rights reserved. But as the density of water ;was so much greater than that of steam, it was easily seen that the weight of water leak- ing past a valve under a given pressure would be 500 or 600 times the weight of steam, and probably in this case one very important effect of superheating the steam in the receiver was to reduce the leakage past the exhaust valve of the low-pressure cylinder. This is further exemplified by the fact that the gain due to supe ’ Proceedings of the Institution of Mechanical Engineers, 1904, p. 1018. 267 PREECE ON SUPERHEATERS. Papers.] heat, as the superheat increases in amount, is less with valves which rest on their seats, such as the drop-valve type, than in those which have rubbing surfaces, where a certain amount of clearance is necessary. The curves in Fig. 1 represent the percentage gain in steam economy as the degree of superheat is increased ; the gain tending to fall off as the superheat rises above 150" F. Therefore, taking into consideration the difficulties of efficient lubrication, expansion, and special precaution in regard to valves, together with the increase capital expenditure incurred by laying down plant suitable for obtaining and using the higher degree of superheat, it is questionable whether it is worth while to go above 200" F. ; and the Author is Fig. 1. S U P E R H E A T : OF of opinion, as the result of his observations, that the most efficient point will be between 150" F. and 200" F. of opinion, as the result of his observations, that the most efficient point will be between 150" F. and 200" F. of opinion, as the result of his observations, that the most efficient point will be between 150" F. and 200" F. I n proceeding with the design of a superheater the essential points to consider are : First, what are the total heat-units required to obtain the necessary superheat ?1 And secondly, what, under the conditions of working, will be the transmission of heat from the flue gases to the steam ? 1 The high-speed engine-curve is from tests by Messrs. Davey Paxman and Co. the drop-vdve tests from tests on engines fitted with the Lentz drop-velve gear. Downloaded by [ University of Sussex] on [17/09/16]. Copyright © ICE Publishing, all rights reserved. The fist point depends on the specific heat of the superheated steam, and it has been generally stated, from the experiments of Regnault, that the specific heat of steam at constant pressure may be taken at 0.48, but from later experiments it has been found that this value changes considerably according to the degree of 1 The high-speed engine-curve is from tests by Messrs. Davey Paxman and Co. the drop-vdve tests from tests on engines fitted with the Lentz drop-velve gear. Downloaded by [ University of Sussex] on [17/09/16]. Copyright © ICE Publishing, all rights reserved. Downloaded by [ University of Sussex] on [17/09/16]. Copyright © ICE Publishing, all rights reserved. Downloaded by [ University of Sussex] on [17/09/16]. Copyright © ICE Publishing, all rights reserved. 268 PIlEECE ON SUPERIIBATERS. [Selectfed [Selectfed superheat and the pressure of the steam. One of the latest series of experiments published are those of Mollier,' who has devised tables and diagrams for finding the specific heat :It nny required pressure or temperature. superheat and the pressure of the steam. One of the latest series of experiments published are those of Mollier,' who has devised tables and diagrams for finding the specific heat :It nny required pressure or temperature. Table I appended shows the results obtained by Mollier, and Fiy. 2 has been prepared from the English equivalents of Mollier's Tables. From the Table it will be seen that the value of the specific TABLE I.-SPECIFIC HEAT OF STEAN AT CORSTAKT PRESSURE FOE SUPERHEATING VROM t' TO t. TABLE I.-SPECIFIC HEAT OF STEAN AT CORSTAKT PRESSURE FOE SUPERHEATING VROM t' TO t. heat decreases for any pressure as the temperature rises, and increases for any given temperature as the pressure rises. The specific heat for any degree of superheat is expressed by the fornlula :- Where H is the total heat in the steam at point, of superheat ; H' ,, ,, ,, ,> ), >, ,, ?, ,, saturation ; t and t' are the the respective total tenlperntures. Where H is the total heat in the steam at point, of superheat ; H' ,, ,, ,, ,> ), >, ,, ?, ,, saturation ; t and t' are the the respective total tenlperntures. Where H is the total heat in the steam at point, of superheat ; t and t' are the the respective total tenlperntures. In Mollier's diagram (Fig. 2) the heat-units appear as ordinates and pressures appear as abscissae ; st series of constant-temperature lines can therefore be derived, from which it is easy to solve equation (1) and to obtain the required spec& heat, Certain tests carried out at Sibley College tend to show the same results as those of Mollier, the experiments covering a range of pressures from 0 to 220 lbs. per square inch by gauge, and a range of superheat for these pressures up to 400" F. E. Hausbrand, “ Evaporating, Condensing and Cooling Apparatus,” chap. i. London, 1903. Downloaded by [ University of Sussex] on [17/09/16]. Copyright © ICE Publishing, all rights reserved. Downloaded by [ University of Sussex] on [17/09/16]. Copyright © ICE Publishing, all rights reserved. p Having obtained the value of the specific heat, the next question is the transmission of heat from the flue- or furnace-gases to the PRERCE ON SUPERHEATERS Papers.] 269 .B Downloaded by [ University of Sussex] on [17/09/16]. Copyright © ICE Publishing, all rights reserved. 270 [Selected PREECE ON SUPERHEATERS. steam. A large number of experiments have been carried out in order to arrive at a satisfactory basis for calculating the probable transmission of heat between hot gases and water, but the informa- tion on the same subject as regards the exchange of heat between gases and dry steam is very slight, and the necessary data can be obtained only from tests taken under ordinary working conditions. The rate of transmission is generally stated per square foot per hour, and depends on the mean temperature difference, the velocity of the steam and the velocity of the gases y y g The rate of transmission is generally stated per square foot per hour, and depends on the mean temperature difference, the velocity of the steam, and the velocity of the gases. The true mean temperature difference (f?.) is given by Mr. Hausbrancl as follows :- Where T, = mean temperature of gases entering; T, = mean temperature of steam entering ; and T, = temperature of gases leaving ; p g g T, = temperature of steam leaving ; p g g T, = temperature of steam leaving ; Then p Then and the rate of transmission is stated to vary directly as the square root of the steam-velocity. and the rate of transmission is stated to vary directly as the square root of the steam-velocity. The Appendix, Tables I and 11, gives various tests from which are worked out, where practicable, the transmission per square foot of heating-surface, and the velocities of the steam. The Author has been unable to obtain sufficient data with regard to the effect of the velocity of the gases, but considers that, for practical design, the effect of this velocity may be neglected until further experiments are carried out. The most useful data obtained are from tests in connection with a Davey-Paxman independently-fired superheater, which is now working in India ; and several tests were carried out before the plant was out of the contractors’ hands. These have been tabulated and are plotted in Figs. 3-8. For each test of a series the rate of combustion was approximately 271 PREECE ON SUPERHEATERS. Papers.] the same, so that several series of tests have been carried out under different and increasing rates of combustion and varying velocities of steam. These results show clearly the effect of the varying steam-velocity on the degree of superheat obtained. Fig. 4 gives efficiency results for increasing velocities ; and Fig. 3 shows the increase in heat, and the variation in efficiency, plotted against the rate of combustion, when the steam-velocity is kept constant ; the mean temperature difference of course varies with the rate of combustion. From the diagrams it appears that the most efficient steam-velocity is between 62 and 67 feet per second. For equal rates of fire, up to this point there seems to be a steady rise in transmission and Fig. 3. b7h.U PER EQF 2000 I S 0 0 1000 10 , RATE OF. COMBUSTION: LBS. PER SQ.FT OF GRATE efficiency, but after passing 67 feet per second, both show a can- siderable falling off. Again from Fig. 3 it is clear that, for a given velocity and varying rates of combustion, there is a decrease in efficiency as the rate of combustion increases. I n other words, for the best fuel-efficiency the velocity of gases must bear some definite ratio to that of the steam ; the best steam-velocity appears to be about 67 feet per second, with a relatively slow rate of combustion. Downloaded by [ University of Sussex] on [17/09/16]. Copyright © ICE Publishing, all rights reserved. Then The maximum transmission obtained was 2,550 B.Th.U. per square foot, but at a low efficiency (see Table I1 and Fig. G and compare with Fig. 7). It is to be hoped that further experiments will be carried out to show the effect of the velocity of the gases. I n order to arrive at R basis for estimating the mean differ- ence of temperature in a superheater, temperatures were taken at Fig. 3. b7h.U PER EQF 2000 I S 0 0 1000 10 , RATE OF. COMBUSTION: LBS. PER SQ.FT OF GRATE Fig. 3. b7h.U PER EQF 2000 I S 0 0 1000 10 , RATE OF. COMBUSTION: LBS. PER SQ.FT OF GRATE Fig. 3. efficiency, but after passing 67 feet per second, both show a can- siderable falling off. Again from Fig. 3 it is clear that, for a given velocity and varying rates of combustion, there is a decrease in efficiency as the rate of combustion increases. I n other words, for the best fuel-efficiency the velocity of gases must bear some definite ratio to that of the steam ; the best steam-velocity appears to be about 67 feet per second, with a relatively slow rate of combustion. The maximum transmission obtained was 2,550 B.Th.U. per square foot, but at a low efficiency (see Table I1 and Fig. G and compare with Fig. 7). It is to be hoped that further experiments will be carried out to show the effect of the velocity of the gases. I n order to arrive at R basis for estimating the mean differ- ence of temperature in a superheater, temperatures were taken at 272 [Selected PREECE ON SUPERHEATERS. [Select PREECE ON SUPERHEATERS. half-hourly intervals on an independently-fired superheater. The readings, extending over 34 hours, averaged :- half-hourly intervals on an independently-fired superheater. The readings, extending over 34 hours, averaged :- O F. Furnace (approx.) . . . . . . . . . . 3,000 Gases leaving superheater . . . . . . . 67'7 Steam entering ,, . . . . . . . 357 ,, leaving ,, . . . . . . . 628 giving a mean difference of temperature of about 850" F. in normal working. The steam was passing at a velocity of 45.5 feet per Fiq. 4. VELOCITY OF STEAM: FT PER SSC. 0 FT RATE OF COMBUSTION 12 TO 16 L B ~ . Then PEP. SQ.' FT. rm From. RATE OF COMBUSTION 12 TO 16 L B ~ . PEP. SQ.' FT. rm From. second, and the total heat transmitted was 1,699 B.Th.U. per square foot of grate-area per hour; this gives a transmission of 2 B.Th.U. per square foot per hour per 1" F. mean difference of temperature. On this basis, for a steam-velocity of 60 to 70 feet per second a transmission of 3 B.Th.U. per square foot per hour per 1" F. mean difference might be expected. g p The heat,ing-surface required may be expressed as W c11 ( t - t') 3 f 111 - square feet; Downloaded by [ University of Sussex] on [17/09/16]. Copyright © ICE Publishing, all rights reserved. Downloaded by [ University of Sussex] on [17/09/16]. Copyright © ICE Publishing, all rights reserved. Downloaded by [ University of Sussex] on [17/09/16]. Copyright © ICE Publishing, all rights reserved. I'REECE ON SUPERHEATERS. SS 60 61 62 VELOCITY OF STEAM ; F? PER SEC. RATE OF c03fBUSTIOX 16 LBS. PER SQ. FT. PER HOUR. Fig. G. l 1 ,b.. 80% zooo 2;;- i l ,so0 70% 150° U l' l l 1 '. ,ooo 60% 100' 1 1 55 60 65 7 0 VELOCITY OF STEAM I FT PER SEC. RATE OB COXBTJSTIOS 16 LBS. PER SQ. FT. PER HOUR, , Fig. 7. B.nl.U. 2500- - - -_ 4 h -944 8%, -?t 2ooo 80% 250" '\ I 273 Papers.] I'REECE ON SUPERHEATERS. SS 60 61 62 VELOCITY OF STEAM ; F? PER SEC. SS 60 61 62 VELOCITY OF STEAM ; F? PER SEC. RATE OF c03fBUSTIOX 16 LBS. PER SQ. FT. PER HOUR. Fig. G. l 1 ,b.. 80% zooo 2;;- i l ,so0 70% 150° U l' l l 1 '. ,ooo 60% 100' 1 1 55 60 65 7 0 VELOCITY OF STEAM I FT PER SEC. RATE OB COXBTJSTIOS 16 LBS. PER SQ. FT. PER HOUR, , Fig. 7. B.nl.U. 2500- - - -_ 4 h -944 8%, -?t 2ooo 80% 250" '\ I 1500, 709. 200. ,ooo BOX m 0 4 68 69 70 71 VELOCITV OF STEAM: F? PER SEC. RATE OF COXBUSTIOS 20 LBS. PER SQ. FT. PER Hor:~. [THE ISST. C.E. VOL. CLSXIII.] T RATE OF c03fBUSTIOX 16 LBS. PER SQ. FT. PER HOUR RATE OF c03fBUSTIOX 16 LBS. PER SQ. FT. PER HOUR. Fig. G. l 1 ,b.. 80% zooo 2;;- i l ,so0 70% 150° U l' l l 1 '. ,ooo 60% 100' 1 1 55 60 65 7 0 VELOCITY OF STEAM I FT PER SEC. , l l l 1 . ,ooo 60% 100' 1 1 55 60 65 7 0 VELOCITY OF STEAM I FT PER SEC. RATE OB COXBTJSTIOS 16 LBS. PER SQ. FT. PER HOUR, , Fig. 7. B.nl.U. 2500- - - -_ 4 h -944 8%, -?t 2ooo 80% 250" '\ I 1500, 709. 200. ,ooo BOX m 0 4 68 69 70 71 VELOCITV OF STEAM: F? PER SEC. RATE OF COXBUSTIOS 20 LBS. PER SQ. FT. PER Hor:~. [THE ISST. C.E. VOL. CLSXIII.] T Downloaded by [ University of Sussex] on [17/09/16]. Copyright © ICE Publishing, all rights reserved. RATE OB COXBTJSTIOS 16 LBS. Downloaded by [ University of Sussex] on [17/09/16]. Copyright © ICE Publishing, all rights reserved. PER SQ. FT. PER HOUR RATE OB COXBTJSTIOS 16 LBS. PER SQ. FT. PER HOUR Fig. 7. B.nl.U. 2500- - - -_ 4 h -944 8%, -?t 2ooo 80% 250" '\ I 1500, 709. 200. ,ooo BOX m 0 4 68 69 70 71 VELOCITV OF STEAM: F? PER SEC. RATE OF COXBUSTIOS 20 LBS. PER SQ. FT. PER Hor:~. RATE OF COXBUSTIOS 20 LBS. PER SQ. FT. PER Hor:~. [THE ISST C E VOL CLSXIII ] RATE OF COXBUSTIOS 20 LBS. PER SQ. FT. PER Hor:~. [THE ISST. C.E. VOL. CLSXIII.] T Downloaded by [ University of Sussex] on [17/09/16]. Copyright © ICE Publishing, all rights reserved. 274 PREECE O N SUPERHEATERS. [Selected to which must be added a,n amount for drying out the vteam, equal to to which must be added a,n amount for drying out the vteam, equal to W (1 - X)L square feet, 6 (T- t') equal to W (1 - X)L square feet, 6 (T- t') taking 6 as the transmission per square foot per hour per 1" F. fro111 gases to moisture, and W denotes the weight of steam ; W denotes the weight of steam ; W denotes the weight of steam ; c, 7 7 ,, specific heat ; T 1 , ,, temperature of gases ; t ;> ,, total temperature of steam ; t' ,; ,, temperature of steam at saturation point ; X ;, ,, dryness fraction of the initial steam ; L ? > ,, latent heat. Fig. 8. 8.n. U 1000 I O W 67 68 69 VELOCITY OC STEAM : F7 PER SEC. Fig. 8. There are many superheaters of different types now in use, :mtl descriptions of a few of these may not be out of place here. There are many superheaters of different types now in use, :mtl descriptions of a few of these may not be out of place here. Bolton Superheater (Figs. g).-The leading principle in this superheater is the use of Field tubes, i.e. one tube inside another ; the steam enters the inlet chamber, passes down the internal tubes of the first section and up the annular space into the box, then down the annular space of the second section, and finally up the internal tubes into the outlet chamber; this enables the steam to be passed over the heating surface in a thin film. Downloaded by [ University of Sussex] on [17/09/16]. Copyright © ICE Publishing, all rights reserved. The tubes being fixed at one end only, are free to expand and contract. The Tinker Superheater (Figs. lO).-This superheater is chiefly used with Lancashire boilers, and is constructed with four headers ant1 two series of looped tubes placed in the down-t'ake ; the tubes are Downloaded by [ University of Sussex] on [17/09/16]. Copyright © ICE Publishing, all rights reserved. 275 PREECE ON SUPERHEATERS. Papers.] Papers.] arranged in a cluster, and steam goes through the two clusters in parallel. The tubes furthest removed from the boiler are subject to a lower temperature than those in front of them, and serve as a store of heat to neutralize the irregular effects of changes of furnace temperature. The Ferguson Superheater (Figs. Il).-The great advantage claimed for this design is that any section can be taken out and replaced without breaking the steam-pipe connections. Each section consist of three tubes connected to a small flange at the ends ; these ar bolted to the main headers, so that they can be easily removed m y section can be blank flanged, and the superheater, as a whole T 2 The Ferguson Superheater (Figs. Il).-The great advantage claimed for this design is that any section can be taken out and replaced without breaking the steam-pipe connections. Each section consists of three tubes connected to a small flange at the ends ; these are bolted to the main headers, so that they can be easily removed ; m y section can be blank flanged, and the superheater, as a whole, T 2 The Ferguson Superheater (Figs. Il).-The great advantage claimed for this design is that any section can be taken out and replaced without breaking the steam-pipe connections. Each section consists of three tubes connected to a small flange at the ends ; these are bolted to the main headers, so that they can be easily removed ; m y section can be blank flanged, and the superheater, as a whole, T 2 T 2 Downloaded by [ University of Sussex] on [17/09/16]. Copyright © ICE Publishing, all rights reserved. Downloaded by [ University of Sussex] on [17/09/16]. Copyright © ICE Publishing, all rights reserved. Downloaded by [ University of Sussex] on [17/09/16]. Copyright © ICE Publishing, all rights reserved. 276 I’GEECIC OK SUPEKHIL4TERS. [Selected Ei; :l by [ University of Sussex] on [17/09/16]. Copyright © ICE Publishing, all rights reserved. Downloaded by [ University of Sussex] on [17/09/16]. Copyright © ICE Publishing, all rights reserved. 277 Papers SECTIONAL ELEVATION SECTIONAL ELEVATION I SECTIONAL PLAN lnches~? 6 0 2 3 ? 5 e ~ ? Feet Scale. I Inch = 4 Feet SECTIONAL ELEVATION I SECTIONAL PLAN lnches~? 6 0 2 3 ? 5 e ~ ? Feet Scale. I Inch = 4 Feet Downloaded by [ University of Sussex] on [17/09/16]. Copyright © ICE Publishing, all rights reserved. Downloaded by [ University of Sussex] on [17/09/16]. Copyright © ICE Publishing, all rights reserved. Downloaded by [ University of Sussex] on [17/09/16]. Copyright © ICE Publishing, all rights reserved. 278 PREECE ON SUPERHEATERS. [Selected [Selected can continue at work. The initial rise of the tubes causes any priming to collect in the headers, where it is drawn off. can continue at work. The initial rise of the tubes causes any priming to collect in the headers, where it is drawn off. The Wotf Superheater (Figs. 12).-This consists of a coil of tubes placed in the smoke-box of either the locomotive or the tubular type of The Wotf Superheater (Figs. 12).-This consists of a coil of tubes placed in the smoke-box of either the locomotive or the tubular type of boiler, the coil being arranged serpentine fashion. The gases leave the boiler-tubes at a temperature of about 750' F., and the superheater has sufficient surface to raise the temperature of the steam to about 630" F. An interesting feature of the Wolff type, is the use of an intermediate superheater for the steam on its passage between the boiler, the coil being arranged serpentine fashion. The gases leave the boiler-tubes at a temperature of about 750' F., and the superheater has sufficient surface to raise the temperature of the steam to about 630" F. An interesting feature of the Wolff type, is the use of an intermediate superheater for the steam on its passage between the Downloaded by [ University of Sussex] on [17/09/16]. Copyright © ICE Publishing, all rights reserved. Downloaded by [ University of Sussex] on [17/09/16]. Copyright © ICE Publishing, all rights reserved. 2 Papers ] PEEECE ON S 279 PEEECE ON SUPERHEATERS. Papers.] Papers.] high- and Iow-pressure cylinders ; this consists of a number of small tubes in parallel, and is shown surrounding the main superheater. The PieZock Superheater.-This superheater, of the type mostly used in locomotive work, consists of a small steel box surrounding part of the boiler-tubes. The pressure inside and outside the box is the same, and it is only necessary to expand the tubes sulficiently tight to prevent leakage. The weight of the box is supported by the tubes, and it is divided into compartments by vertical plates, which lead the steam round them ; the boiler- tubes are made slightly taper from the smoke-box end to the fire-box so as to admitof removal without difliculty tubes in parallel, and is shown surrounding the main superheate The PieZock Superheater.-This superheater, of the type mo used in locomotive work, consists of a small steel box surround part of the boiler-tubes. The pressure inside and outside box is the same, and it is only necessary to expand the tube tubes in parallel, and is shown surrounding the main superheater. The PieZock Superheater.-This superheater, of the type mostly used in locomotive work, consists of a small steel box surrounding part of the boiler-tubes. The pressure inside and outside the box is the same, and it is only necessary to expand the tubes sulficiently tight to prevent leakage. The weight of the box is supported by the tubes, and it is divided into compartments by vertical plates, which lead the steam round them ; the boiler- tubes are made slightly taper from the smoke-box end to the fire-box so as to admit of removal without difliculty. sulficiently tight to prevent leakage. The weight of the box is supported by the tubes, and it is divided into compartments by vertical plates, which lead the steam round them ; the boiler- tubes are made slightly taper from the smoke-box end to the fire-box so as to admitof removal without difliculty. y The Babcock- Wilcox Superheater (Figs. 13 and 14).-This super- heater is placed above the tubes of the well-known water-tube boiler Downloaded by [ University of Sussex] on [17/09/16]. Copyright © ICE Publishing, all rights reserved. Downloaded by [ University of Sussex] on [17/09/16]. Copyright © ICE Publishing, all rights reserved. 280 PREECE ON SGPERHEATEKS [Selectell 280 PREECE ON SGPERHEATEKS. [Selectell and in this position it is asserted that 150" F. of superheat can he easily obtained. Freedom from the ill effects caused by expansion is secured by Pi:ls. 14. LONQITUDINAL SECTlON I SECTIONAL PLAN AA Scale: I Inch = 8 Feet FEET .CL% fO.-.-- - 7 ____ J 10 FEET BABCOCK-TTIIXOX Sl:PI%,RHIi.4TTX. Pi:ls. 14. LONQITUDINAL SECTlON Pi:ls. 14. BABCOCK-TTIIXOX Sl:PI%,RHIi.4TTX. the tubes being free at one end, and also through the headers having no pipe-connections between them. A flooding arrangement is provided to prevent overheating when steam is being raised. In another type (Figs. 14), the superheater consists of R isel-ies of the tubes being free at one end, and also through the headers having no pipe-connections between them. A flooding arrangement is provided to prevent overheating when steam is being raised. In another type (Figs. 14), the superheater consists of R isel-ies of Downloaded by [ University of Sussex] on [17/09/16]. Copyright © ICE Publishing, all rights reserved. Downloaded by [ University of Sussex] on [17/09/16]. Copyright © ICE Publishing, all rights reserved. 281 PRFECE ON SUPERHEATERS. Papers.] Papers.] boxes connected by U tubes, the steam passing in series from one box to the next, and so on to the steam main. The gases rising - from the furnace pass through a perforated wall which compmatively constant temperature. ensures a The Pnxnzan Superheater (Figs. 15 and 16).-This superheater, when arranged as an independently fired heater, consists of a series of mild-steel headers connected together by means of looped tubes, the number and arrangement of the boxes varying with the amount of steam, and the degree of superheat to be dealt with. Fig. L5 shows The Pnxnzan Superheater (Figs. 15 and 16).-This superheater, when arranged as an independently fired heater, consists of a series of mild-steel headers connected together by means of looped tubes, the number and arrangement of the boxes varying with the amount of steam, and the degree of superheat to be dealt with. Fig. L5 shows Downloaded by [ University of Sussex] on [17/09/16]. Copyright © ICE Publishing, all rights reserved. Downloaded by [ University of Sussex] on [17/09/16]. Copyright © ICE Publishing, all rights reserved. 282 PREECE OS SUPERHEATERS [S l d ERHEATERS. uperheat, and Fig. 1 h t UPERHEATERS. f superheat, and Fig. 16 f ow superheat. asses in a single run thro op; it is carried thence to 282 [Selected PREECE OS SUPERHEATERS. one arranged for high degrees of superheat, and Fig. 16 for large quantities sfid s correspondingly low superheat. I n the former case the steam passes in a single run through the tubes, entering at the bottom loop ; it is carried thence to the top loop and then downwards passing and repassing through the loops loop, and then downwards, passing and repassing through the loops, until it reaches the outlet. For the moderate temperatures, the steam is split into two or three courses, as indicated in the Figure. By the arrangement of the steam-path described the gases will leave at a temperature of 420" F. Downloaded by [ University of Sussex] on [17/09/16]. Copyright © ICE Publishing, all rights reserved. 283 Papers.] PREECE ON SUPERHEATERS. while the steam temperature can be raised to 680" F. The gases escape by cavity walls on either flank, and are under full control. while the steam temperature can be raised to 680" F. The gases escape by cavity walls on either flank, and are under full control. A similar type of superheater is supplied for use with the Davey- Paxman " Economic " boiler (Fig. 17). I n this a supplementary chamber is provided behind the combustion chamber, which can be completely isolated by means of a firebrick damper, and the gases Fig. 17. PAXMAN SUPERHEATER WITH DAVEY-PAXDIAN '' ECONOMIC " BOILER. Fig. 17. can be passed to the main flue when in use, thus enabling the super- heater to be easily regulated. can be passed to the main flue when in use, thus enabling the super heater to be easily regulated. The McPhail-Simpson Superheater (Figs. lS).-The most recent type of this superheater consists of a series of looped tubes of cold- drawn steel, and a header of mild steel, which is dished to form a cover to the tube-plate ; a division plate is fixed to divide the box Downloaded by [ University of Sussex] on [17/09/16]. Copyright © ICE Publishing, all rights reserved. Downloaded by [ University of Sussex] on [17/09/16]. Copyright © ICE Publishing, all rights reserved y [ University of Sussex] on [17/09/16]. Copyright © ICE Publishing, all rights reserved. [Selectetl LONGITUDINAL SECTION. SECTIONAL P L A N . Scale. 3/ls Inch = I Foot FEET5 4 3 2 I 0 5 FEET MCPHAIL-STMPSON SUPKRHEATER. LONGITUDINAL SECTION. LONGITUDINAL SECTION. LONGITUDINAL SECTION. LONGITUDINAL SECTION. SECTIONAL P L A N . Scale. 3/ls Inch = I Foot FEET5 4 3 2 I 0 5 FEET MCPHAIL-STMPSON SUPKRHEATER. Downloaded by [ University of Sussex] on [17/09/16]. Copyright © ICE Publishing, all rights reserved. 285 PREECE ON SUPERHEATEW. Papers.] Papers.] into inlet and outlet compartments. The ends of the tubes are turned to a shoulder fitting exactly in the holes of the tube-plate, where they are expanded and beaded over. This superheater is also formed for independent firing, the required number of headers being coupled up in parallel. It is also made with two inlet and outlet headers as separate boxes, this type being used generally with water-tube boilers. into inlet and outlet compartments. The ends of the tubes are turned to a shoulder fitting exactly in the holes of the tube-plate, where they are expanded and beaded over. This superheater is also formed for independent firing, the required number of headers being coupled up in parallel. It is also made with two inlet and outlet headers as separate boxes, this type being used generally with water-tube boilers. The foregoing are the principal superheaters now in use, and the modern practice is to make the tubes of solid drawn steel, and the headers or boxes of mild steel. The size of tube most generally used appears to be about l& inch in external diameter and 1 inch to 18 inch in internal diameter, expanded and beaded over in the header. Various methods of making the mild-steel header are used ; in some cases it consists of tube-plates with a dished cover, in others the box is pressed out of steel plate and the ends and top are welded together to form a complete box. Table I1 gives the estimated gain obtained by using super- heat of varying degrees ; the calculation is based on the use of coal of about 13,500 B.Th.U., valued at 15s. per ton, and charges for depreciation, renewals, etc., on the superheater (i.e. 15 per cent. on the cost) have been reduced to a coal basis. The assumption relates to a power-station using on an average 9,000 lbs. y [ University of Sussex] on [17/09/16]. Copyright © ICE Publishing, all rights reserved. of dry steam per hour, with a load-factor of 20 per cent., and this would necessitate a superheater capable of dealing with a maximum load of about 45,000 lbs. per hour. TABLE 11-ESTIMATED GAIN FROM USIXG SUPERHEATED STEAM. TABLE 11-ESTIMATED GAIN FROM USIXG SUPERHEATED STEAM. ~ ~~ ~ Superheat. . . . . . . 1 I IOU' F. , 150° F. ~ 2000 F. I 250° F. Estimated saving in steam . . Per cent. Total coal per annum . . . . Tons . . Approximate cost of superheater . . . 1,000 ,, coal per hour (boilers). . ,, 9,000 Average steam per hour . . . Lbs. .. Depreciation, etc., on superheater in tons 3,910 of coal per annum. . . . . .)i " Total equivalent coal per annum . Tons 13,910 Xet saving . . . . . . Per cent. I . . ,, ,, ,, (superheater) ,, 13.6 ~ 19 ' 22 766 810 1 780 864 6,900 7,290 7,020 7,780 23.5 3,632 3,528 13 498 3,575 7 . 1 1 9.75 ~16.55 1 10.1 From the foregoing it may be seen that the greatest net saving occurs in using a superheat of 200" F. From the foregoing it may be seen that the greatest net saving occurs in using a superheat of 200" F. The Paper is accompanied by nineteen drawings, from which the Figures in the text have been selected, and by an Appendix. p [APPENDIX. Downloaded by [ University of Sussex] on [17/09/16]. Copyright © ICE Publishing, all rights reserved. 286 PlZEECE ON SUPERHEATERS. [Selected Y ;I .. . ta 0 0 0 t, i 0 0 '2 - 0 X 1 F- 0 0 0 0 1 0 0 0 0 0 ersity of Sussex] on [17/09/16] Copyright © ICE Publishing all rights reserved Downloaded by [ University of Sussex] on [17/09/16]. Copyright © ICE Publishing, all rights reserved. Downloaded by [ University of Sussex] on [17/09/16]. Copyright © ICE Publishing, all rights reserved. I'REEUE ON SUPERHEATEHS. Papers.] 287 . . . . . . . . . . . . 0 W 5 : z R ?2 O d O * r - w ' w w W O O o d 3 F- i ~ m m o w m ' w m > m c o m m r - o m . ? ? y [ University of Sussex] on [17/09/16]. Copyright © ICE Publishing, all rights reserved. " ? s o ? ? i 034 3 N 3 3 . . " 0 m m m m m m w r - 0 0 F ? ? 9 0 N ? ? m o w m o o o m d + n * m m d F - i M d i d Downloaded by [ University of Sussex] on [17/09/16]. Copyright © ICE Publishing, all rights reserved. 288 PKEECE OX SUPERHEATERS. m r. 0 . . 4 0 v > P - ri . . . . o w W W 4 r i m 3 La W ? ? m 0 W W m r i r ( m . . - -~ ~ m v 0 0 1 - m w T u ) m m m m m m . . - I " W W U. La 0 0 . . . . . . . . ^ r ^ ^ - - . . . . . . l Downloaded by [ University of Sussex] on [17/09/16]. Copyright © ICE Publishing, all rights reserved.
https://openalex.org/W1995213831	https://zenodo.org/records/2229117/files/article.pdf	German	null	Über die direkte Umwandlung von Nitrilen in Ester	Berichte der Deutschen Chemischen Gesellschaft	1,918	public-domain	293	I> E. 51, 296 [1918]. 2) B. 44, 1115 [1911]. Dem Titel der Arbeit: ~ Z u r Kenntnie der Stilben- o-carbonsiiuren., lie13 sich allerdings nicht entnehmen, daD in ihr unter anderem von der direkten Veresterung der Nitrile die Rede ist. 9 B. 48, 1800 [1915]; A. 411, 123 [1916]. dieser Arbeit ermoglicht wurde , meinen besten Dank ehrerbietigst auszusprechen. p Chem. Laborat. Beiersdorf & Co. und Eppendorfer Krankenheue in Hamburg. 805 805 dieser Arbeit ermoglicht wurde , meinen besten Dank ehrerbietigst auszusprechen. (Eingegangen am 16. Februar 1918.) Uber diesen Gegenstand erschien im vorletzten HeM der ,Berichtea eine Arbeit von Hrn. L. Spiegel'). Der Autor zeigt, da13 man Ni- trile bequem durch Erhitzen mit Alkohol und konz. Schwefel- siiure im geschlossenen Rohr auf 130-140° in die zugehorigen Ester verwandeln kann. Hrn. Spiegel scheint nun entgangen zu sein, da13 auch ich vor einigen Jahren mit Erfolg die direkte Veresterung der Nitrile untersucht habe. Ich konstatierte in einer gemeinsam mit Hrn. Matt o n durchgefiihrten Arbeit 3, daI3 Nitrile leicht in Ester ubergehen, wenn man in ihre alkoholischen Losungen auf dem Wasser- bad Chlorwasserstoff einleitet. Auf diese Weise konnten z. B., o-Nitro-p-tolunitril (I.) und das Stilbenderivat (11.) glatt in die ent- I-\. CH: CH.\/.CN /7 11. L/ NO, NO2 H~c./-\.cN I. \?/ sprechenden Ester verwandelt werden. Spater habe ich diese Reak- tion auf weitere Nitrile, z. B. auf solche der Isatogen-Reihe ubertragen'). Die von Matton und mir gefundene Tatsache, da8 sich solche Ni- trile, die in o-Stellung zur Nitrilgruppe einen Methylrest oder eine Athylenliicke enthalten, auf die angegebene Weise nicht verestern lassen, stimmt gut zu der Angabe von Hrn. Spiegel, daI3 sich eine o-stiindige Methylgruppe auch bei der Veresterung der Nitrile mit Alkohol und Schwefelsiiure als hinderlich erweist. Unsere Arbeitea ergiinzen sich also aufs beste.
https://openalex.org/W4383560134	https://nvlvet.com.ua/index.php/journal/article/download/4867/4982	English	null	The state of the immune system of rats under conditions of oxidative stress and the influence of the feed additive “Sylimevit”	Naukovij vìsnik Lʹvìvsʹkogo nacìonalʹnogo unìversitetu veterinarnoï medicini ta bìotehnologìj ìmenì S.Z. G̀žicʹkogo	2,023	cc-by	5,254	The state of the immune system of rats under conditions of oxidative stress and the influence of the feed additive “Sylimevit” B. V. Gutyj1 , R. V. Voloshyn1, V. V. Stybel1, B. M. Verveha2, R. M. Sachuk3, I. S. Starostenko4, R. V. Mylostyvyi5, V. I. Kushnir1,6, I. Ya. Mazur1, I. I. Khariv1, Ya. I. Turko1, V. I. Khalak7, V. R. Magrelo1 1Stepan Gzhytskyi National University of Veterinary Medicine and Biotechnologies, Lviv, Ukraine 2Danylo Halytsky Lviv National Medical University, Lviv, Ukraine 3Rivne State University for the Humanities, Rivne, Ukraine 4Bila Tserkva National Agrarian University, Bila Tserkva, Ukraine 5Dnipro State Agrarian and Economic University, Dnipro, Ukraine 6State Scientific-Research Control Institute of Veterinary Medicinal Products and Feed Additives, Lviv, Ukraine 7State Institution Institute of Grain Crops NAAS of Ukraine, Dnipro, Ukraine Scientific-Research Control Institute of Veterinary Medicinal Products and Feed Additives, Lviv, Ukrain Institution Institute of Grain Crops NAAS of Ukraine, Dnipro, Ukraine Gutyj, B. V., Voloshyn, R. V., Stybel, V. V., Verveha, B. M., Sachuk, R. M., Starostenko, I. S., Mylostyvyi, R. V., Kushnir, V. I., Mazur, I. Ya., Khariv, I. I., Turko, Ya. I., Khalak, V. I., & Magrelo, V. R. (2023). The state of the immune system of rats under conditions of oxidative stress and the influence of the feed additive “Sylimevit”. Scientific Messenger of Lviv National University of Veterinary Medicine and Biotechnologies. Series: Veterinary sciences, 25(110), 131–136. doi: 10.32718/nvlvet11022 Gutyj, B. V., Voloshyn, R. V., Stybel, V. V., Verveha, B. M., Sachuk, R. M., Starostenko, I. S., Mylostyvyi, R. V., Kushnir, V. I., Mazur, I. Ya., Khariv, I. I., Turko, Ya. I., Khalak, V. I., & Magrelo, V. R. (2023). The state of the immune system of rats under conditions of oxidative stress and the influence of the feed additive “Sylimevit”. Scientific Messenger of Lviv National University of Veterinary Medicine and Biotechnologies. Series: Veterinary sciences, 25(110), 131–136. doi: 10.32718/nvlvet11022 Gutyj, B. V., Voloshyn, R. V., Stybel, V. V., Verveha, B. M., Sachuk, R. M., Starostenko, I. S., Mylostyvyi, R. V., Kushnir, V. I., Mazur, I. Ya., Khariv, I. I., Turko, Ya. I., Khalak, V. I., & Magrelo, V. R. (2023). The state of the immune system of rats under conditions of oxidative stress and the influence of the feed additive “Sylimevit”. Scientific Messenger of Lviv National University of Veterinary Medicine and Biotechnologies. Series: Veterinary sciences, 25(110), 131–136. doi: 10.32718/nvlvet11022 Received 20.04.2023 Received in revised form 22.05.2023 Accepted 23.05.2023 Stepan Gzhytskyi National University of Veterinary Medicine and Biotechnologies Lviv, Pekarska Str., 50, Lviv, 79010, Ukraine. Науковий вісник ЛНУВМБ імені С.З. Ґжицького. Серія: Ветеринарні науки, 2023, т 25, № 110 Науковий вісник ЛНУВМБ імені С.З. Ґжицького. Серія: Ветеринарні науки, 2023, т 25, № 110 Науковий вісник Львівського національного університету ветеринарної медицини та біотехнологій імені С.З. Ґжицького. Серія: Ветеринарні науки Scientific Messenger of Lviv National University of Veterinary Medicine and Biotechnologies. Series: Veterinary sciences ISSN 2518–7554 print doi: 10.32718/nvlvet11022 ISSN 2518–1327 online https://nvlvet.com.ua/index.php/journal doi: 10.32718/nvlvet11022 https://nvlvet.com.ua/index.php/journal State Scientific-Research Control Institute of Veterinary Medicinal Products and Feed Additives, Donetska Str., 11, Lviv, 79019, Ukraine. Keywords: milk thistle, oxidative stress, immune system, tetrachloromethane. State Institution Institute of grain crops of NAAS, V. Vernadsky Str., 14, Dnipro, 49027, Ukraine. Bila Tserkva National Agrarian University, pl. Soborna 8/1, Bila Tserkva, 09117, Ukraine. Rivne State University for the Humanities, Plastova Str., 29-a, Rivne, 33028, Ukraine. Introduction The study was conducted on young white laboratory rats of the Wistar line with a body weight of 180 to 200 g. These rats were kept in standard conditions of the vivari- um of the State Research Control Institute of Veterinary Medicines and Feed Additives. The rats were fed a bal- anced diet throughout the experiment containing all the necessary components. Animals had unlimited access to drinking water. The animals were divided into three groups of 20 individuals each: 1st group (C) intact ani- mals; 2nd group (R1) – rats affected by tetrachloro- methane; The 3rd group (R2) – rats affected by tetrachlo- romethane, which were fed with the feed additive “Syli- mevit”. The homeostasis of the internal environment of the an- imal body depends on the interrelationship of individual links of metabolic processes and the ability of the compo- nents that participate in the overall system (Gutyj et al., 2022; 2023). Blood, as one of the body's biological fluids, responds with quantitative and qualitative changes in its composition to any exogenous or endogenous influences. Therefore, it is a biomarker that allows for determining the general state of organs and systems and assessing the course of the main metabolic processes (Zhang et al., 2021; Lesyk et al., 2022; Kushnir et al., 2023). Therefore, studying morphological and biochemical indicators of blood is one informative method that allows for establish- ing the transition from the body's physiological state to the pathological one (Kisera et al., 2021; Kuljaba et 2022). Intragastric administration of tetrachloromethane twice (with an interval of 48 hours) in a dose of 0.1 ml per 100 g of body weight in a 50 % oil solution was used for the experimental intoxication of rats. The animals of the second experimental group were fed the feed additive “Sylimevit” for 30 days together with feed at a dose of 0.1 g per 100 g of body weight. This supplement contained milk thistle fruits, selenium, methiphene, and vitamins A, E, and C. The problem of the influence of adverse environmen- tal factors on the immune system has gained particular importance since it plays a leading role in maintaining health and is recognized as one of the most sensitive fac- tors, even in relatively low concentrations (Khariv et al., 2017; Krempa et al., 2021; Varkholiak et al., 2021). Introduction The immune system is one of the essential homeostat- ic systems of the body, which determines the degree of health of animals and their adaptive capabilities (Müller et al., 2019; Wang et al., 2021; Radzykhovskyi et al., 2022). As an indicator of the body's physiological state, it reacts to changes in environmental conditions. Violation of its function is considered one of the pathogenetic mecha- nisms of the pathological process (Netea et al., 2020; Place & Kanneganti, 2020; Daëron, 2022). Immunotoxici- ty is defined as the property of a toxicant to cause im- pairment of the function of the immune system, which is manifested by inadequate immune reactions. Immunotox- icity is considered in two aspects: the direct damaging effect of the substance on the immune system and the participation of the immune system in the implementation of the mechanisms of their toxic action (McComb et al., 2013; 2019; Hillion et al., 2020). Using ether anesthesia, blood for biochemical and hematological studies in rats was collected from the jugu- lar vein on the experiment's fifth, tenth, twentieth, twenty- fifth, and thirtieth days. Lysozyme activity of blood serum was determined us- ing a daily culture of Micrococcus lysodeicticus strain VKM-109 as a test microbe by the nephelometric method; optical density was measured at a wavelength of 540 nm. Bactericidal activity in blood serum samples was studied according to this method by Yu. M. Markov (1968) using a daily culture of E. coli strain VKM-125. Photocolorime- try was performed before and after a 3-hour incubation. Determination of the content of circulating immune complexes in blood serum was carried out using a borate buffer. Selective precipitation of antigen-antibody com- plexes occurred under the influence of high molecular weight PEG with a mass of 6000 Da. The results were calculated by photocolorimetry of the density of the pre- cipitate at a wavelength of 450 nm. To improve the immune and antioxidant status of an- imals with toxic liver damage, new drugs and feed addi- tives based on plant raw materials have been widely used in recent years. The phagocytic reaction of blood neutrophils was as- sessed by PhA and phagocytic index (PhI) according to the method of V. S. Gostev (1950). Stabilized blood was incubated with a daily culture of E. coli strain VKM-125. Smears were examined under a microscope in an immer- sion system. Introduction PhA was determined by the number of active neutrophils from 100 counted cells and PhI – by the num- ber of phagocytosed microbial bodies by one active neu- trophil. The intensive development of animal husbandry at the current stage requires new approaches to the organization of feeding farm animals and the introduction of modern feed additives, which are usually not used in their pure form as feed but are purposefully added to feed or water to improve their quality, increase productivity and well- being of animals (Martyshuk et al., 2020; 2021; Martyshuk & Hutyi, 2021). Housing, feeding, care, and all manipulations with an- imals were carried out following the European Conven- tion “On the Protection of Vertebrate Animals Used for Experimental and Scientific Purposes” (Strasbourg, 1986) and “General Ethical Principles of Animal Experiments”, adopted by the First National Congress on bioethics (Ky- iv, 2001). The experiments were carried out in compli- ance with the principles of humanity outlined in the di- rective of the European Community. The state of the immune system of rats under conditions of oxidative stress and the influence of the feed additive “Sylimevit” Series: Veterinary scie 131 ies: Veteri 131 131 Науковий вісник ЛНУВМБ імені С.З. Ґжицького. Серія: Ветеринарні науки, 2023, т 25, № 110 The state of the immune system of rats under conditions of oxidative stress and the influence of the feed additive “Sylimevit” Tel.: +38-068-136-20-54 E-mail: bvh@ukr.net The immune system plays a crucial role in maintaining the body's homeostasis, determining the state of health of animals and their ability to adapt. The work aimed to investigate the effect of a feed additive based on milk thistle fruits, selenium, metiphene, and vitamins A, E, and C on rats' immune status under experimental tetrachloromethane poisoning conditions. The study was conducted on young white male Wistar laboratory rats. Intragastric administration of tetrachloromethane twice (with an interval of 48 hours) in a dose of 0.1 ml per 100 g of body weight in a 50 % oil solution was used for the experimental intoxication of rats. The animals of the second experimental group were fed the feed additive “Sylimevit” for 30 days together with feed at a dose of 0.1 g per 100 g of body weight. The introduction of tetrachlo- romethane in experimental groups of rats led to the development of oxidative stress, which occurs due to specific chemical processes in the body of experimental animals. It was found that the development of oxidative stress caused by tetrachloromethane leads to suppression of the humoral and nonspecific link of the immune system of rats. This is manifested in a decrease in the bactericidal and lysozyme activity of the blood serum, a decrease in the phagocytic index, and the phagocytic activity of neutrophils. In addi- tion, an increase in the number of circulating immune complexes was observed. It was also established that feeding the feed additive “Sylimevit” strengthens the immune defense of the body of rats poisoned with tetrachloromethane. This feed additive helps to strengthen the body's defense mechanisms, increas- ing the immune response and helping to resist the toxic effects of tetrachloromethane. Danylo Halytsky Lviv National Medical University, Pekarska St., 69, Lviv, 79010 Ukraine. Rivne State University for the Humanities, Plastova Str., 29-a, Rivne, 33028, Ukraine. Bila Tserkva National Agrarian University, pl. Soborna 8/1, Bila Tserkva, 09117, Ukraine. Dnipro State Agrarian and Economic University, Yefremov Str., 25, Dnipro, 49027, Ukraine. Keywords: milk thistle, oxidative stress, immune system, tetrachloromethane. State Scientific-Research Control Institute of Veterinary Medicinal Products and Feed Additives, Donetska Str., 11, Lviv, 79019, Ukraine. State Institution Institute of grain crops of NAAS, V. Vernadsky Str., 14, Dnipro, 49027, Ukraine. State Institution Institute of grain crops of NAAS, V. Vernadsky Str., 14, Dnipro, 49027, Ukraine. B. Scientific Messenger LNUVMB. Series: Veterinary sciences, 2023, vol. 25, no 110 132 The aim of the research The work aimed to investigate the effect of the feed additive “Sylimevit” on the state of the immune system of rats under the conditions of tetrachloromethane poisoning Scientific Messenger LNUVMB. Series: Veterinary sciences, 2023, vol. 25, no 110 Scientific Messenger LNUVMB. Series: Veterinary sciences, 2023, vol. 25, no 110 132 Науковий вісник ЛНУВМБ імені С.З. Ґжицького. Серія: Ветеринарні науки, 2023, т 25, № 110 Науковий вісник ЛНУВМБ імені С.З. Ґжицького. Серія: Ветеринарні науки, 2023, т 25, № 110 Науковий вісник ЛНУВМБ імені С.З. Ґжицького. Серія: Ветеринарні науки, 2023, т 25, № 110 bactericidal activity of blood serum was established in the rats of the first experimental group. Thus, on the 20th day of the experiment, LABS decreased by 7.6 % and BABS – by 5.69 % compared to the tenth day. Results and discussion When evaluating the activity of the immune system in the animal body, it is essential to consider that immuno- logical parameters are subject to significant fluctuations both in the presence of oxidative stress and under the influence of the Silimevit feed additive. The reaction of the immune system is the first to receive toxic substances. Through a general assessment of the indicators of the immune system and the antioxidant protection system of the animal body, it is possible to develop an optimal scheme for the prevention of the development of oxida- tive stress. A decrease in the bactericidal and lysozyme activity of blood serum indicates inhibition of the functioning of the humoral link of immunity. The lowest values of bacteri- cidal activity (BABS) and lysozyme activity (LABS) were found in rats of the first experimental group on the 25th day of the experiment, where BABS was 25.41 ± 0.75 %, and LABS was 31.2 ± 0.63 %, respectively. In the rats of the second experimental group fed with the feed additive “Sylimevit”, a probable increase in the lysozyme activity of the blood serum was established throughout the experiment. Thus, on the 5th and 10th day of the experiment, LABS increased by 7.3 and 7.8 % compared to the control group. It is worth noting that the highest lysozyme activity of blood serum was on the second experimental group's 10th day of the experiment. On the 25th and 30th day of the experiment, the lysozyme activity of the blood serum of the second experimental group of rats increased by 6.8 and 6.2 % compared to the control. When studying the antimicrobial activity of the blood serum of rats under the conditions of experimental devel- opment of oxidative stress, it was established that on the 5th and 10th day of the experiment, there was a slight increase in the activity of lysozyme and bactericidal activ- ity of the cow. In particular, the lysozyme activity of the blood serum of the rats of the first experimental group increased by 6.2 % and 5.8 %, respectively (Table 1), and the bactericidal activity of the blood serum increased by 2.81 % and 2.23 % (Table 2). compared to the control group. Subsequently, a decrease in both lysozyme and Scientific Messenger LNUVMB. Series: Veterinary sciences, 2023, vol. 25, no 110 Table 1 Table 1 Lysozyme activity of blood serum of rats under conditions of oxidative stress and action of Sylimevit, % (M ± m; n = 5) Day of Research Group of animals Control Research 1 Research 2 Fifth 35.4 ± 0.76 41.6 ± 1.06 42.7 ± 0.68* Tenth 40.9 ± 0.85 43.2 ± 0.67* Twentieth 33.3 ± 1.00 42.7 ± 0.84*** Twenty-fifth 31.2 ± 0.63* 42.2 ± 0.99* Thirtieth 31.3 ± 0.95 41.6 ± 0.63* ctivity of blood serum of rats under conditions of oxidative stress and action of Sylimevit, % (M ± m; n = Lysozyme activity of blood serum of rats under conditions of oxidative stress and action of Sylimevi Table 2 Table 2 Bactericidal activity of blood serum of rats under conditions of oxidative stress and action of Sylimevit, % (M ± m; n = 5) Day of Research Group of animals Control Research 1 Research 2 Fifth 30.74 ± 1.22 33.55 ± 0.84 37.68 ± 1.21 Tenth 32.97 ± 1.11 39.21 ± 0.88 Twentieth 27.28 ± 0.98 41.54 ± 0.76** Twenty-fifth 25.41 ± 0.75* 42.36 ± 1.15* Thirtieth 25.37 ± 1.18 41.14 ± 1.22 vity of blood serum of rats under conditions of oxidative stress and action of Sylimevit, % (M ± m; n = 5) able 2 actericidal activity of blood serum of rats under conditions of oxidative stress and action of Sylimevit, % (M During experimental tetrachloromethane intoxication in rats of the second research group, feeding Sylimevit increased the bactericidal activity of blood serum. A probable increase in this indicator was observed from the fifth day, when it increased by 6.94 % compared to the control group. Subsequently, a gradual increase in the bactericidal activity of blood serum was observed, where it increased by 8.47 % on the 10th day of the experiment and by 10.8 % on the 20th day, relative to the control group. It is worth noting that the bactericidal activity of blood serum in rats of the second experimental group was the highest on the 25th day of the experiment. to the penetration of antigens. Circulating immune com- plexes trigger successive chains of pathological changes since the long-term circulation of even a tiny amount of these complexes in body fluids can lead to their accumu- lation in tissues. With the development of oxidative stress caused by the introduction of tetrachloromethane in the rats of the first experimental group, the number of circulating im- mune complexes probably increased from the fifth day of the experiment. Thus, on the 5th and 10th day of the ex- periment, the number of circulating immune complexes in the blood of the first experimental group increased by 59.7 % and 73.2 %, respectively, compared to the number in the control group of rats. The highest number of circu- Under physiological conditions, the formation and presence of circulating immune complexes in fluids is a manifestation of the immune response of the animal body Scientific Messenger LNUVMB. Series: Veterinary sciences, 2023, vol. 25, no 110 133 Науковий вісник ЛНУВМБ імені С.З. Ґжицького. Table 2 Серія: Ветеринарні науки, 2023, т 25, № 110 lating immune complexes was observed on the 25th and 30th day of the experiment (Table 3). imental group of rats was lower than that of the first ex- perimental group throughout the experiment. However, compared to the control group of animals, the level of the studied indicator in the blood of the second experimental group of rats remained high, where on the 5th and 10th day of the experiment, it increased by 41.4 and 30.7 %, respectively. On the 25th and 30th day of the experiment, the level of circulating immune complexes in the blood of rats of the second experimental group decreased to 52.39 ± 1.99 and 51.68 ± 1.24 %. At the same time, this indica- tor was significantly higher in the first experimental group. Detection of a high number of circulating immune complexes in the blood serum of rats of the first research group indicates suppression of the body's immunoreactive system. This results from binding specific antibodies to metabolic products in tetrachloromethane poisoning. Feeding the feed additive “Sylimevit” to rats of the second experimental group during experimental tetrachlo- romethane poisoning contributed to a decrease in the level of circulating immune complexes in their blood compared to sick rats not fed the feed additive. The level of circulat- ing immune complexes in the blood of the second exper- Table 4 Table 4 Phagocytic activity of neutrophils in the blood of rats under conditions of oxidative stress and the effect of the feed additive “Sylimevit”, % (M ± m; n = 5) Day of Research Group of animals Control Research 1 Research 2 Fifth 20.4 ± 1.56 19.1 ± 1.75 19.4 ±1.95 Tenth 17.1 ± 1.50 19.6 ± 2.15 Twentieth 15.2 ± 0.87 19.4 ± 1.27 Twenty-fifth 12.7 ± 1.31* 19.9 ± 1.40 Thirtieth 13.0 ± 0.87* 19.9 ± 1.34 Table 5 The phagocytic index of the blood of rats under conditions of oxidative stress and the effect of the feed additive “Sylimevit”, units (M ± m; n = 5) Day of Research Group of animals Control Research 1 Research 2 Fifth 10.5 ± 1.49 9.2 ± 1.35 10.1 ± 1.12 Tenth 8.4 ± 1.76 9.8 ± 1.81 Twentieth 7.2 ± 1.62 9.4 ± 0.81 Twenty-fifth 6.5 ± 0.95 9.9 ± 0.78 Thirtieth 6.8 ± 0.71 10.3 ± 0.56 Table 4 Phagocytic activity of neutrophils in the blood of rats under conditions of oxidative stress and the effect of the feed additive “Sylimevit”, % (M ± m; n = 5) Day of Research Group of animals Control Research 1 Research 2 Fifth 20.4 ± 1.56 19.1 ± 1.75 19.4 ±1.95 Tenth 17.1 ± 1.50 19.6 ± 2.15 Twentieth 15.2 ± 0.87 19.4 ± 1.27 Twenty-fifth 12.7 ± 1.31* 19.9 ± 1.40 Thirtieth 13.0 ± 0.87* 19.9 ± 1.34 Table 4 Phagocytic activity of neutrophils in the blood of rats under conditions of oxidative stress and the effect of the feed additive “Sylimevit”, % (M ± m; n = 5) Phagocytic activity of neutrophils in the blood of rats under conditions of oxidative stress and the effect of the feed additive “Sylimevit”, % (M ± m; n = 5) Table 3 Table 3 Circulating immune complexes in the blood of rats under conditions of oxidative stress and the effect of the feed additive “Sylimevit”, mmol/l (M ± m; n = 5) Circulating immune complexes in the blood of rats under conditions of oxidative stress and the effect of the feed additive “Sylimevit”, mmol/l (M ± m; n = 5) y , ( ; ) Day of Research Group of animals Control Research 1 Research 2 Fifth 42.31±1.14 67.58 ± 2.19* 59.81 ± 1.35* Tenth 73.26 ± 3.19* 55.31 ± 1.57 Twentieth 73.89 ± 2.18* 53.47 ± 1.92 Twenty-fifth 74.67 ± 2.27* 52.39 ± 1.99 Thirtieth 74.55 ± 2.11* 51.68 ± 1.24*** of the experiment. Compared with the indicators of the control group, the phagocytic activity of neutrophils in the blood of the first experimental group decreased by 7.7 and 7.4 %, respectively. In sick rats of the first research group, in addition to a decrease in the activity of the immune system's humoral link, suppression of the immune system and the nonspe- cific link of the immune system were also detected. This is manifested in a decrease in the phagocytic activity of neutrophils and a decrease in the phagocytic index (tables 4 and 5). When feeding the feed additive “Sylimevit” to the rats of the second experimental group, an increase in the phagocytic activity of neutrophils was established, where, accordingly, on the 20th day of the experiment, this indi- cator increased by 4.2 %, and on the 25th day – by 7.2 % compared to the first experimental group. ) It was established that in rats experimentally induced to develop oxidative stress, the phagocytic activity of neutrophils probably decreased on the 25th and 30th day Scientific Messenger LNUVMB. Series: Veterinary sciences, 2023, vol. 25, no 110 Conclusion Kisera, Y. V., Martyniv, Y. V., & Gutyj, B. V. (2021). Dynamics of morphological, immunological and his- tological changes in microsporіа in guinea pigs. Regu- latory Mechanisms in Biosystems, 12(2), 206–211. DOI: 10.15421/022129. The introduction of tetrachloromethane in experi- mental groups of rats led to the development of oxidative stress caused by specific chemical processes occurring in the body. During the development of oxidative stress in rats caused by the introduction of carbon tetrachloride, suppression of the humoral and nonspecific links of the immune system was established, which indicates a de- crease in the bactericidal and lysozyme activity of blood serum, the phagocytic index, and the phagocytic activity of neutrophils. At the same time, an increase in the num- ber of circulating immune complexes was observed. Krempa, N. Y., Kozenko, O. V., Chornyj, M. V., Gutyj, B. V., & Martyshuk, T. V. (2021). Immune status of young pigs different methods of their breeding using means Globigen® Pig Doser and Globigen® Jump Start. Scien- tific Messenger of Lviv National University of Veteri- nary Medicine and Biotechnologies. Series: Veterinary sciences, 23(104), 23–29. DOI: 10.32718/nvlvet10404 Kuljaba, O., Stybel, V., Gutyj, B., Peleno, R., Semaniuk, V., Busol, L., Leskiv, K., Semaniuk, N., Pryima, O., Mazur, I., & Turko, Y. (2022). The effect of butaselmevit and closaverm A on the immune status of cows with experi- mental fasciolosis sensitized by atypical mycobacteria. Scientific Messenger of LNU of Veterinary Medicine and Biotechnologies. Series: Veterinary Sciences, 24(108), 82–85. DOI: 10.32718/nvlvet10812. In addition, it was established that the feeding of the feed additive “Sylimevit” led to the strengthening of the immune defense of the body of rats that were poisoned with tetrachloromethane. “Sylimevit” feed additive con- tributed to the increase of the body's immune response, helping to strengthen the protective mechanisms against tetrachloromethane poisoning. Kushnir, V., Kushnir, I., Gutyj, B., Kutsan, O., Nychyk, S., Simonov, M., & Guta, Z. (2023). Comparative as- sessment of different methods of studying skin toxici- ty of powder for wounds. Scientific Messenger of LNU of Veterinary Medicine and Biotechnologies. Series: Veterinary Sciences, 25(109), 13–18. DOI: 10.32718/nvlvet10903. Table 5 Table 5 The phagocytic index of the blood of rats under conditions of oxidative stress and the effect of the feed additive “Sylimevit”, units (M ± m; n = 5) Day of Research Group of animals Control Research 1 Research 2 Fifth 10.5 ± 1.49 9.2 ± 1.35 10.1 ± 1.12 Tenth 8.4 ± 1.76 9.8 ± 1.81 Twentieth 7.2 ± 1.62 9.4 ± 0.81 Twenty-fifth 6.5 ± 0.95 9.9 ± 0.78 Thirtieth 6.8 ± 0.71 10.3 ± 0.56 index of the blood of rats under conditions of oxidative stress and the effect of the feed additive ts (M ± m; n = 5) 134 134 Науковий вісник ЛНУВМБ імені С.З. Ґжицького. Серія: Ветеринарні науки, 2023, т 25, № 110 (2017). The influence of brovitatoxide in conjunction with milk thistle fruits on the immune system of tur- keys for eimeriozic invasion. Scientific Messenger of LNU of Veterinary Medicine and Biotechnologies. Series: Veterinary Sciences, 19(73), 163–168. DOI: 10.15421/nvlvet7334. (2017). The influence of brovitatoxide in conjunction with milk thistle fruits on the immune system of tur- keys for eimeriozic invasion. Scientific Messenger of LNU of Veterinary Medicine and Biotechnologies. Series: Veterinary Sciences, 19(73), 163–168. DOI: 10.15421/nvlvet7334. When examining the phagocytic index in experimental rats injected with tetrachloromethane, a decrease of 20 and 31.4 % compared to the control group was established on the 10th and 20th day of the experiment. The lowest phagocytic index was in the blood of rats of the first ex- perimental group on the 25th and 30th day of the experi- ment, where relative to the control group, it decreased by 38.1 and 35.2 % (Table 5). Khariv, M., Gutyj, B., Ohorodnyk, N., Vishchur, O., Khariv, I., Solovodzinska, I., Mudrak, D., Grymak, C., Bodnar, P. (2017). Activity of the T- and B-system of the cell immunity of animals under conditions of oxi- dation stress and effects of the liposomal drug. Ukrainian Journal of Ecology, 7(4), 536–541. URL: https://www.ujecology.com/articles/activity-of-the-t-and- bsystem-of-the-cell-immunity-of-animals-under- conditions-of-oxidation-stress-and-effects-of-the-li.pdf. When rats were fed Sylimevit under conditions of ox- idative stress, a slight decrease in the phagocytic index was established on the 20th and 25th days of the experi- ment. On the 30th day of the experiment, PhI in the blood of rats of the second experimental group reached physio- logical limits. conditions-of-oxidation-stress-and-effects-of-the-li.p Conflict of interest The authors declare that there is no conflict of interest. References Daëron, M. (2022). The immune system as a system of relations. Front Immunol, 13, 984678. DOI: 10.3389/ fimmu.2022.984678. Lesyk, Y. V., Dychok-Niedzielska, A. Z., Boiko, О. V., Honchar, О. F., Bashchenko, М. І., Kovalchuk, І. І., & Gutyj, B. V. (2022). Hematological and biochemical pa- rameters and resistance of the organism of mother rabbits receiving sulfur compounds. Regulatory Mechanisms in Biosystems, 13(1), 60–66. DOI: 10.15421/022208. Gutyj, B. V., Varkholiak, I. S., Verveha, B. M., Mar- tyshuk, T. V., & Leskiv, K. Y. (2023). The antioxidant protection system state of rats under experimental doxorubicin intoxication and the effects of correcting factors. Medical and Clinical Chemistry, 1, 34–41. DOI: 10.11603/mcch.2410-681X.2023.i1.13714. Martyshuk, T. V., & Hutyi, B. V. (2021). Imunofiziolo- hichnyi stan ta antyoksydantnyi potentsial orhanizmu porosiat za umov oksydatsiinoho stresu ta dii koryhui- uchykh chynnykiv: monohrafiia. Lviv: SPOLOM (in Ukrainian). Gutyj, B., Martyshuk, T., Khariv, I., & Guta, Z. (2022). The immune status of the organism of bulls under cadmium load and the effects of correcting factors. EUREKA: Life Sciences, 4, 3–9. DOI: 10.21303/ 2504-5695.2022.002622. Martyshuk, T. V., Gutyj, B. V., & Khalak, V. I. (2021). System of antioxidant protection of the body of piglets under the action of feed additive “Butaselmevit-plus”. Ukrainian Journal of Veterinary and Agricultural Sci- ences, 4(2), 38–43. DOI: 10.32718/ujvas4-2.07. Hillion, S., Arleevskaya, M. I., Blanco, P., Bordron, A., Brooks, W. H., Cesbron, J. Y., Kaveri, S., Vivier, E., & Renaudineau, Y. (2020). The Innate Part of the Adap- tive Immune System. Clin Rev Allergy Immunol, 58(2), 151–154. DOI: 10.1007/s12016-019-08740-1. Martyshuk, T. V., Gutyj, B. V., Zhelavskyi, M. M., Midyk. S. V., Fedorchenko, A. M., Todoriuk, V. B., Nahirniak, T. B., Kisera, Ya. V., Sus, H. V., Chem- erys, V. A., Levkivska, N. D., & Iglitskej, I. I. (2020). Khariv, I., Gutyj, B., Hunchak, V., Slobodyuk, N., Vynyarska, A., Sobolta, A., Todoriuk, V., & Seniv, R. Scientific Messenger LNUVMB. Series: Veterinary sciences, 2023, vol. 25, no 110 135 Науковий вісник ЛНУВМБ імені С.З. Ґжицького. Серія: Ветеринарні науки, 2023, т 25, № 110 Effect of Butaselmevit-Plus on the immune system of piglets during and after weaning. Ukrainian Journal of Ecology, 10(2), 347‒352. DOI: 10.15421/2020_106. Place, D. E., & Kanneganti, T. D. (2020). The innate immune system and cell death in autoinflammatory and autoimmune disease. Curr Opin Immunol, 67, 95– 105. DOI: 10.1016/j.coi.2020.10.013. 105. DOI: 10.1016/j.coi.2020.10.013. McComb, S., Thiriot, A., Akache, B., Krishnan, L., & Stark, F. (2019). Scientific Messenger LNUVMB. Series: Veterinary sciences, 2023, vol. 25, no 110 136 References Introduction to the Immune System. Methods in Molecular Biology, 2024, 1–24. DOI: 10.1007/978-1-4939-9597-4_1 Radzykhovskyi, M., Sokulskiy, I., Dyshkant, O., Antoniuk, A., Gutyj, B., & Sachuk, R. (2022). Experimental study of tropism of cultivated canine parvovirus in the immu- nogenesis organs of puppies. Regulatory Mechanisms in Biosystems, 13(3), 241–246. DOI: 10.15421/022231. 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https://openalex.org/W2608690647	http://bura.brunel.ac.uk/bitstream/2438/14460/5/Fulltext.pdf	English	null	A generalisation of the Hill's quadratic yield function for planar plastic anisotropy to consider loading direction	International journal of mechanical sciences	2,017	cc-by	16,160	1. Introduction crystallographic slip systems, which is basically a consequence of the movement of dislocations. In FCC materials, compressive and tensile strengths are virtually identical and yielding is not inﬂuenced by the hydrostatic pressure as well. The yield surface of such materials is usually represented adequately by an even function of the principal values of the deviatoric stresses (e.g. Hershey [19] and Hosford [21]). Hexagonal Close-Packed (HCP) materials, such as magnesium and titanium alloys, have less active slip systems at low/room temperatures but they have additional twinning systems that accommodate plastic deformation by a diﬀerent mechanism known as twinning or distortion of the lattice. Twinning is a polar deformation mechanism (it only develops in one direction) and this is the main reason for the asymmetric behaviour observed on HCP alloys in tension-compression. Material modelling is very important not only for the development of new metal alloys but also for the simulation of manufacturing processes. Within material modelling, plasticity plays a fundamental role and its description is essential for the accurate design of manu- facturing processes. In plasticity, yield functions are critical because they provide the yielding point of the material and also when used within an associated ﬂow rule scheme, they describe the plastic ﬂow of the metal accurately. One of the ﬁrst yield functions for plastic anisotropy was developed in the pioneer work by Hill (1948) [20]. Hill developed a quadratic yield function with anisotropic coeﬃcients that could either predict the r-values or directional ﬂow stresses, but never both simultaneously. Moreover, Hill's original yield function does not include the eﬀect of the biaxial symmetric stress and so it is not accurate in the modelling of aluminium alloys. Many posteriori yield functions [2–6] were developed after Hill's and the coeﬃcients of these yield functions were designed to include the biaxial symmetric stress eﬀect. Whilst the equi-biaxial ﬂow stress has been deﬁned in these functions, none of them have characterised the coeﬃcients for the unsymmetric biaxial stress state between pure uniaxial loading and equi-biaxial loading. For the description of incompressible plastic anisotropy, many yield functions have been suggested based on the isotropic hardening assumption (Hill [20], Barlat and Lian [2], Barlat et al. [3], Karaﬁllis and Boyce [23]). Among them, Cazacu and Barlat [10] introduced a general formulation which originated from the rigorous theory of representation of tensor functions. A R T I C L E I N F O Keywords: Quadratic yield function NURBS Planar anisotropy R-values Flow stresses Earing proﬁle Cup drawing In this work, a new generalised quadratic yield function for plane stress analysis that is able to describe the plastic anisotropy of metals and also the asymmetric behaviour in tension-compression typical of the Hexagonal Closed-Pack (HCP) materials, is developed. The new yield function has a quadratic form in the stress tensor and it simultaneously predicts the r-values and directional ﬂow stresses, which is shown to agree very well with experimental results. It also accurately describes the biaxial symmetric stress state which is fundamental for the accurate modelling of aluminium alloys. The new quadratic yield function represents the non-symmetric biaxial stress state by performing a linear interpolation from pure uniaxial loading to a biaxial symmetric stress state. The main advantages of this new yield function is that it can be used for the modelling of metals with any crystallographic structure (BCC, FCC or HCP), it only has ﬁve anisotropic coeﬃcients and also that it is a simple quadratic yield criterion that is able to accurately reproduce the plastic anisotropy of metals whilst using an associated ﬂow rule. A generalisation of the Hill's quadratic yield function for planar plastic anisotropy to consider loading direction ARK R.P.R. Cardosoa,⁎, O.B. Adetorob R.P.R. Cardosoa,⁎, O.B. Adetorob a Brunel University London, Uxbridge, UB8 3PH London, UK b University of the West of England, BS16 1QY Bristol, UK ⁎ Corresponding author. International Journal of M echanical Sciences 128–129 (2017) 253–268 International Journal of M echanical Sciences 128–129 (2017) 253–268 Contents lists available at ScienceDirect http://dx.doi.org/10.1016/j.ijmecsci.2017.04.024 Received 31 January 2017; Received in revised form 28 March 2017; Accepted 24 April 2017 Available online 27 April 2017 0020-7403/ © 2017 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecom m on e Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecom m ons.org/licenses/BY/4.0/). 1. Introduction However with this approach, the conditions for the convexity of the yield surface are diﬃcult to derive and impose. The convexity has a physical basis and, in addition, this property ensures numerical stability in computer simulations. For this reason, a particular case of this general theory, which is based on linearly transformed stress components has received more interest from The predominant deformation mechanism in Face-Cubic-Centred metals (FCC) such as aluminium alloys, is deformation by slip in the R.P.R. Cardoso, O.B. Adetoro International Journal of M echanical Sciences 128–129 (2017) 253–268 Most of the early developed phenomenological yield potentials (e.g. von-Mises [38] and Hill's [20]) are quadratic in the stress tensor. These yield potentials were mainly designed from distortion energy balance equations, and they were developed primarily for steel alloys, with Hill's 1948 [20] going a step further by including plastic anisotropy in the potential. It is widely accepted that these potentials ﬁt the yield locus very well for steel, but are unable to accurately predicting the anomalous behaviour of aluminium alloys (Dodd and Caddell [16]), especially in reproducing the yield locus on the vicinity of the symmetric biaxial stress state. There are two ways of accurately model aluminium alloys: i) by using non-quadratic yield functions with associated ﬂow rules; ii) by using quadratic yield functions with non- associated ﬂow rules (Stoughton and Yoon [37]), where in this case a plastic potential needs to be deﬁned for the plastic ﬂow. The use of non- associated ﬂow rules allows for the use of simpler yield potentials, such as the quadratic potential of Hill 1948 [20], but a second plastic potential needs to be used for the plastic ﬂow. The use of two diﬀerent potentials in the non-associated ﬂow rule can however lead to diﬃculties during return mapping procedures, especially if the loci of the two potentials (yield and plastic potential) are of considerably diﬀerent shapes. Considering however the ﬂexibility in phenomenolo- gical modelling, it must be possible to develop a quadratic generalised yield function for simultaneously predicting r-values and directional ﬂow stresses accurately as opposed to the individual treatment that has been adopted so far. It also follows to say that it must be possible to simultaneously match the r-values and directional ﬂow stresses for any stress state, as for example under planar anisotropy assumption. 1. Introduction This generalised yield function must be able to accurately predict the anomalous aluminium behaviour [16] and the symmetric biaxial stress state. Certainly, the yield function must be accurate for a wide range of cases and valid if, and only if, it is proven to be convex in the principal stress space. Therefore, the main ideas for the new yield function proposed in this paper are as follows: the metal forming and material modelling communities in general. Barlat et al. [3] applied this method to a full stress state in an orthotropic material and Karaﬁllis and Boyce [23] generalised it as the so-called isotropic plasticity equivalent theory with a more general yield function and a linear transformation that can accommodate lower material symmetry. Cazacu et al. [11] proposed a criterion based on a linear transfor- mation that accounts for the strength-diﬀerential eﬀect, particularly prominent in Hexagonal Closed-Pack (HCP) materials, with the work being extended in Plunkett et al. [31] and Plunkett et al. [30] by including the eﬀect of texture development in the yield function. Barlat et al. [5,6] later introduced two linear transformations which were applied on the sum of two yield functions in the case of plane and general stress states, in order to improve the accuracy of the functions by Cazacu et al. [11], Plunkett et al. [31] and Plunkett et al. [30] in the modelling of the anisotropic behaviour of aluminium sheets. Bron and Besson [8] further extended Karaﬁllis and Boyce's approach to two linear transformations. These recently proposed yield functions include more anisotropy coeﬃcients and therefore give a better description of the anisotropic properties of a material. Although the mathematical formulations are complex and very heavy from a computational point of view. These reported developments in yield functions have been particularly important for the study of the formability of sheet metals as shown in the works of Kuroda and Tvergaard [25], Stoughton and Yoon [36], Lou et al. [28] and Dasappa et al. [14]. Apart from the phenomenological studies listed above, another approach for the prediction of plastic anisotropy and strain hardening is the one based on polycrystal plasticity models. Commercial aluminium and magnesium alloys such as the aluminium AA6022 and AA2090 and the magnesium AZ31B used in this paper and used generally in forming operations, are polycrystalline materials composed of numerous grains each with a given lattice orientation with respect to macroscopic axes. 1. Introduction At low temperatures, metals and alloys deform by dislocation glide or slip on given crystallographic planes and directions thereby producing microscopic shear deformations (Kocks et al. [24]). Therefore, the distribution of grain orientations and crystallographic texture in gen- eral, play an important role in the study and in the modelling of plasticity. Due to the geometrical nature of slip deformations, strain incompatibilities usually arise between grains thereby producing micro- residual stresses, which from a macroscopic point of view, can lead to the well-known Bauschinger eﬀect. Slip results in gradual lattice rotation where dislocations accumulate at micro-structural barriers, increasing the slip resistance and consequently strain hardening. There is also another crystallographic deformation mechanism that is very typical of HCP materials which is the twinning. Proust et al. [32] developed the modelling of texture, twinning and hardening evolution of hexagonal materials by using the well-known Visco-Plastic Self- Consistent (VPSC) approach (Lebensohn and Tome [26]), where the interaction between a grain and its surrounding eﬀective medium is taken into account. Polycrystal models can be used in multi-scale simulations of metal forming operations, but they are usually expensive in computational time. • A new quadratic yield function is developed for the simultaneous prediction of r-values and directional ﬂow stresses and its convexity is proven in Appendix A for the case of proportional loading; • This new model can simultaneously predict the r-values and directional ﬂow stress accurately for any given angle from the rolling direction; • The biaxial symmetric ﬂow stress is incorporated in this new quadratic yield function (this is detailed in Section 2.1). 1. Introduction However, the biaxial r-value is not included in this formulation; the biaxial r-value is not included in this formulation; • It is postulated that the stress tensor changes in a linear manner between symmetric biaxial stress state and unaxial stress state, hence it is included in the new quadratic yield function in an interpolatory manner (details given in Section 2.1); • Consequently due to this new quadratic yield function, it is possible to simultaneous predict r-values and directional ﬂow stress from the use of an associated ﬂow rule; The main objective of this research work is therefore to develop the yield function for plane stress analysis as general as possible so that it can work with associated ﬂow rules for the modelling of planar anisotropy for both FCC and HCP materials and also that it is able to describe the asymmetric behaviour in tension-compression typical of HCP materials. Some noteworthy studies that use dislocation motion, micro-struc- tural grain size and shape data for the prediction of the yield strength of metallic alloys include the studies by Esmaeili et al. [17] and Balogh et al. [1], while some other studies perform a macroscopic study on the anisotropy of aluminium sheets from the consideration of morphologi- cal texture and crystallographic texture evolution (Choi et al. [12]). Polycrystal modelling aspects have been treated in a large number of publications and books such as in Kocks et al. [24], Gambin [18] and Dawson [15]. More recently, crystallographic plasticity has been extensively used in several numerical simulations, because it naturally predicts texture evolution and anisotropy, Bauschinger eﬀect, transient behaviour and permanent softening. However, their computational cost is still prohibitive when compared to the use of phenomenological constitutive models. 2.1. Incorporation of the biaxial symmetric ﬂow stress H r r G r N r r r r r = 2 1 + = 2 1 + = 2( + ) (2 + 1) 2 (1 + ) 0 0 0 0 90 45 90 0 (3) H r r G r N r r r r r = 2 1 + = 2 1 + = 2( + ) (2 + 1) 2 (1 + ) 0 0 0 0 90 45 90 0 (3) H r r G r N r r r r r = 2 1 + = 2 1 + = 2( + ) (2 + 1) 2 (1 + ) 0 0 0 0 90 45 90 0 (3) (3) The r-value anisotropic coeﬃcient F u ( ) will be calculated from the use of a Non-Uniform Rational B-Spline (NURBS) approximation on the parametric coordinate u. For that purpose, it will be necessary to calculate ﬁrst the r-value anisotropic coeﬃcient F at every 15° from the rolling direction and then use the NURBS approximation to build a function F u ( ) that can generate the F-coeﬃcient for any loading orientation θ. The Hill's 1948 [20] formulae for this coeﬃcient can be used as presented in the following equation: The material calibration in most of well-known yield functions requires several mechanical tests: uniaxial tests for r-values and directional ﬂow stresses and mechanical tests for the equi-biaxial stress (or bulge test) and the disk compression for the equi-biaxial r-value. The results from these tests are then included in optimisation algo- rithms for the calculation of the anisotropic coeﬃcients, a procedure that is very common for example with the Barlat yield functions for aluminium [5,6]. The stress tensor as one transits from a symmetric biaxial state through an unsymmetric biaxial state to a uniaxial stress state remains unknown, however it is postulated in this work that a linear variation is valid. Hence an interpolation scheme is thus proposed between a uni-axial stress and a biaxial symmetric ﬂow stress. The Mohr circle from Fig. 2 shows how far the stress state is from uniaxial stress conditions, or alternatively, how close it is to symmetric biaxial stress state. If σ = 0 2 we have uniaxial stress state and if σ σ = 2 1 we then have symmetric biaxial stress state. 2.1. Incorporation of the biaxial symmetric ﬂow stress The model deﬁned so far does not consider the biaxial symmetric yield stress. If the stress tensor deviates considerably from the uniaxial stress state (deﬁned by the principal stresses σ > 0 1 and σ = 0 2 for tension or σ < 0 2 and σ = 0 1 for compression), the diﬀerences in the accuracy can be substantial and this is more severe when the stress tensor is closer to the biaxial symmetric stress state. So, the incorporation of the biaxial symmetric ﬂow stress in the model is important. A generalised yield function in the normalised principal space, σ σ σ σ / − / 1 2 is shown in Fig. 1 and for each quadrant, the Mohr's circle with the possible loading directions is depicted. In the ﬁrst quadrant (σ > 0 1 and σ > 0 2 ) it is possible to have a uniaxial tensile stress tensor deﬁned with angle θ from the rolling direction, a biaxial tensile stress state and a biaxial symmetric tensile stress state, which is not represented by a circle but rather by a dot (σ σ = 1 2). In the second and forth quadrant, the applied loading leads to a shear stress state (σ > 0 1 and σ < 0 2 or σ < 0 1 and σ > 0 2 ) and in the third quadrant it is possible to have a uniaxial compressive stress tensor deﬁned with angle θ from the rolling direction, a biaxial compressive stress state and a biaxial symmetric compressive stress state. 2. Model formulation This parametric variable u exists within the limits u 0.0 ≤ ≤1.0, with u=0.0 deﬁning the rolling direc- tion, u=0.125 being 45° from the rolling direction, u=0.25 deﬁning 90° with the rolling direction and ﬁnally u=1.0 again deﬁning the rolling direction. The coeﬃcient C u ( ) a is designed to ﬁt the yield stresses while the coeﬃcient F u ( ) is designed to ﬁt the r-values. In this work, the functions for these two anisotropic coeﬃcients are described from the use of Non-Uniform Rational B-Splines (NURBS) but they can also be described from the use of any other type of functions as far as the accurate values for the coeﬃcients are conserved. The main reasons for the use of NURBS are essentially related to the local compact support of NURBS which is explained in more detail later in this manuscript. It is also demonstrated in Appendix A that the new yield potential for plane stress analysis delivers a convex yield locus, which is a fundamental prerequisite for a stable stress integration procedure in elasto-plastic material modelling. The Hill's coeﬃcients H, G and N are obtained from the experimental r-values at 0°, 45° and 90° from the rolling direction [20]: C u σ σ G H θ F u H θ N H θ θ F u G H ( ) = 3 2 ( + ) cos + [ ( ) + ] sin + 2 ( − ) cos sin ( ) + + a θ 4 4 2 2 (9) where σ σ θ is the normalised ﬂow stress at direction θ from the rolling direction obtained from experimental data. where σ σ θ is the normalised ﬂow stress at direction θ from the rolling direction obtained from experimental data. 2. Model formulation (2) the following is obtained for coeﬃcient C u ( ) a : and after replacing Eq. (8) into Eq. (2) the following is obtained for coeﬃcient C u ( ) a : where C u ( ) a is a new coeﬃcient which deﬁnes the anisotropy in the yield stresses and the anisotropy for the r-values is conserved from the adaptation of coeﬃcient F from the original Hill's model by making it variable. Both coeﬃcients are a function of a parametric coordinate u, which represents the orientation of the loading direction when mea- sured from the rolling direction. This parametric variable u exists within the limits u 0.0 ≤ ≤1.0, with u=0.0 deﬁning the rolling direc- tion, u=0.125 being 45° from the rolling direction, u=0.25 deﬁning 90° with the rolling direction and ﬁnally u=1.0 again deﬁning the rolling direction. The coeﬃcient C u ( ) a is designed to ﬁt the yield stresses while the coeﬃcient F u ( ) is designed to ﬁt the r-values. In this work, the functions for these two anisotropic coeﬃcients are described from the use of Non-Uniform Rational B-Splines (NURBS) but they can also be described from the use of any other type of functions as far as the accurate values for the coeﬃcients are conserved. The main reasons for the use of NURBS are essentially related to the local compact support of NURBS which is explained in more detail later in this manuscript. It is also demonstrated in Appendix A that the new yield potential for plane stress analysis delivers a convex yield locus, which is a fundamental prerequisite for a stable stress integration procedure in elasto-plastic material modelling. The Hill's coeﬃcients H, G and N are obtained from the experimental r-values at 0°, 45° and 90° from the rolling direction [20]: where C u ( ) a is a new coeﬃcient which deﬁnes the anisotropy in the yield stresses and the anisotropy for the r-values is conserved from the adaptation of coeﬃcient F from the original Hill's model by making it variable. Both coeﬃcients are a function of a parametric coordinate u, which represents the orientation of the loading direction when mea- sured from the rolling direction. 2. Model formulation In this formulation, the well-established Hill's [20] yield potential is used and extra ﬂexibility is introduced in some coeﬃcients in order to achieve a fully generalised function. Hill [20] proposed a yield function that can be used for the study of the planar anisotropy of metals and for which the equivalent stress is deﬁned as: 254 International Journal of M echanical Sciences 128–129 (2017) 253–268 R.P.R. Cardoso, O.B. Adetoro and the angle θ can be easily obtained from the Mohr's circle or from the equation for the principal directions from plane stress analysis: σ G H σ F H σ Hσ σ Nτ F G H = 3 2 ( + ) + ( + ) −2 + 2 + + xx yy xx yy xy 2 2 2 (1) (1) θ τ σ σ tan (2 ) = 2 − xy xx yy (7) θ τ σ σ tan (2 ) = 2 − xy xx yy The coeﬃcients F, G, H and N are designed to ﬁt the r-values or, alternatively, the directional ﬂow stresses but never both in simulta- neous. This is a major limitation in Hill's yield potential because both ﬁtting is required for the accurate modelling of planar plastic aniso- tropic metals. Hill's yield potential has however some great advantages which are its quadratic form and the simplicity of the model for the description of plastic anisotropy. (7) The coeﬃcient C u ( ) a will be calculated from the new yield function from Eq. (2) and from the yield stresses σθ deﬁned at every 15° from the rolling direction. From the stress transformation of the uniaxial loading to the anisotropic axes we can get: σ σ θ σ σ θ τ σ θ θ = cos = sin = cos sin xx θ yy θ xy θ 2 2 (8) Therefore, a new yield function (YldParam) is deﬁned as: σ C u G H σ F u H σ Hσ σ Nτ F u G H = 1 ( ) 3 2 ( + ) + [ ( ) + ] −2 + 2 ( ) + + a xx yy xx yy xy 2 2 2 (2) (8) (2) and after replacing Eq. (8) into Eq. (2) the following is obtained for coeﬃcient C u ( ) a : and after replacing Eq. (8) into Eq. 2.2. Iso-Shear contours for the yield locus Another coeﬃcient C u ( ) c can be introduced for greater ﬂexibility of the yield locus for diﬀerent (non-zero) iso-shear contour levels. This new coeﬃcient can be added to the linear interpolation for the biaxial symmetric ﬂow stress as described in Section 2.1. It is associated with the shear stress τxy in the following way: σ C u β C u = 1 ( , ) + ( ) 3 2 × × c τ σ G H σ F u H σ Hσ σ Nτ F u G H ( + ) + [ ( ) + ] −2 + 2 ( ) + + xy xx yy xx yy xy 0 2 2 2 (14) σ C u β C u = 1 ( , ) + ( ) 3 2 × × c τ σ G H σ F u H σ Hσ σ Nτ F u G H ( + ) + [ ( ) + ] −2 + 2 ( ) + + xy xx yy xx yy xy 0 2 2 2 (14) (14) where σ0 is the initial yield stress at 00 with the rolling direction. An example for the coeﬃcient C u ( ) c for the Al2090 aluminium alloy used in the example of Section 5.1.2 is depicted in Fig. 3. Fig. 2. Mohr's circle for a generalised biaxial stress state. C u β β C u β C u ( , ) = · ( ) + (1 − ) · ( ) a b C u β β C u β C u ( , ) = · ( ) + (1 − ) · ( ) a b (10) Fig. 3. Characterisation for the coeﬃcient C u ( ) c for the Al2090 aluminium alloy. Fig. 3. Characterisation for the coeﬃcient C u ( ) c for the Al2090 aluminium alloy. where: where: β σ σ σ = − 1 2 1 (11) for biaxial tension and: for biaxial tension and: β σ σ σ = − 2 1 2 (12) (12) for biaxial compression. For β = 0 we have symmetric biaxial stress state and for β = 1 we have uniaxial stress. For β 0 < < 1 we have a stress state somewhere between uniaxial and symmetric biaxial. Thus, the new quadratic yield function from Eq. 2.1. Incorporation of the biaxial symmetric ﬂow stress We can therefore inter- polate between these two stress states by introducing a parameter β deﬁned in Eqs. (11) and (12) that represents the deviation from a symmetric biaxial stress state. F θ H θ θ G θ θ r θ N θ θ θ θ r θ ( ) = (1 −4 sin cos ) − (sin cos + cos ) + 2 sin cos sin cos + sin θ θ 2 2 2 2 2 2 2 2 2 2 (4) This function for F θ ( ) is singular at 0° and so the following alternative function was used for the calculation of the coeﬃcient at 0°: F r r r (0°) = 2 (1 + ) 0 90 0 (5) F r r r (0°) = 2 (1 + ) 0 90 0 (5) For the NURBS approximation for both C u ( ) a and F u ( ) the following relation between the angle from the rolling direction and the para- metric coordinate u is necessary: For the NURBS approximation for both C u ( ) a and F u ( ) the following relation between the angle from the rolling direction and the para- metric coordinate u is necessary: Therefore, we can deﬁne a coeﬃcient C u ( ) b for the biaxial sym- metric stress state and the coeﬃcient C u ( ) a becomes: θ πu = 2 (6) (6) θ πu = 2 θ πu = 2 255 International Journal of M echanical Sciences 128–129 (2017) 253–268 R.P.R. Cardoso, O.B. Adetoro Fig. 1. Yield function and Mohr's circles at diﬀerent quadrants representing diﬀerent loading directions. Fig. 1. Yield function and Mohr's circles at diﬀerent quadrants representing diﬀerent loading directions. Fig. 2. Mohr's circle for a generalised biaxial stress state. Fig. 2. Mohr's circle for a generalised biaxial stress state. 3. Non-uniform rational B-spline (NURBS) for the representation of the non-constant coeﬃcients The basis functions are non-negative, that is, u N u ∀ → ( ) ≥0 J p ; 4. Aﬃne invariance. 4. Aﬃne invariance. 4. Aﬃne invariance. (22) In Eq. (22), N u ( ) I are the NURBS basis functions which are used to approximate the coeﬃcients F u ( ), C u ( ) a and C u ( ) b . FI, CaI and CbI are the control points, whose deﬁnition can be found in the works of Piegl and Tiller [29]. A detailed description on how these control points are obtained for the current work can be found in Appendix B. The use of the same basis functions for all coeﬃcients is the ﬁrst advantage of the use of NURBS. Another important advantage of using NURBS is that they have local compact support, i.e. for a particular u in the parametric domain, only p + 1 control points in the domain of inﬂuence need to be used because outside of this domain of inﬂuence all other NURBS approximation functions are zero. In this work, p=2 is used for the NURBS basis functions. In Eq. (22), N u ( ) I are the NURBS basis functions which are used to approximate the coeﬃcients F u ( ), C u ( ) a and C u ( ) b . FI, CaI and CbI are the control points, whose deﬁnition can be found in the works of Piegl and Tiller [29]. A detailed description on how these control points are obtained for the current work can be found in Appendix B. The use of the same basis functions for all coeﬃcients is the ﬁrst advantage of the use of NURBS. Another important advantage of using NURBS is that they have local compact support, i.e. for a particular u in the parametric domain, only p + 1 control points in the domain of inﬂuence need to be used because outside of this domain of inﬂuence all other NURBS approximation functions are zero. In this work, p=2 is used for the NURBS basis functions. 3.1. Knot vector An open knot vector is a set of non-negative parametric coordinates which are repeated p + 1 times at the beginning and at the end of the vector (p is the order of the polynomial basis functions). For one- dimensional basis functions of order p, the following generic open knot vector can be deﬁned: U u u u u = { ,…, ,…, ,…, } p m m p 1 +1 +1 + +1 (16) (16) where m is the number of control points or basis functions. The basis functions of order p have p −1 continuous derivatives. More than one knot can be considered at the same parametric coordinate and it is thus referred as a repeated knot. An important property of repeated knots is that the continuous derivative of their basis functions is decreased by the number of times the knot is repeated. Also, the basis functions are interpolatory only if the knot's multiplicity is the same as the polynomial's order p [29]. For this quadratic yield function, the knot vector is deﬁned for the limits u 0.0 ≤ ≤1.0, where u=0.0 corresponds to an angle θ = 0° with the rolling direction and u=1.0 corresponds to an angle of θ = 360° with the rolling direction. For instance, for a quadratic degree in the NURBS basis functions (p=2) we have the following knot vector: 2.2. Iso-Shear contours for the yield locus (2) becomes: σ C u β G H σ F u H σ Hσ σ Nτ F u G H = 1 ( , ) 3 2 ( + ) + [ ( ) + ] −2 + 2 ( ) + + xx yy xx yy xy 2 2 2 (13) Fig. 3. Characterisation for the coeﬃcient C u ( ) c for the Al2090 aluminium alloy. Fig. 3. Characterisation for the coeﬃcient C u ( ) c for the Al2090 aluminium alloy. 256 International Journal of M echanical Sciences 128–129 (2017) 253–268 International Journal of M echanical Sciences 128–129 (2017) 253–268 R.P.R. Cardoso, O.B. Adetoro ∑ ∑ ∑ F u N u W F W C u N u W C W C u N u W C W ( ) = ( ) ( ) = ( ) ( ) = ( ) I I I I a I I I aI b I I I bI (20) 4. Return mapping procedure From a phenomenological point of view, the plastic ﬂow can be interpreted as an irreversible process in a material body, typically a metal, characterised in terms of the history of the strain tensor ϵ and two additional variables: the plastic strain ϵp and a suitable set of internal variables α often referred to as hardening parameters. Conventional constitutive laws which represent plastic deformation of metals are typically described by considering three parts: yield func- tions, stress-strain (or hardening) functions and the associated normal- ity ﬂow rule. The yield function describes yield stresses in general deformation states, which are relative values measured with respect to a reference yield stress. The stress-strain function represents the work- hardening behaviour of the reference stress, which is usually a uniaxial or balanced biaxial tension stress. U = {0.0, 0.0, 0.0, 1.0, 1.0, 1.0} (17) (17) 3. Non-uniform rational B-spline (NURBS) for the representation of the non-constant coeﬃcients 3. Non-uniform rational B-spline (NURBS) for the representation of the non-constant coeﬃcients NURBS have been used extensively in geometric modelling because it is able to represent curves and/or surfaces with high complexity in their shape. Piegl [29], Hughes et al. [22] and Bazilevs et al. [7] addressed the most fundamental properties of the NURBS basis func- tions, however the most important attributes for this work are listed below: (20) with: ∑ W N u W = ( ) I I I ∑ W N u W = ( ) I I I (21) (21) 1. They form a partition of unity, i.e.: 1. They form a partition of unity, i.e.: We will use the weights WI equal to 1.0 and because of the partition of unity property of the NURBS basis functions ( N u ∑ ( ) = 1 I I ), the NURBS approximations are reduced to: We will use the weights WI equal to 1.0 and because of the partition of unity property of the NURBS basis functions ( N u ∑ ( ) = 1 I I ), the NURBS approximations are reduced to: ∑N u u U u u ( ) = 1, ∈ = [ , ]; J m J p m p =1 1 + +1 (15) (15) ∑ ∑ ∑ F u N u F C u N u C C u N u C ( ) = ( ) ( ) = ( ) ( ) = ( ) I I I a I I aI b I I bI (22) 2. The support of each N u ( ) J p is compact and contained in u u [ , ] J J p + +1 ; 2. The support of each N u ( ) J p is compact and contained in u u [ , ] J J p + +1 ; 3 The basis functions are non-negative that is u N u ∀ → ( ) ≥0 J p ; 2. The support of each N u ( ) J p is compact and contained in u u [ , ] J J p + +1 ; p 3. The basis functions are non-negative, that is, u N u ∀ → ( ) ≥0 J p ; p 3. 5.1. r-values and directional ﬂow stresses for FCC materials In the forward-Euler scheme for return mapping, the stress tensor can be corrected from the predictor stage as follows: σ σ ϵ σ σ γ σ D D = − = − Δ ∂ ∂ t t trial t p trial +Δ Δ (30) (30) The r-value is by deﬁnition obtained from the plastic strain in the width direction over the plastic strain along the thickness direction, i.e.: The r-value is by deﬁnition obtained from the plastic strain in the width direction over the plastic strain along the thickness direction, i.e.: In Fig. 6, the yield locus contours for every 0.5 values of shear stress is shown, where the shape of the diﬀerent yield locus is projected at diﬀerent shear stress planes. Fig. 7 shows a plot of coeﬃcients F u ( ), G, H, N, C u ( ) a and C u ( ) b as a function of the angle with the rolling direction. The coeﬃcients G, H and N are constants but the coeﬃcients F u ( ), C u ( ) a and C u ( ) b are not constant, they are function of the parametric variable u which in turn represents the angle measured from the rolling direction. The plots for the non-constant coeﬃcients is symmetric about 180°, as expected for this alloy because there is no diﬀerence in its behaviour when in tension compared to when in compression. Another important aspect that is noteworthy from the plot of the coeﬃcients in Fig. 7 is the comparison between C u ( ) a and C u ( ) b . It can be seen that these two coeﬃcients are the same at every orientation except at orientations in the vicinity of the symmetric biaxial stress region. This was also expected considering the coeﬃcient C u ( ) a was designed to ﬁt the uniaxial yield (ﬂow) stresses, while the coeﬃcient C u ( ) b was designed for the symmetric biaxial stress state. r = ϵ −(ϵ + ϵ ) θ θ π p θ p θ π p + /2 + /2 (31) (31) and using Eq. (26) and the stress transformation Eq. (8), the following result for the r-value can be obtained: and using Eq. (26) and the stress transformation Eq. 5.1. r-values and directional ﬂow stresses for FCC materials (8), the following result for the r-value can be obtained: r H N F u G H θ θ F u θ G θ = + [2 − ( ) − −4 ] sin cos ( ) sin + cos θ 2 2 2 2 (32) (32) where it can clearly be seen that the r-value can be matched by adjusting the parameter F u ( ) accordingly. σ α γf ̇ ( , ) = 0 ⎛ ⎝⎜ ⎞ ⎠⎟ ϵ σ σ σ γ σ σ C u C u u u σ F u F u u u = Δ ∂ ∂ + ∂ ∂ ( ) ∂ ( ) ∂ ∂ ∂ + ∂ ∂( ) ∂( ) ∂ ∂ ∂ p t Δ (27) where: ∑ ∑ C u u dN u du C F u u dN u du F ∂ ( ) ∂ = ( ) ∂ ( ) ∂ = ( ) I I I J J J (28) ⎛ ⎝⎜ ⎞ ⎠⎟ ϵ σ σ σ γ σ σ C u C u u u σ F u F u u u = Δ ∂ ∂ + ∂ ∂ ( ) ∂ ( ) ∂ ∂ ∂ + ∂ ∂( ) ∂( ) ∂ ∂ ∂ p t Δ (27) (27) where: ∑ ∑ C u u dN u du C F u u dN u du F ∂ ( ) ∂ = ( ) ∂ ( ) ∂ = ( ) I I I J J J (28) (28) σ α γf ̇ ( , ) = 0 unloaded to eliminate the elastic deformation, since r-value is a plastic property. An additional boundary condition to impose equal vertical displacement was also considered for the nodes on the top of the element square. The consistency requirement allows the unloading to an elastic stress state ( σ α f ̇ ( , ) < 0 and consequently γ = 0) and it also demonstrates that the stress tensor is always located at the yield surface ( σ α f ̇ ( , ) = 0 and so γ > 0). The predicted r-value for an angle θ with the rolling direction is deﬁned as: When using associated ﬂow rule, the plastic strain tensor can be obtained directly from the yield potential as follows: r = − ϵ ϵ + ϵ θ 22 22 11 (33) where: r = − ϵ ϵ + ϵ θ 22 22 11 (33) r = − ϵ ϵ + ϵ θ 22 22 11 (33) ϵ σ γ σ = Δ ∂ ∂ p t Δ (26) ϵ σ γ σ = Δ ∂ ∂ p t Δ (26) where: ⎛ ⎝⎜ ⎞ ⎠⎟ ⎛ ⎝⎜ ⎞ ⎠⎟ dx x dy y ϵ = ln 1 + ϵ = ln 1 + 11 22 (34) When using the forward-Euler scheme for return mapping proce- dures [9,34,39,40], the time steps are assumed to be small enough for the coeﬃcients C u ( ) a and F u ( ) to be considered constant during return mapping. However, for larger time steps, the derivative of the equivalent yield stress obtained from the following chain rule can be used: ⎛ ⎝⎜ ⎞ ⎠⎟ ⎛ ⎝⎜ ⎞ ⎠⎟ dx x dy y ϵ = ln 1 + ϵ = ln 1 + 11 22 (34) This simple one-element test is going to be used with the diﬀerent yield criteria listed above for the assessment of the accuracy of the diﬀerent yield models for plastic anisotropy. These validations are going to be performed for three diﬀerent alloys (case studies): two aluminium alloys AA6022 and AA2090, for weak and strong plastic anisotropy validations, and also for the AZ31B Mg alloy, where the main objective is to demonstrate the generality and accuracy of the new yield function in describing the plastic anisotropy as well as the asymmetry in tension-compression. 5.1. r-values and directional ﬂow stresses for FCC materials 5.1. r-values and directional ﬂow stresses for FCC materials and σ u ∂/∂ is obtained from the derivative of Eqs. (6) and (7). If an associated ﬂow rule is used and if the return mapping of the trial stress state to the yield surface is considered to be along the path with the closest distance to the yield function then the derivative σ u ∂/∂ can be assumed to be zero and so the plastic strain tensor from Eq. (27) reduces to: and σ u ∂/∂ is obtained from the derivative of Eqs. (6) and (7). If an associated ﬂow rule is used and if the return mapping of the trial stress state to the yield surface is considered to be along the path with the closest distance to the yield function then the derivative σ u ∂/∂ can be assumed to be zero and so the plastic strain tensor from Eq. (27) reduces to: 5.1.1. The AA6022 aluminium alloy 5.1.1. The AA6022 aluminium alloy The Young's modulus and Poisson's ratio used were: E = 70000.0 MPa and ν = 0.3, respectively. The following Voce's curve was used for strain hardening: σ = 328.36 −194.5 · exp (−10.941 · ϵ ) p (35) σ = 328.36 −194.5 · exp (−10.941 · ϵ ) p ϵ σ γ σ = Δ ∂ ∂ p t Δ (29) (35) (29) In Fig. 5, the yield locus projected on the zero shear stress plane, τ = 0 xy , is shown for Hill's 1948, Barlat yld2000 and the new quadratic yield function (the stresses are normalised from the uniaxial stress at 0 degrees (σ0)). It can be seen that the new yield function delivers a yield locus which is almost coincident with the yield locus from Barlat et al. [5], yld2000, but considerably diﬀerent from the yield locus of Hill's 1948 yield criterion. It can be also seen that the symmetric biaxial yield stress was captured very well with this new yield function. which simpliﬁes considerably the return mapping procedure. 3.2. Basis functions, control points and approximation for the coeﬃcients 3.2. Basis functions, control points and approximation for the coeﬃcients The notion of irreversibility of plastic ﬂow is expressed by the following equations of evolution for the set of internal variables ϵ α { , } p , called ﬂow rule and hardening law, respectively: For a speciﬁc local parametric coordinate u from an open knot vector and for a degree p of the polynomial, the basis functions are obtained recursively from the following formulae [13,22,29]: ϵ σ α α σ α γ γ r H ̇ = ̇ ( , ) ̇ = ̇ ( , ) p (23) (23) N u u u u u N u u u u u N u ( ) = − − ( ) + − − ( ), I p I I p I I p I p I p I I p + −1 + +1 + +1 +1 −1 (18) where σ α r( , ) and σ α H( , ) are prescribed functions which deﬁne the direction of plastic ﬂow and the type of hardening. The parameter γ̇ is a non-negative function, called the consistency parameter, which is assumed to obey the following Kuhn-Tucker complementary conditions: (18) where I is the index for the basis functions. The formula for the basis functions in Eq. (18) must be initialised from piecewise basis functions corresponding to the polynomial order p=0, i.e.: σ α σ α γ f γf ̇ ≥0 ( , ) ≤0 ̇ ( , ) = 0 (24) ⎧⎨ ⎩ N u if u u u otherwise ( ) = 1 ≤ < 0 I I I 0 +1 (19) σ α σ α f γf ( , ) ≤0 ̇ ( , ) = 0 (24) (24) (19) where σ α f σ ρ ( , ) = − (ϵ ) p , with ρ being the stress value from the uni- axial stress-strain curve. In addition to the Kuhn-Tucker complementary conditions there are the consistency requirement, i.e.: In this work, NURBS approximation functions are deﬁned for the coeﬃcients C u ( ) a , C u ( ) b and F u ( ) as follows: 7 257 R.P.R. Cardoso, O.B. Adetoro International Journal of M echanical Sciences 128–129 (2017) 253–26 σ α γf ̇ ( , ) = 0 (25) (25) σ α γf ̇ ( , ) = 0 5.1.2. The Al2090 aluminium alloy The aluminium alloy AA6022 does not represent an alloy with a 5. Validations and discussion For validating the proposed quadratic yield function, single element uniaxial simulations are performed along every 15 degrees from the rolling direction for diﬀerent case studies. The predicted r-values and ﬂow stresses are compared with experimental results and with predic- tions from diﬀerent yield criteria such as Hill's 1948, “yld91” (Barlat and Lian [3]), “yld96” (Barlat et al. [4]), “yld2000” (Barlat et al. [5]), “CPB06ex2” (Cazacu et al. [11]) and the new quadratic yield function, “YldParam”, proposed in this paper. Fig. 8 shows the prediction for the r-values and a comparison with experimental results. It can be seen that the new quadratic yield function predicts the r-values for every direction very well. Hill's 1948 shows a good agreement at 0°, 45° and 90° and the same can be said for the Barlat yield functions yld96 and yld2000. The prediction of r-values for the Barlat yield functions at every 15° from the rolling direction is not as good as the new yield function because the Fig. 4 shows the procedure used for the prediction of r-values. As shown in Fig. 4, a 1 × 1 × 0.1 mm single element is elongated and then 258 coeﬃcients for Barlat yield functions were designed to ﬁt r-values at 0°, Fig. 4. Deﬁnitions for r-value calculation. Fig. 5. Yield locus for AA6022: comparison between Hill's 1948, yld2000 and the new yield function (τ = 0 xy ). Stresses normalised with the yield stress at 0 degrees (σ0). Fig. 6. Yield locus contours for AA6022 projected on shear planes for every 0.5 shear stress. R.P.R. Cardoso, O.B. Adetoro International Journal of M echanical Sciences 128–129 (2017) 253–268 International Journal of M echanical Sciences 128–129 (2017) 253–268 R.P.R. Cardoso, O.B. Adetoro Fig. 4. Deﬁnitions for r-value calculation. Cardoso, O.B. Adetoro International Journal of M echanical Sciences 128–129 (2017) 253–268 Fig. 4. Deﬁnitions for r-value calculation. Fig. 6. Yield locus contours for AA6022 projected on shear planes for every 0.5 shear stress. Fig. 5. Yield locus for AA6022: comparison between Hill's 1948, yld2000 and the new yield function (τ = 0 xy ). Stresses normalised with the yield stress at 0 degrees (σ0). Fig. 5. Yield locus for AA6022: comparison between Hill's 1948, yld2000 and the new yield function (τ = 0 xy ). Stresses normalised with the yield stress at 0 degrees (σ0). Fig. 5. 5. Validations and discussion Yield locus for AA6022: comparison between Hill's 1948, yld2000 and the new yield function (τ = 0 xy ). Stresses normalised with the yield stress at 0 degrees (σ0). Fig. 6. Yield locus contours for AA6022 projected on shear planes for every 0.5 shear stress. Fig. 6. Yield locus contours for AA6022 projected on shear planes for every 0.5 shear stress. coeﬃcients for Barlat yield functions were designed to ﬁt r-values at 0°, 45° and 90° only and so we cannot expect a perfect prediction for all other directions. On the contrary, the coeﬃcient F u ( ) for the new yield function was designed to ﬁt r-values at every direction and this explains the better accuracy of the newly proposed model. Fig. 7. Coeﬃcients F u ( ), G, H, N, C u ( ) a and C u ( ) b for the AA6022 aluminium alloy. In Fig. 9 the predictions for the normalised ﬂow stresses at every15° from the rolling direction are compared. Again, the comparison is made for the same yield models used for the r-values prediction and a comparison is also made with experimental results. The ﬂow stresses for the new quadratic yield function were obtained following the deriva- tion from Eq. (9), i.e.: σ σ C u F u G H G H θ F u H θ N H θ θ = ( ) 2 3 ( ) + + ( + ) cos + [ ( ) + ] sin + 2 ( − ) cos sin θ a 4 4 2 2 (36) σ σ C u F u G H G H θ F u H θ N H θ θ = ( ) 2 3 ( ) + + ( + ) cos + [ ( ) + ] sin + 2 ( − ) cos sin θ a 4 4 2 2 (36) All yield criteria deliver normalised ﬂow stresses very close to experimental results with the exception of Hill's 1948 yield criterion. It is also fair to say that the disparity for the Hill's results for the normalised yield stresses is expected because in this work the Hill coeﬃcients were designed to ﬁt the r-values and not the normalised ﬂow stresses. Fig. 7. Coeﬃcients F u ( ), G, H, N, C u ( ) a and C u ( ) b for the AA6022 aluminium alloy. 5. Validations and discussion Fig. 7. Coeﬃcients F u ( ), G, H, N, C u ( ) a and C u ( ) b for the AA6022 aluminium alloy. strong plastic anisotropy. This can be clearly seen from Figs. 8 and 9 for the low amplitude for both the r-values and for the normalised ﬂow stresses. The aluminium alloy AA2090, on the contrary, shows a very strong plastic anisotropy with high amplitudes or range for both r- 259 International Journal of M echanical Sciences 128–129 (2017) 253–268 R.P.R. Cardoso, O.B. Adetoro Fig. 8. Comparison between measured and predicted r-values for the AA6022 aluminium alloy. Fig. 8. Comparison between measured and predicted r-values for the AA6022 aluminium alloy. Fig. 8. Comparison between measured and predicted r-values for the AA6022 aluminium alloy. ween measured and predicted r-values for the AA6022 aluminium allo g. 8. Comparison between measured and predicted r-values for the AA values and normalised ﬂow stresses and thus it represents a higher challenge for the newly proposed quadratic yield function. from the use of quadratic yield functions with an associated ﬂow rule. It is well-agreed up till now that if an associated ﬂow rule is used with aluminium alloys than the yield criterion needs to be non-quadratic, otherwise erroneous predictions for r-values and/or normalised yield stresses should be expected. Alternatively, if a non-associated ﬂow rule is to be used, then a quadratic yield potential, such as Hill's 1948, should be able to accurately represent the aluminium's behaviour. The main reason is that with a non-associated ﬂow rule the yield potential can be used to match the normalised yield stresses while the plastic potential can be used to match the r-values. Barlat yield functions (yld91, Barlat et al. [3], yld96, Barlat et al. [4] and yld2000, Barlat et al. [5]) were all designed to be non-quadratic yield functions as well as the CPB06ex2 yield function of Cazacu et al. [11]. The Young's modulus and Poisson's ratio used in this validation were: E = 70000.0 MPa and ν = 0.3, respectively. The following Power law was used for strain hardening: σ = 646.0 · (0.025 + ϵ ) p 0.227 (37) σ = 646.0 · (0.025 + ϵ ) p 0.227 (37) σ = 646.0 · (0.025 + ϵ ) p 0.227 σ = 646.0 · (0.025 + ϵ ) p 0.227 In Fig. 5. Validations and discussion 10, the yield locus projected on the zero shear stress plane, τ = 0 xy , for the Hill's 1948 and the new quadratic yield function is shown together with some experimental results. The stresses were normalised with the uniaxial stress at 0 degrees (σ0). It can be seen that the new yield function delivers a yield locus which matches excellently well with the experimental results. It is quite interesting to see the diﬀerence in the yield locus when compared with the Hill's 1948 yield function; Hill's 1948 does not ﬁt the biaxial symmetric ﬂow stress accurately as expected because it does not have included the equi- biaxial stress in his model. On the contrary, the equi-biaxial yield stress was very well captured with this new quadratic yield function. Another noteworthy point is the ability of predicting the aluminium's behaviour In Fig. 11 the yield locus contours for every 0.5 values of shear stress is shown. In Fig. 12, a plot of the coeﬃcients F u ( ), G, H, N, C u ( ) a and C u ( ) b is shown as a function of the angle from the rolling direction. Again, the coeﬃcients G, H and N are constant but the coeﬃcients F u ( ), C u ( ) a and C u ( ) b are not. The plots of these two non-constant coeﬃcients show again a symmetry at 180°. It is also worth noting for this example Fig. 9. Comparison between measured and predicted ﬂow stresses for the AA6022 aluminium alloy. Fig. 9. Comparison between measured and predicted ﬂow stresses for the AA6022 aluminium alloy. 260 International Journal of M echanical Sciences 128–129 (2017) 253–268 R.P.R. Cardoso, O.B. Adetoro Fig. 10. Yield locus for Al2090: comparison between Hill 1948, the new parametric yield function and experimental results. the comparison between C u ( ) a and C u ( ) b as discussed in the previous section but, although, the deviation is not as prominent for this alloy because the eﬀect of the symmetric biaxial stress is much less when compared to that of the AA6022 aluminium alloy. Fig. 13 shows the comparison between the predicted r-values and experimental results. It can be seen that the predictions for the r-values from the new quadratic yield function match the experimental results for every orientation very well, with a slight deviation at 60°. 5. Validations and discussion Hill's 1948 gives good agreement at 0°, 45° and 90° and the same can be said for the Barlat yield functions yld91, yld96 and yld2000. The prediction of r-values for the Barlat yield functions at every 15° from the rolling direction is not as good as the new quadratic yield function because the coeﬃcients for Barlat yield functions were designed to ﬁt r-values at 0°, 45° and 90° only and so we cannot expect a perfect prediction for all other directions. On the contrary, the coeﬃcient F u ( ) for the new quadratic yield function was designed to ﬁt r-values at every direction and this explains the better accuracy of the proposed model. All yield criteria shown deliver normalised ﬂow stresses very close to experiments with the exception of Hill's 1948 yield criterion as seen in Fig. 14. It can also be said that the Barlat yield criteria (yld91 and yld96) struggles a bit to match the experimental normalised ﬂow stress at 30°. As discussed in the previous section, it is fair to say that the disparity for the Hill's results for the normalised yield stresses is expected because Hill coeﬃcients in this work were designed to ﬁt the r-values, not the normalised ﬂow stresses. If the Hill coeﬃcients had been ﬁtted for the normalised yield stresses we should then expect a much better prediction of the Hill model for the normalised yield stresses but then the prediction for the r-values would be worst. Fig. 10. Yield locus for Al2090: comparison between Hill 1948, the new parametric yield function and experimental results. Fig. 11. Yield locus contours for Al2090 projected on shear planes for every 0.5 shear stress. 5.2. Cup drawing for earing prediction The new yield function was also tested for a cup drawing simulation for the prediction of the cup earing proﬁle for an Al 2090-T3 aluminium alloy. Fig. 15 depicts the geometry for the die tools and for the blank sheet. The following dimensions were used in our analysis: • Punch diameter: D = 97.46 mm p • Punch proﬁle radius: r = 12.70 mm p • Die opening diameter: D = 101.48 mm d • Die proﬁle radius: r = 12.70 mm d • Blank radius: D = 158.76 mm b • Punch diameter: D = 97.46 mm p p • Punch proﬁle radius: r = 12.70 mm p • Die opening diameter: D = 101.48 mm d • Die opening diameter: D = 101.48 mm d • Die proﬁle radius: r = 12.70 mm d • Blank radius: D = 158.76 mm b The material properties used in the analysis are given below: The material properties used in the analysis are given below: In their work, Yoon et al. [41] said that since the stresses in the ﬂange area are nearly compressive in nature, the cup drawing simulations should also account for the compressive test results. In other words, they are suggesting that there is a strength diﬀerential eﬀect that should have been considered during the material characterisation of the Al2090 aluminium alloy. It was shown in Yoon et al. [41] that if the strength diﬀerential eﬀect was considered (Yld96 with translation of the yield surface) then a better agreement could be obtained with the experi- mental results. It is therefore more important to compare the results obtained with the new yield function with the results from the use of Yld96 i h l i f h i ld f (b h l d i Fi 17) Fig. 13. Predictions and experimental results for the r-values of the Al2090 aluminium alloy. Fig. 15. Al2090 cup forming example. Deﬁnition of tools and blank geometry. Fig. 13. Predictions and experimental results for the r-values of the Al2090 aluminium alloy. Fig. 13. Predictions and experimental results for the r-values of the Al2090 aluminium alloy. Fig. 13. Predictions and experimental results for the r-values of the Al2090 aluminium alloy. the measured result, but however, both Yld96 and the new yield function do not lead to the correct trend: the experimental cup height at 0 degrees is larger than that at 90 degrees, whereas the simulated results predict the reverse. Yoon et al. [41] and Yoon et al. [43] reported this to be a consequence of the cup drawing simulations being performed from coeﬃcients based on the tensile test results. In their work, Yoon et al. [41] said that since the stresses in the ﬂange area are nearly compressive in nature, the cup drawing simulations should also account for the compressive test results. In other words, they are suggesting that there is a strength diﬀerential eﬀect that should have been considered during the material characterisation of the Al2090 aluminium alloy. It was shown in Yoon et al. [41] that if the strength diﬀerential eﬀect was considered (Yld96 with translation of the yield surface) then a better agreement could be obtained with the experi- mental results. It is therefore more important to compare the results obtained with the new yield function with the results from the use of Yld96 without translation of the yield surface (both plotted in Fig. The material properties used in the analysis are given below: Fig. 11. Yield locus contours for Al2090 projected on shear planes for every 0.5 shear stress. Fig. 11. Yield locus contours for Al2090 projected on shear planes for every 0.5 shear stress. • Stress-strain curve characteristics: σ MPa = 646(0.025 + ϵ) ( ) 0.227 • Stress-strain curve characteristics: σ MPa = 646(0.025 + ϵ) ( ) 0.227 • Initial sheet thickness: t = 1.6 mm 0 • Coulomb coeﬃcient of friction: 0.1 Fig. 12. Coeﬃcients F u ( ), G, H, N, C u ( ) a and C u ( ) b for the Al2090 aluminium alloy. • Blank holding force: 22.2 kN The cup drawing simulation was carried out in the commercial software ABAQUS and by using a user material subroutine “VUMAT” for the new yield function. Fig. 16 depicts the deformed conﬁguration and the earing proﬁle for the cup after the cup drawing operation. In Fig. 17 the cup earing proﬁle is compared for the experimental results from Yoon et al. [41], Yld96 without translation [41] (or without consideration of the strength diﬀerential eﬀects) and the new yield function. For an orthotropic material, the cup height proﬁle between 0 and 90 degrees should be the mirror image of the cup height proﬁle between 90 and 180 degrees with respect to the 90 degrees axis. However, the measured earing proﬁle slightly deviates from this condition and, according to Yoon et al. [41], this deviation might have occurred because the center of the blank was not aligned properly with the centers of the die and the punch during processing. The earing magnitude is in good agreement with the simulations of Yoon et al. [41] for the Yld96 without translation and also in reasonable agreement with Fig. 12. Coeﬃcients F u ( ), G, H, N, C u ( ) a and C u ( ) b for the Al2090 aluminium alloy. 261 International Journal of M echanical Sciences 128–129 (2017) 253–268 R.P.R. Cardoso, O.B. Adetoro the measured result, but however, both Yld96 and the new yield function do not lead to the correct trend: the experimental cup height at 0 degrees is larger than that at 90 degrees, whereas the simulated results predict the reverse. Yoon et al. [41] and Yoon et al. [43] reported this to be a consequence of the cup drawing simulations being performed from coeﬃcients based on the tensile test results. The material properties used in the analysis are given below: 17) the measured result, but however, both Yld96 and the new yield function do not lead to the correct trend: the experimental cup height at 0 degrees is larger than that at 90 degrees, whereas the simulated results predict the reverse. Yoon et al. [41] and Yoon et al. [43] reported this to be a consequence of the cup drawing simulations being performed from coeﬃcients based on the tensile test results. In their work, Yoon et al. [41] said that since the stresses in the ﬂange area are nearly compressive in nature, the cup drawing simulations should also account for the compressive test results. In other words, they are suggesting that there is a strength diﬀerential eﬀect that should have been considered during the material characterisation of the Al2090 aluminium alloy. It was shown in Yoon et al. [41] that if the strength diﬀerential eﬀect was considered (Yld96 with translation of the yield surface) then a better agreement could be obtained with the experi- mental results. It is therefore more important to compare the results obtained with the new yield function with the results from the use of Yld96 without translation of the yield surface (both plotted in Fig. 17) Fig. 15. Al2090 cup forming example. Deﬁnition of tools and blank geometry. Fig. 14. Comparison for predictions of ﬂow stresses for the Al2090 aluminium alloy. 262 Fig. 14. Comparison for predictions of ﬂow stresses for the Al2090 aluminium alloy. 262 262 Fig. 16. Al2090 deformed cup after drawing. R.P.R. Cardoso, O.B. Adetoro International Journal of M echanical Sciences 128–129 (2 International Journal of M echanical Sciences 128–129 (2017) 253–268 R.P.R. Cardoso, O.B. Adetoro Fig. 16. Al2090 deformed cup after drawing. as they properly reﬂect the material characterisation employed in the simulation. Three points on the yield surface were considered for the plotting of the iso-error maps: point A, corresponding to the uniaxial stress state; point B, which is a biaxial stress state; point C, which corresponds to the pure shear stress state. All of these points are schematically represented in Fig. 18. For the Al2090-T3 alloy, Yoon et al. [42] demonstrated that a more accurate prediction of the r-value at 75 degrees should be reﬂected into a small ear around 15 degrees and so the earing proﬁle should include six ears instead of four ears. The material properties used in the analysis are given below: This behaviour was not observed with the current model maybe due to the slight deviation of the r-value prediction obtained for 75 degrees from the rolling direction. Without any loss of generality, the initial stress state for points A, B and C is the one corresponding to locations at the yield locus for the initial yield point. Subsequently, incremental stresses in the principal directions “S1” and “S2” are added and the return mapping scheme is applied for the assessment of the iso-error contours. There is the need to calculate the exact stress tensor for points A, B and C after return mapping and that is done in this current work by applying 10,000 inﬁnitesimal stress increments between the yield locus and the yield locus for the trial stress state. The iso-error contour E is constructed from the following equation: 5.3. Iso-error maps In this section we assess the accuracy of the return mapping procedure used in this work by means of the iso-error maps, as detailed in Simo and Hughes [34]. The new yield function developed in this work was designed to work with the semi-implicit or forward-Euler return mapping scheme, as detailed in Section 4 and as derived in a previous work of Cardoso and Yoon [9], where it is assumed that the trial stress state is not too far away from the yield locus or, in other words, the incremental time steps are considered to be suﬃciently small enough. The iso-error maps presented in this section were constructed for the Al2090 alloy, whose yield locus has the shape as described in Fig. 10, including regions with high curvature, making the accuracy and even the convergence of the return mapping scheme much more complicated. σ σ σ σ σ σ E = ( − ) : ( − ) * : * × 100 (38) (38) where σ* is the exact stress tensor and σ is the stress tensor after return mapping. The plots for the iso-error contours for points A, B and C in Figs. 19, 20 and 21, respectively, were constructed for S Sy 1/ ≤1.75 and S Sy 2/ ≤1.75, where Sy is the initial yield stress of Al2090 alloy. Because the return mapping scheme used in this work is a forward-Euler scheme Fig 17 Cup earing proﬁle for the Al2090 aluminium alloy Fig. 17. Cup earing proﬁle for the Al2090 aluminium alloy. Fig. 17. Cup earing proﬁle for the Al2090 aluminium alloy. 263 263 International Journal of M echanical Sciences 128–129 (2017) 253–268 R.P.R. Cardoso, O.B. Adetoro 468 10 12 14 16 18 20 20 22 22 24 24 26 26 28 28 30 30 32 32 34 34 36 36 38 38 40 40 42 42 44 44 46 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 S1/Sy 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 -S2/Sy Fig. 21. Iso-error plot for point C (pure shear stress state). Fig. 18. Points A (uniaxial), B (biaxial) and C (pure shear) for the iso-error maps. Fig. 21. Iso-error plot for point C (pure shear stress state). Fig. 18. Points A (uniaxial), B (biaxial) and C (pure shear) for the iso-error maps. for the forward-Euler scheme in the work of Cardoso and Yoon [9]. 5.3. Iso-error maps 4 8 12 16 16 20 20 24 24 24 28 28 28 32 32 32 36 36 36 36 40 40 40 44 44 48 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 S1/Sy 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 S2/Sy Fig. 20. Iso-error plot for point B (biaxial stress state). 2 2 4 4 6 6 8 8 10 10 12 12 14 14 16 16 18 18 20 20 22 22 24 24 26 26 28 28 30 30 32 32 34 34 36 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 S1/Sy 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 S2/Sy Fig. 19. Iso-error plot for point A (uniaxial stress state). 2 2 4 4 6 6 8 8 10 10 12 12 14 14 16 16 18 18 20 20 22 22 24 24 26 26 28 28 30 30 32 32 34 34 36 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 S1/Sy 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 S2/Sy Fig. 19. Iso-error plot for point A (uniaxial stress state). 4 8 12 16 16 20 20 24 24 24 28 28 28 32 32 32 36 36 36 36 40 40 40 44 44 48 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 S1/Sy 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 S2/Sy 4 8 12 16 16 20 20 24 24 24 28 28 28 32 32 32 36 36 36 36 40 40 40 44 44 48 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 S1/Sy 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 S2/Sy Fig. 20. Iso-error plot for point B (biaxial stress state). 5.3. Iso-error maps Another aspect that is worth discussing is the computational performance of the return mapping scheme used. This was addressed by Cardoso and Yoon [9], however the return mapping scheme now comes together with a new yield function so it is worth considering the computational costs or beneﬁts added by the new yield function to the return mapping procedure. When compared with the yld96 function used for the cup drawing for the earing proﬁle of previous section, it can be said that there is the need to calculate the loading direction as in Eq. (7), however this is computationally inexpensive. The other major diﬀerence of this new yield function is the calculation of the plastic ﬂow direction, where in this case we calculate the normal to the yield surface (derivative of the yield potential) by using B-Splines basis functions and the derivative of the B-Spline's basis functions for the anisotropic coeﬃcients. When comparing with the yld96 yield function, the normal to the yield surface also has to be calculated and the derivatives of the yield potential, however, for the approximation of the coeﬃcients for a particular loading direction, only the coeﬃcients inside the local compact support of the B-Spline basis functions need to be considered. If the polynomials used for the B-Splines basis function have degree “p” then the size of the local compact support is p + 1, so for a quadratic degree only three coeﬃcients in the local compact support of the loading direction “u” need to be used. If we compare with more elaborated yield functions such as Barlat's yld2000, there are the advantages of the straightforwardness of obtaining the anisotropic coeﬃcients as well as the simplicity for the calculation of the plastic ﬂow tensor for the normal to the yield surface. In the comparison study carried out for the Al2090 alloy, the diﬀerences for the computational performance of the return mapping scheme used for the new yield function and for the yld96 and yld2000 yield functions are barely undetectable. 2 2 4 4 6 6 8 8 10 10 12 12 14 14 16 16 18 18 20 20 22 22 24 24 26 26 28 28 30 30 32 32 34 34 36 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 S1/Sy 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 S2/Sy Fig. 19. Iso-error plot for point A (uniaxial stress state). 5.4.1. The AZ31B Mg alloy The last case study presented in this paper is for a HCP material, the AZ31B Mg alloy. Many yield functions were developed to account for the asymmetry in tension-compression for these alloys and amongst them we can distinguish the works of Cazacu et al. [10,11] and more recently the work of Soare and Benzerga [35] on the modelling of asymmetric yield functions. The main objective of this case study is to demonstrate that the newly proposed yield function for plane stress analysis is generalised enough to accurately predict the plastic aniso- tropy for this alloy as well as its asymmetric behaviour in tension- compression. The Young's modulus and Poisson's ratio used were: E = 42000.0 MPa and ν = 0.35, respectively. The following Voce curve Fig. 20. Iso-error plot for point B (biaxial stress state). (traditionally used for explicit analysis), then if larger time steps (or larger S Sy 1/ ratio) are used there is the chance of divergence in the return mapping scheme and that is even more critical for the high curvature yield locus of the Al2090 aluminium alloy. This was also proved to be the case when the Barlat yld2000 yield function was used 264 International Journal of M echanical Sciences 128–129 (2017) 253–268 R.P.R. Cardoso, O.B. Adetoro Fig. 22. Yield locus for AZ31B Mg alloy: comparison between Hill 1948, Plunkett et al. [31] for the CPB06ex2 yield function, the new parametric yield function and experiments from Lou et al. [27]. Fig. 24. Coeﬃcients F u ( ), G, H, N and C u ( ) a for the AZ31B Mg alloy. Fig. 24. Coeﬃcients F u ( ), G, H, N and C u ( ) a for the AZ31B Mg alloy. Fig. 24. Coeﬃcients F u ( ), G, H, N and C u ( ) a for the AZ31B Mg alloy. Fig. 24. Coeﬃcients F u ( ), G, H, N and C u ( ) a for the AZ31B Mg alloy. Fig. 22. Yield locus for AZ31B Mg alloy: comparison between Hill 1948, Plunkett et al. [31] for the CPB06ex2 yield function, the new parametric yield function and experiments from Lou et al. [27]. Fig. 24 shows a plot of coeﬃcients F u ( ), G, H, N, C u ( ) a and C u ( ) b as a function of the angle from the rolling direction. was used for strain hardening: σ = 361.43 −158.6 · exp (−9.74 · ϵ ) p (39) (39) σ = 361.43 −158.6 · exp (−9.74 · ϵ ) p σ = 361.43 −158.6 · exp (−9.74 · ϵ ) p In Fig. 22, the yield locus for the Hill 1948 yield function, for Cazacu et al. [11] CPB06ex2 yield potential, for the newly proposed quadratic yield criterion (YldParam) and for the experimental results from Lou et al. [27] are presented. It can be seen that the proposed new yield function is able to predict accurately the asymmetry in tension- compression that is very typical of this alloy. The CPB06ex2 yield criterion is also extremely accurate but, as reported by Plunkett et al. [31], it requires the calculation of 18 anisotropic coeﬃcients plus 2 additional coeﬃcients to describe the asymmetry in tension-compres- sion, it is not a quadratic yield potential and it requires at least two linear transformations for the yield potential. In Fig. 23 it is shown the yield locus contours for every 0.5 values of shear stress. It shows the shape of the diﬀerent yield locus when projected at diﬀerent shear stress planes. Fig. 25 shows the r-values predictions and comparisons with experimental results for both the tension and compression regions. The prediction from the new yield function is compared with Plunkett et al. [31] predictions based on the yield criterion CPB06ex2 of Cazacu et al. [11]. It can be seen that the CPB06ex2 yield criterion is just perfect in accurately predicting the r-values for both tension and compression regions. The new yield function is also great for the prediction of the r-values in tension and some slight deviations are seen for the r-values in compression, mostly for 45° and 75°. However, it is reasonable to say that these minor deviations are still good if we consider the fact that these predictions were obtained from the use of a quadratic yield potential. In regards to the normalised yield stresses from the plots in Fig. 26, the conclusion is that both Cazacu et al. [11] CPB06ex2 yield potential and the newly proposed formulation deliver excellent agreement with the normalised yield stresses from experi- ments. Fig. 23. Yield locus contours for the AZ31B Mg alloy projected on shear planes for every 0.5 shear stress. In this paper, the general deﬁnition or classiﬁcation of a “quadratic planar anisotropic yield function” is adopted. 5.4.1. The AZ31B Mg alloy It is important to notice that for this case study the coeﬃcients G, H and N have diﬀerent values for tension and compression and so are not constant for the entire range of loading directions. The reason for the diﬀerent distribution for these coeﬃcients is due to the asymmetric behaviour of HCP alloys, which requires a separated calibration for the tension and compression domains. The plot for the coeﬃcients F u ( ), C u ( ) a and C u ( ) b is not symmetric at 180° due to the asymmetric behaviour in tension-compression of this particular Mg alloy. Also, regarding the coeﬃcients C u ( ) a and C u ( ) b from Fig. 24, and in particular their comparison in the vicinity of the symmetric biaxial stress state, it can be said that the diﬀerence between the coeﬃcients is higher in tension than it is in compression. was used for strain hardening: Comparison between the predicted Normalised Flow Stress in Tension and Compression for the new yield function, Plunkett et al. [31] and experimental values for the AZ31B Mg alloy. for the distribution of these anisotropic parameters are deﬁned as a B- Spline function of the local parametric variable, however they can also be represented by any other function with any degree as far as an accurate ﬁtting of the coeﬃcient is achieved. prediction of directional ﬂow stresses. Quadratic NURBS basis functions were used for the mathematical description of these two coeﬃcients, making the method computationally eﬀective. It was shown in the discussion/validations section that the yield locus, r-values and directional ﬂow stresses predictions were almost perfectly matched for two aluminium alloys (AA6022 and AA2090), with weak and strong plastic planar anisotropy, and also for a HCP magnesium alloy (AZ31B Mg), where the asymmetric behaviour in tension-compression was also shown to be very well captured. In addition, FE simulations of the cup drawing of a circular blank was conducted, where the predicted earing proﬁle matches the experimen- tal results satisfactorily. These prove that the newly developed yield potential is generalised enough for the prediction of plastic planar anisotropy and for the accurate description of the asymmetry in tension-compression of HCP materials. was used for strain hardening: This commonly refers to the exponent used directly in the stress components inside the yield criterion, which in the case of this new yield function is of degree two as in the original Hill's yield criterion [20]. All non-quadratic (higher- order) yield functions in the literature are deﬁned as such because of the higher exponent of the stress components and this invariably results in the more accurate ﬁtting of the yield surface. This higher-order ﬁtting however comes at such costs as diﬃculties in converging during return mapping procedures and also higher number of coeﬃcients that must be obtained experimentally, amongst other costs. However, in this new function the same accurate ﬁtting is achievable through the introduction of the variable anisotropic parameters C and F which are both functions of the angle between rolling and loading directions, which is one of the main advantages of this yield function. The curves Fig. 23. Yield locus contours for the AZ31B Mg alloy projected on shear planes for every 0.5 shear stress. 265 International Journal of M echanical Sciences 128–129 (2017) 253–268 R.P.R. Cardoso, O.B. Adetoro Fig. 25. Comparison between the predicted r-values (Tension and Compression) for the new yield function, Plunkett et al. [31] and experimental results for the AZ31B Mg alloy. Fig. 25. Comparison between the predicted r-values (Tension and Compression) for the new yield function, Plunkett et al. [31] and experimental results for the AZ31B Mg alloy. Fig. 25. Comparison between the predicted r-values (Tension and Compression) for the new yield function, Plunkett et al. [31] and exper redicted r-values (Tension and Compression) for the new yield function, Plunkett et al. [31] and experimental results for the AZ31B Mg all Fig. 25. Comparison between the predicted r-values (Tension and Compression) for the new yield function, Plunkett et al. [31] and experimental results for the AZ31B Mg alloy. Fig. 26. Comparison between the predicted Normalised Flow Stress in Tension and Compression for the new yield function, Plunkett et al. [31] and experimental values for the AZ31B Mg alloy. Fig. 26. Comparison between the predicted Normalised Flow Stress in Tension and Compression for the new yield function, Plunkett et al. [31] and experimental values for the AZ31B Mg alloy. Fig. 26. Comparison between the predicted Normalised Flow Stress in Tension and Compression for the new yield function, Plunkett et al. alloy. Fig. 26. Appendix A. Proof of convexity in the principal stress space (τ = 0 xy ) and for the case of proportional loading Appendix A. Proof of convexity in the principal stress space (τ = 0 xy ) and for the case of proportional dix A. Proof of convexity in the principal stress space (τ = 0 xy ) and for the case of proportional loading Appendix A. Proof of convexity in the principal stress space (τ = 0 xy ) and for the case of proportional loading A yield function is convex if its Hessian matrix H deﬁned as: σ σ σ H = ∂ ∂∂ i j 2 (A.1) is positive semi-deﬁnite, i.e. if its eigenvalues are not negative (Rockafellar [33]). The analysis is going to be done initially for the yield function projected on the zero shear stress plane, i.e. for τ = 0 xy . In this case the Hessian matrix is deﬁned as follows: is positive semi-deﬁnite, i.e. if its eigenvalues are not negative (Rockafellar [33]). The analysis is going to b projected on the zero shear stress plane, i.e. for τ = 0 xy . Appendix A. Proof of convexity in the principal stress space (τ = 0 xy ) and for the case of proportional loading (A.9), A ≥0 means the coeﬃcient C u ( ) needs to be positive and the coeﬃcients F u ( ), G and H also need to be positive. From Eq. (A.9), A ≥0 means the coeﬃcient C u ( ) needs to be positive and the coeﬃcients F u ( ), G and H From Eq. (A.9), A ≥0 means the coeﬃcient C u ( ) needs to be positive and the coeﬃcients F u ( ), G and H also need to be positive. Appendix A. Proof of convexity in the principal stress space (τ = 0 xy ) and for the case of proportional loading In this case the Hessian matrix is deﬁned as follows: ⎡ ⎣ ⎢ ⎢ ⎢⎢ ⎤ ⎦ ⎥ ⎥ ⎥⎥ H = σ σ σ σ σ σ σ σ σ σ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ xx yy xx xx yy yy 2 2 2 2 2 2 (A.2) ⎡ ⎣ ⎢ ⎢ ⎢⎢ ⎤ ⎦ ⎥ ⎥ ⎥⎥ H = σ σ σ σ σ σ σ σ σ σ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ xx yy xx xx yy yy 2 2 2 2 2 2 (A.2) The ﬁrst derivatives are deﬁned as: σ σ A G H σ Hσ G H σ F u H σ Hσ σ σ σ A F u H σ Hσ G H σ F u H σ Hσ σ ∂ ∂ = ( + ) − ( + ) + [ ( ) + ] −2 ∂ ∂ = ( ( ) + ) − ( + ) + [ ( ) + ] −2 xx xx yy xx yy xx yy yy yy xx xx yy xx yy 2 2 2 2 where: A C u F u G H = 1 ( ) 3 2( ( ) + + ) (A.3) (A.4) The second derivatives can be obtained from the following equations: σ σ A G F u G H H F u σ G H σ F u H σ Hσ σ σ σ σ A G F u G H H F u σ σ G H σ F u H σ Hσ σ σ σ A G F u G H H F u σ G H σ F u H σ Hσ σ ∂ ∂ = [ · ( ) + · + · ( )] {( + ) + [ ( ) + ] −2 } ∂ ∂ ∂ = − [ · ( ) + · + · ( )] {( + ) + [ ( ) + ] −2 } ∂ ∂ = [ · ( ) + · + · ( )] {( + ) + [ ( ) + ] −2 } xx yy xx yy xx yy xx yy yy xx xx yy xx yy yy yy xx xx xx yy 2 2 2 2 2 3/2 2 2 2 3/2 2 2 2 2 2 3/2 and so the Hessian matrix can be written as: ⎡ ⎣ ⎢ ⎢ ⎤ ⎦ ⎥ ⎥ A G F u G H H F u G H σ F u H σ Hσ σ σ σ σ σ σ σ H = [ · ( ) + · + · ( )] {( + ) + [ ( ) + ] −2 } − − xx yy xx yy yy xx yy xx yy xx 2 2 3/2 2 2 The eigenvalues for this Hessian matrix are: α = 0 1 α = 0 1 α A G F u G H H F u G H σ F u H σ Hσ σ σ σ = [ · ( ) + · + · ( )] {( + ) + [ ( ) + ] −2 } ( + ) xx yy xx yy xx yy 2 2 2 3/2 2 2 which means that the new yield function is convex if: A G F u G H H F u G H σ F u H σ Hσ σ [ · ( ) + · + · ( )] {( + ) + [ ( ) + ] −2 } ≥0 xx yy xx yy 2 2 3/2 or: or: A G F u G H H F u [ · ( ) + · + · ( )] ≥0 A G F u G H H F u [ · ( ) + · + · ( )] ≥0 From Eq. 6. Concluding remarks In this work, a new generalised quadratic yield function was developed for the description of planar plastic anisotropy in metallic alloys. The new yield function delivers a good prediction of both r- values and directional ﬂow stresses and it also accurately describes the biaxial symmetric ﬂow stress and the unsymmetric biaxial stress state. One coeﬃcient F u ( ) was made function of a directional parameter that represents the angle between the loading direction and the rolling direction. An additional coeﬃcient C u ( ) was added for the accurate 266 International Journal of M echanical Sciences 128–129 (2017) 253–268 R.P.R. Cardoso, O.B. Adetoro References Int J Plast 2008;24:847–66 [9] Cardoso Rui PR, Yoon Jeong Whan. Stress integration method for a nonlinear kinematic/isotropic hardening model and its characterization based on polycrystal plasticity. Int J Plast 2009;25:1684–710. [32] Proust G, Tome CN, Kaschner GC. 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Orthotropic yield criteria for description of the anisotropy in tension and compression of sheet metals. Appendix B. Control points for coeﬃcients C u ( ) and F u ( ) The NURBS control points for the coeﬃcients C u ( ) and F u ( ) were generated from the algorithm described in the work by Piegl and Tiller [29] where the major equations used are described here. The NURBS control points for the coeﬃcients C u ( ) and F u ( ) were generated from the algorithm described in the work by Piegl and Tiller [29] where the major equations used are described here. Given a set of points k n F ( = 0,…, ) k , the aim is to interpolate these points with a second-degree nonrational B-spline curve. From the parameter u 0 ≤ ≤1 that represents the angle between the loading and rolling directions, we can deﬁne a parameter uk for each Fk and select an appropriate knot vector U u u = { ,…, } m 0 so that the following n n ( + 1) × ( + 1) system of linear equations can be deﬁned: ∑N u F P = ( ) k I n I k I =0 ,2 (B.1) where N u ( ) i k ,2 stands for the quadratic NURBS basis function of control point PI, evaluated at the parametric coordinate uk. The control points PI are the n + 1 unknowns in the linear system of equations. There are diﬀerent methods for the deﬁnition of the parametric coordinates uk. In this work, we ﬁrst deﬁned the total chord length d as follows: 267 International Journal of M echanical Sciences 128–129 (2017) 253–268 R.P.R. Cardoso, O.B. Adetoro ∑ d F F = − k n k k =1 −1 ∑ d F F = − k n k k =1 −1 (B.2) and then the parametric coordinates were deﬁned as: and then the parametric coordinates were deﬁned as: u u u u d k n F F = 0 = 1 = + − = 1,…, −1 n k k k k 0 −1 −1 u u = 0 = 1 n 0 u u u d k n F F 1 = + − = 1,…, −1 n k k k k −1 −1 (B.3) For more details on these, refer to the work by Piegl and Tiller [29]. [22] Hughes TJR, Cottrel JA, Bazilevs Y. Isogeometric analysis: CAD, ﬁnite elements, NURBS, exact geometry and mesh reﬁnement. Comput Methods Appl Mech Eng 2005;194:4135–95. International Journal of M echanical Sciences 128–129 (2017) 253–268 References Int J Mech Sci 1995;37:733. [17] Esmaeili S, Lloyd DJ, Poole WJ. A yield strength model for the Al-Mg-Si-Cu alloy AA6111. Acta Mater 2003;51:2243–57. y [41] Yoon JW, Barlat F, Chung K, Pourboghrat F, Yang DY. Earing predictions based on asymmetric nonquadratic yield function. Int J Plast 2000;16:1075–104. [18] Gambin W. Plasticity and texture. 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https://openalex.org/W3177247126	https://www.mdpi.com/2223-7747/10/7/1348/pdf?version=1625148478	English	null	Therapeutic Promises of Medicinal Plants in Bangladesh and Their Bioactive Compounds against Ulcers and Inflammatory Diseases	Plants	2,021	cc-by	21,464	Citation: Ahmed, S.R.; Rabbee, M.F.; Roy, A.; Chowdhury, R.; Banik, A.; Kubra, K.; Hassan Chowdhury, M.M.; Baek, K.-H. Therapeutic Promises of Medicinal Plants in Bangladesh and Their Bioactive Compounds against Ulcers and Inﬂammatory Diseases. Plants 2021, 10, 1348. https:// doi.org/10.3390/plants10071348 Received: 21 May 2021 Accepted: 28 June 2021 Published: 1 July 2021 Keywords: medicinal plants; anti-ulcer; inﬂammation; bioactive compounds; metabolites; therapeutic uses Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Therapeutic Promises of Medicinal Plants in Bangladesh and Their Bioactive Compounds against Ulcers and Inﬂammatory Diseases Sheikh Rashel Ahmed 1,†, Muhammad Fazle Rabbee 2,†, Anindita Roy 1 , Rocky Chowdhury 3 , Anik Banik 1 , Khadizatul Kubra 4, Mohammed Mehadi Hassan Chowdhury 5,* and Kwang-Hyun Baek 2,* 1 Department of Plant and Environmental Biotechnology, Faculty of Biotechnology and Genetic Engineering, Sylhet Agricultural University, Sylhet 3100, Bangladesh; rashel.peb@sau.ac.bd (S.R.A.); krisarpita@gmail com (A R ); anikbanik bgesau@gmail com (A B ) y g y y g p krisarpita@gmail.com (A.R.); anikbanik.bgesau@gmail.com (A.B.) 2 Department of Biotechnology, Yeungnam University, Gyeongsan 38541, Korea; rabbi_biotech@ynu.ac.kr 3 h l f d k d d d lb l 2 Department of Biotechnology, Yeungnam University, Gyeongsan 38541, Korea; rabbi_biotech@ynu.ac.kr 3 School of Medicine, Deakin University, 75 Pigdons Rd, Waurn Ponds, Melbourne, VIC 3216, Australia; p gy g y y g y 3 School of Medicine, Deakin University, 75 Pigdons Rd, Waurn Ponds, Melbourne, VIC 3216, Australia; rocky.mbcu@gmail.com 4 Department of Biotechnology and Genetic Engineering, Faculty of Science, Noakhali Science and Technology University, Noakhali 3814, Bangladesh; khadiza@nstu.edu.bd 5 Department of Microbiology, Faculty of Science, Noakhali Science and Technology University, Noakhali 3814, Bangladesh g * Correspondence: mmhc.nstu@gmail.com (M.M.H.C.); khbaek@ynu.ac.kr (K.-H.B.); Tel : +610-414456610 (M M H C ); +82-53-810-3029 (K -H B ) g * Correspondence: mmhc.nstu@gmail.com (M.M.H.C.); khbaek@ynu Tel.: +610-414456610 (M.M.H.C.); +82-53-810-3029 (K.-H.B.) * Correspondence: mmhc.nstu@gmail.com (M.M.H.C.); khbaek@ynu.ac.kr (K.-H.B.); Correspondence: mmhc.nstu@gmail.com (M.M.H.C.); khbae Tel.: +610-414456610 (M.M.H.C.); +82-53-810-3029 (K.-H.B.) † These authors contributed equally to this work. Abstract: When functioning properly, the stomach is the center of both physical and mental satis- faction. Gastrointestinal disorders, or malfunctioning of the stomach, due to infections caused by various biological entities and physiochemical abnormalities, are now widespread, with most of the diseases being inﬂammatory, which, depending on the position and degree of inﬂammation, have different names such as peptic or gastric ulcers, irritable bowel diseases, ulcerative colitis, and so on. While many synthetic drugs, such as non-steroidal anti-inﬂammatory drugs, are now extensively used to treat these diseases, their harmful and long-term side effects cannot be ignored. To treat these diseases safely and successfully, different potent medicinal plants and their active components are considered game-changers. In consideration of this, the present review aimed to reveal a general and comprehensive updated overview of the anti-ulcer and anti-inﬂammatory activities of medicinal plants. To emphasize the efﬁcacy of the medicinal plants, various bioactive compounds from the plant extract, their experimental animal models, and clinical trials are depicted. plants plants plants plants plants 1. Introduction Although the regulations of these conventional medicines are very limited in many countries of the world, the World Health Organization (WHO) has de- veloped a web-based network to ensure its safe and rational use [13]. Due to the role of medicinal plants in the development of powerful therapeutic agents, over 1.5 million practitioners of the traditional medicinal system are using medicinal plants in preventive, promotional, and curative applications [14]. In Europe, it is estimated that the demand for the phytochemical and plant extract-based market has grown from $833.7 million in 2014 to $1.25 billion in 2019, which also indicates the current situation of plant-based consumers’ health awareness in the country [15]. Ulcers and different inﬂammatory diseases of the gastrointestinal tract are very much in need of effective therapeutic methods, since many individuals, regardless of age and gender, are still victims of these inﬂammatory diseases and remain under continuous medication without any hope of permanent recovery. Many chemically synthesized drugs are now being used to treat ulcers; however, they leave a range of side effects in the long run. For example, non-steroidal anti-inﬂammatory drugs (NSAIDs) are widely used in the treatment of inﬂammatory diseases, which may raise the risk of blood clotting that results in heart attacks and strokes. Therefore, the search for plant-mediated drugs is intended to counter these harmful diseases [16–18]. More speciﬁcally, anti-inﬂammatory drugs extracted from plants are being considered [19]. In addition, the application of herbal therapy to treat inﬂammatory bowel disease (IBD) is preferred worldwide because of its effectiveness and safety, although the relevant clinical trials are relatively limited to date [20]. Drugs such as amino-salicylates, corticosteroids, and immune-modulators are used in the treatment of ulcerative colitis, however, medicinal plants may be an effective and safe alternative to such medications [21]. The encouraging and ensuring health beneﬁts of various clinical trials increased the acceptance of plant medicines among common people. Further research and investigations into the diverse active components of herbs and their clinical roles will illuminate and instigate the therapeutic use of plant-based medicines in the future [22]. This review focuses on supporting the therapeutic use of medicinal plants available in Bangladesh in the treatment of various inﬂammatory diseases of the gastrointestinal tract. 1. Introduction For thousands of years, humans have been using medicinal plants, also referred to as healthy herbs, and have a long history of use in primitive medicines [1]. Traditional medicine prepared from these plants is still recognized as a preferred method in the health care system in many parts of the world because of its usefulness and affordability in the treatment of diseases [2]. The history of the Sumerian civilization ﬁrst provided the instances of hundreds of medicinal herbs (e.g., opium), which were listed on clay tablets. Moreover, in 1550 B.C., the Egyptian Ebers Papyrus (an ancient medical document) depicted more than 882 herbal remedies of illness and injuries [3]. For many centuries, miscellaneous bioactive compounds emerging from the medicinal arbor have been used as a precursor to treating various diseases [4]. Owing to the existence of diversity, these organic molecules may be used as models for different synthetic drugs [5]. In addition, medicinal 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/plants Plants 2021, 10, 1348. https://doi.org/10.3390/plants10071348 Plants 2021, 10, 1348 2 of 30 plants have justiﬁed their abilities to deal with several life-threatening diseases like cancer, hepatitis, and acquired immunodeﬁciency syndrome (AIDS) [6–8]. Hence, drug researchers are investigating the abundant curing substances found in nature using ethnobotany and have succeeded in discovering hundreds of pioneer compounds and drugs like aspirin, digoxin, quinine, and opium. The four major branches of phytochemicals include alkaloids, glycosides, polyphenols, and terpenes [9]. The widespread use of bioactive compounds is now evident in various applied branches of science such as agrochemicals, geo-medicine, modern pharmacology, plant science, food industry, cosmetics, and nano-bioscience [10]. In developing and underdeveloped societies, plant medicines are preferred over modern synthetic medicines due to their easy availability and affordability [11]. More- over, the demand for plant-originated products is growing throughout the world. More than 85% of people in Asia, Africa, Latin America, the Middle East, and approximately 100 million people in the European Union rely on traditional herbal medicine for health issues. Approximately 90% of people in certain countries still practice in, and use, plant- based medicines [12]. 2. Inﬂammatory Diseases and Ulcers Inﬂammation is a natural reaction in the defense of tissues against various injuries caused by physical stress combined with harmful chemicals or microbes [23]. Inﬂammation tends to be one of the prime causes for the occurrence of various diseases such as cancer, obesity, cardiovascular disease, rheumatoid arthritis, osteoporosis, asthma, IBD, central nervous system (CNS) depression, diabetes, and Parkinson disease [24]. Inﬂammation can be triggered by many stimuli including pathogens or cytokines (i.e., interleukin-6 or IL-6, tumor necrosis factor-alpha or TNF-α, neutrophils, and monocytes). These stimulating agents can be differentiated into macrophages, which are subsequently attracted to injured tissue sites by chemotaxis and intensify the inﬂammatory reactions to the damaged areas as well as initiate phagocytosis [25]. Inﬂammation can be characterized by swelling, joint pain, and redness. Inﬂammation leads to several chronic diseases including arthritis, au- toimmune disease, coeliac disease, colitis, and asthma, which are often associated with the increased risk of development of cardiovascular diseases, diabetes, cancer, and osteo- porosis [26]. Phytochemicals derived from medicinal plants can be used in the alleviation of inﬂammatory reactions by inhibiting different forms of enzymes e.g., lipoxygenase (LOX), cyclooxygenase (COX), phospholipase A2, and proteins (e.g., inhibition of the pro- inﬂammatory cytokines) [23]. In medical science, Crohn’s disease, and ulcerative colitis (UC) are two types of IBD that cause inﬂammation in the entire gastrointestinal tract and colonic mucosa, raising the risk of colon cancer as well [27]. An ulcer is the condition of corrosion in the linings of the stomach and the duodenum. Thus, ulcers in the gastrointestinal tract are subdivided into ulcerative colitis (lower) and peptic ulcers (upper) depending on the location of the infection [28,29]. Peptic ulcers, also known as gastric and duodenal ulcers, can be characterized as submucosal damage of the digestive tract caused by the disruption of the balance between the hostile factors (i.e., gastric acid, Helicobacter pylori, and anti-inﬂammatory drugs) and protective factors (i.e., mucus, bicarbonate, prostaglandins, and blood ﬂow towards the mucosa) [30]. The common symptoms of peptic ulcers include a burning sensation and pain in the middle or upper stomach, bloating, heartburn, nausea or vomiting, and weight loss [31]. Excessive consumption of alcohol, smoking, chewing tobacco, serious illness, and the intake of NSAIDs increase the risk of ulcer development [28,32]. Gastric ulcers and duodenal ulcers, which are more prevalent in the Eastern and Western countries, cause morbidity and mortality worldwide [33], and H. 1. Introduction Here, we have considered and reviewed the published articles using the keywords: medicinal plants, bioactive compounds, ulcers, inﬂammation, anti-ulcers, and anti-inﬂammatory diseases since 2000, from different databases like PubMed (https://pubmed.ncbi.nlm.nih.gov/) (accessed on 21 May 2021), Scopus Database (https://scopus.com) (accessed on 21 May 2021), and Google Scholar (https://scholar.google.com/) (accessed on 21 May 2021). The research ﬁndings of relevant medicinal plants, native or cultivated, in Bangladesh were taken into consideration. Plants 2021, 10, 1348 3 of 30 2. Inﬂammatory Diseases and Ulcers pylori are considered as one of the most important factors in the development of this disease [34]. 3.1. Aegle marmelos A. marmelos of the Rutaceae family is known most often as the ‘bael’ tree in subcontinent regions and as the ‘wood apple’ in other parts of the world [46,47]. All parts of this plant (leaf, root, seed, bark, and fruit) contain medicinal properties and have many active chemi- cal constituents, such as ﬂavonoids (e.g., ﬂavone and rutin) [48,49], tannins (e.g., skimmia- nine) [50], phenylpropanoids (e.g., lignans and phenylpropenes) [51], saponins, luvangetin, coumarins (e.g., xanthotoxol, marmelosin, and scoparone) [52], carbohydrates (e.g., galac- tose, L-rahamanos, etc.), terpenoids, essential oils (e.g., phellandrene, limonene, ocimene, and pinene) [53], ester, and alkaloids (e.g., skimmiarepin and cinnamamide) [54,55]. The ripe and unripe fruits of this plant have historically been used in the treatment of ulcers, chronic diarrhea, dysentery, rectal inﬂammation, and certain cancers [56]. Additionally, leaves from A. marmelos soaked in water overnight can control peptic ulcers [37] and root decoction is used to treat fevers and colds [56]. Extensive experimental studies have demonstrated that A. marmelos may prevent ul- cers and inﬂammation effectively. An aqueous extract of A. marmelos resulted in decreasing the ulcer score and ulcer index in rats’ stomachs, which was successfully achieved after the administration of 400 mg/kg of an aqueous extract from A. marmelos [57]. In other studies, ethanol, and an aqueous extract from A. marmelos, provided 56.33% and 37.2% protection of ulcers in vivo and the ethanol-induced gastric ulcer model, respectively, compared to standard drugs such as omeprazole (50.4% protection) (20 mg/kg body weight) [57,58]. Ramakrishna et al. investigated the effect of the oral administration of methanol extract from unripe A. marmelos in rats against H. pylori LPS-induced gastric ulcers which showed that this extract (500 mg/kg dose) caused a 93.98% reduction in the gastric ulcers [59]. This improvement in gastric ulcers was due to the inhibition of parameters of gastric secretion, including free and total acidity, pepsin concentration, acid release, and the volume of gastric juice [59]. Similarly, from another study, the extract from the unripe fruit of A. marmelos at a dose of 50 and 100 mg/kg (intraperitoneal injection) also exhibited gastro-protective action against ethanol-induced gastric mucosal damage [60]. These ﬁndings suggest that the A. marmelos and the active compounds in it trigger the prostaglandin-independent pathway as a gastro-protective mechanism. 3. Plant Mediated Treatment of Ulcer and Inﬂammatory Diseases Medicinal plants are the blessings of nature that humans have been using since prehistoric times as the precursor of most drugs. The treatment of ulcers and IBD is facilitated by the intensive use of many medicinal plants [35]. In addition, in recent years, numerous studies on plant extracts have substantially demonstrated anti-ulcer and anti-inﬂammatory activities in both in vitro and in vivo models. Drugs derived from medicinal plants are effective against inﬂammation of the di- gestive tract primarily in two ways, either by reducing acid and pepsin secretion or by assisting the cytoprotection via mucosal defense factors [36]. The mode of action of these drugs differs according to their function. These medications maintain a balance between several aggressive factors (i.e., pepsin, acid, bile salts, and H. pylori) and defensive factors (i.e., cellular mucus, mucin secretion, mucosal blood ﬂow, bicarbonate secretion, and cell turnover) [36,37]. For instance, the methanol extract of drumstick leaves, and ﬂower buds inhibited aspirin-induced gastric lesion formation in rats [38]. It has been reported that several compounds, such as cavidine, chelerythrine, quercetin, hesperidin, α-pinene, and garcinol, present in medicinal plants were used against ulcer diseases [39]. Additionally, prominent anti-inﬂammatory compounds such as resveratrol, colchicine, epigallocatechin- 3-gallate (EGCG) capsaicin, phytosterols, saponins, and curcumin derived from plants were used to treat inﬂammatory diseases [40,41]. Plants 2021, 10, 1348 4 of 30 In addition to the use of traditional medicines such as NSAIDs, the use of proton pump inhibitors (e.g., pantoprazole, omeprazole, lansoprazole, and rabeprazole), his- tamine receptor blockers (e.g., famotidine and ranitidine), synthetic prostaglandin E1 (e.g., misoprostol), antacids (e.g., aluminum hydroxide and magnesium trisilicate combination), corticosteroids (e.g., dexamethasone), immune-suppressants, immune-modulators (e.g., azathioprine and 6-mercapto-purine), antibiotics (e.g., clarithromycin and metronidazole), and biologic agents (e.g., TNF-α) for the treatment of inﬂammatory diseases is in practice on a large scale and has signiﬁcant adverse and undesirable side effects including gas- trointestinal and hepatic toxicity, renal and cardiovascular malfunctions, and hematologic effects such as hemophilia and thrombocytopenia [32,42–45]. As a result, the use of natural products such as plants and herbal derivatives is increasingly growing among ulcerated and inﬂammatory patients having minimal or no side effects as they are the products of nature. Different types of plants which are currently considered to be effective to treat ulcers and inﬂammatory diseases are discussed below: 3.1. Aegle marmelos Bioactive compounds exerting the gastro-protective properties were not speciﬁcally investigated, but the major compounds like eugenol, lu- vangetin, marmin, marmelosin, mucilage, aegelin, dictamine, and auroptene might trigger gastro-protective activities [59,61,62]. Marmelosin (molecular formula C16H14O4) (Figure 1) showed the ability to ﬁght against cellular and DNA damage which are vital events that trigger inﬂammation. Moreover, the downregulation of the pro-inﬂammatory marker (TNF-α) and the transcription factor (NFkB) by marmelosin inhibits chronic inﬂammation in cancer [63]. Eugenol (molecular formula C10H12O2) (Figure 1), another major bioactive compound of A. marmelos, can help to cure ulcers as it is signiﬁcantly effective in reducing myeloperoxidase (MPO) activity, which is responsible for the neutrophilic inﬁltration dur- Plants 2021, 10, 1348 5 of 30 5 of 30 ing ulcer formation [64]. Moreover, eugenol inhibits the production of superoxide anion by neutrophils which results in the reduction of MPO substrates, hydrogen peroxides, and ultimately triggers the decline of MPO activity in neutrophils [65]. Figure 1. Chemical structures of different bioactive compounds derived from the medicinal plants. Figure 1. Chemical structures of different bioactive compounds derived from the medicinal plants. 3.4. Annona squamosa Additionally, the literature suggested that leaf extract of A. squamosa has both thyroid inhibitory and anti- peroxidative properties [91], which are also beneﬁcial during the proper functioning of lipid and carbohydrate metabolism [92]. 3.3. Alpinia nigra and A. species The decocted roots of A. nigra (Zingiberaceae family), locally known as ‘bhulchengi’ or ‘khetranga’, are used as a treatment of gastric ulcers [77]. Natural bioactive compounds as well as crude hydroalcoholic fractions isolated from the Alpinia spp. exhibits anti- inﬂammatory and analgesic activities. Hydroalcoholic and aqueous extracts from the rhi- zomes and leaves of different Alpinia spp. have bioactive components with anti-nociceptive and anti-allergic characteristics [78]. Recently, platelet-activating factor antagonists, named diarylheptanoids, have been reported from Alpinia ofﬁcinarum rhizome extract [79], which also exhibited analgesic actions with 80% ethanoic extract [80]. Fruit extract of A. nigra at a concentration of 250 mg/kg and 500 mg/kg exhibited dose-dependent potential against inﬂammatory paw edema induced by carrageenan. These healing properties may be due to the existence of a large amount of terpenoid, tannins, alkaloids, and ﬂavonoids [81]. Diarylheptanoid, ﬂavonoids, and some volatile oils (e.g., cineole alpha-pinene, sesquiter- pene, linalool, lactones) which are the major bioactive components isolated from Alpinia species have several biological activities including anti-oxidant and anti-cancerous due to the presence of functional aldehyde (–CHO) and keto (C=O) groups in their structure [82]. 3.2. Aloe vera A. vera from Liliaceae is known as ‘ghritkumari’ in Bangladesh and India, and ‘Aloe vera’ worldwide [66]. The leaves, mainly, of A. vera have great medicinal importance as they have active chemical compounds such as saponins, essential amino acids (e.g., cysteine, alanin, arginine, and histidine); anthraquinones (e.g., aloetic acid, aloin A and B (or collectively known as barbaloin), anthracine, anthranon, emodin, etc.); enzymes (e.g., alkaline phosphotase, amylase, catalase, cellulase, cyclooxidase, and lipase); hormones (e.g., auxin and gibberllins); chromones, lignin, minerals, salicylic acid, sterols, carbohydrates (e.g., lignins and sugars); dietary ﬁbers, protein, organic acids, lipids, and vitamins [67]. For the treatment of ulcers, rural people take the inner gel layer of ﬂeshy leaves orally with water [68]. This plant has great potential for curing and preventing gastric ulcers by stimulating its anti-inﬂammatory and healing function and by regulating the mucus and gastric secretion [69]. A. vera can successfully treat various illnesses and conditions including duodenal ulcers, peptic ulcers, mouth ulcers, and sore throats [70]. A. vera, coupled with silver nanoparticles, was effective for ulcer healing by their anti-inﬂammatory enhanced re-epithelialization as well as ﬁbroblast activation effects [71]. A. vera extract pre- treated animals (dose of 200 mg/kg bwt for ﬁve days) had reduced signs of mucosal injury relative to untreated controls, although the incidence was not as high as in omeprazole- treated rats [72]. On the contrary, A. vera extract pre-treatment was ineffective against gastric lesion formation [73]. Furthermore, the extract of A. vera has been shown to cause dose-dependent amelioration in the severity, as well as incidence, of acetic acid- induced gastric lesions when used as a preventive measure in rats [74]. Aloin A and B (molecular formula: C21H22O9), collectively known as barbaloin (Figure 1), is considered Plants 2021, 10, 1348 6 of 30 one of the prominent constituents isolated from A. vera which showed potency to treat ulcers and inﬂammatory diseases [75]. To treat ulcerative colitis, barbaloin effectively increases the mRNA expression of IL-4 and IL-10 in tissues and simultaneously decreases the expression of IFN-γ, IL-6, IL-1β, and TNF-α [76]. Furthermore, barbaloin can prevent the ulcer-mediated myosin light chain kinase (MLCK) signaling pathway by activating the 5’ adenosine monophosphate-activated protein kinase (AMPK) signaling pathway. 3.4. Annona squamosa A. squamosa of the Annonaceae family is generally referred to as the ‘custard apple’ [83]. Based on the phytochemical analysis, alkaloids, total phenolic compounds (TPCs), includ- ing ﬂavonoids, saponins, tannins, and phenols, are most abundant in Annona squamosa. It has been reported that these phenolic compounds have regulatory effects on several physiological and biochemical processes (e.g., signal transduction pathways, cell prolifer- ation, enzymatic activity, and cellular redox potential) to cure chronic diseases [84]. The bark, leaves, stems, roots, seeds, and fruits of A. squamoza contain active alkaloid com- pounds (e.g., annosqualine, dihydrosinapoyltyramine, and liriodenin) [85]; sugar (e.g., quercetin-3-O-glucosid) [86]; phenolics, ﬂavonoids, and tocopherol [87]; acetogenins (e.g., squamostatins, squamoxinone, mosinone, and bullacin) [88], essential oils rich in ent- kaurane type diterpenoids and sequeterpenes (e.g., β-caryophyllene, and β-cedrene) [88]. yp p q p ( g β y p y β ) [ ] A. squamoza had anti-tumor, anti-oxidant, anti-diabetic, anti-lipidimic, anti-ulcer, and anti-inﬂammatory properties due to its cyclic peptide contents [89]. In this phytochemical and in vivo pharmacological research, twelve known compounds were isolated and, among them, 1-(4-β-D-glucopyranosyloxyphenyl)-2-(β-D-glucopyranosyloxy)-ethane was isolated from natural sources for the ﬁrst time. It has been shown that some isolated compounds provided gastroprotection through the inhibition of H+ K+-ATPase (proton pump) activity and the boosting of the mucosal defense mechanism simultaneously. A. squamosa leaves have been shown to have substantial analgesic as well as anti-inﬂammatory activities compared to synthetic drugs such as aceclofenac and pentozocine, indicating a mixed central as well as a peripheral mechanism for its effectiveness [90]. Additionally, the literature suggested that leaf extract of A. squamosa has both thyroid inhibitory and anti- peroxidative properties [91], which are also beneﬁcial during the proper functioning of lipid and carbohydrate metabolism [92]. A. squamoza had anti-tumor, anti-oxidant, anti-diabetic, anti-lipidimic, anti-ulcer, and anti-inﬂammatory properties due to its cyclic peptide contents [89]. In this phytochemical and in vivo pharmacological research, twelve known compounds were isolated and, among them, 1-(4-β-D-glucopyranosyloxyphenyl)-2-(β-D-glucopyranosyloxy)-ethane was isolated from natural sources for the ﬁrst time. It has been shown that some isolated compounds provided gastroprotection through the inhibition of H+ K+-ATPase (proton pump) activity and the boosting of the mucosal defense mechanism simultaneously. A. squamosa leaves have been shown to have substantial analgesic as well as anti-inﬂammatory activities compared to synthetic drugs such as aceclofenac and pentozocine, indicating a mixed central as well as a peripheral mechanism for its effectiveness [90]. 3.6. Aristolochia indica This plant (family: Aristolochiaceae) has been applied against both ulcers and anti- inﬂammatory diseases and so on [100]. Aristolochic acids and esters, aristolactams, ter- penoids, and ﬂavonoids are the major groups of phytoconstituents that are common in all species of Aristolochia including Aristolochia indica. Among them, aristolactams (molec- ular formula: C17H11NO4) (Figure 1) are synthesized as biogenetic intermediates which are normally supposed to be generated from the reduction products of aristolochic acids through the cyclization condensation reaction. From the Aristolochia species, twelve aristo- lactams have been reported, and among them, there were also six compounds having the 3,4-methylenedioxy substitution groups [101]. Anti-inﬂammatory effects of the isolated compounds aristolactam I and hinokinin from A. indica have been reported against TNF-α and IL-6 respectively [102]. (-) Hinokinin exhibited its anti-inﬂammatory effects through the nuclear factor kappa-light-chain-enhancer of the activated B cells (NF-κB) dependent mechanism whereas aristolactam I followed the NF-κB independent mechanism. The ethanol extract of the root bark of A. indica successfully inhibited inﬂammatory activity induced by a hyposensitive agent, named “compound 48/80” (a condensation product of N-methoxyphenethylamine and formaldehyde), which has an almost identical inhibitory pattern of synthetic anti-inﬂammatory drugs such as ketotifen fumarate [103]. 3.7. Artocarpus heterophyllus A. heterophyllus Lam. from the Moraceae family is locally called ‘kanthal’. The leaf ash is taken orally to treat ulcers and young leaves in combination with roots are often beneﬁcial for skin problems, respiratory diseases, and diarrhea (oral medication) [104]. The methano- lic extract of A. heterophyllus at a concentration of 500 mg/kg inhibited the indomethacin- induced gastric ulceration, decreased gastric acid concentration, and increased gastric pH concentration [105]. In another experiment, the natural phenolic compound Moracin-C (molecular formula: C19H18O4) (Figure 1) was isolated from A. heterophyllus, and has been proven to have considerable anti-inﬂammatory effects by blocking the release of lipopolysaccharide (LPS), activated nitric oxide (NO), and reactive oxygen species (ROS) without showing detectable cytotoxicity [106]. Moreover, the compound Moracin-C signiﬁ- cantly decreased LPS-stimulated mRNA up-regulation and protein expression of inducible cyclooxygenase-2 (COX-2), nitric oxide synthase (iNOS), and several pro-inﬂammatory cytokines (e.g., IL-1, IL-6, and TNF-α). The anti-inﬂammatory activity of Moracin-C was linked with the activation of the mitogen-activated protein kinases (e.g., p38, ERK, and JNK) as well as NF-κB pathways by decreasing the nuclear translocation of NF-κB p65 subunit as observed by confocal microscopy and nuclear separation experiment [106]. 3.5. Anthocephalus cadamba Various parts of A. cadamba (Family: Rubiaceae) are used in the treatment of numerous conventional health problems, including mouth ulcers, stomatitis, subdermal inﬂammatory deposits, and gastric disturbances [93]. The anti-ulcer action of methanolic and aqueous Plants 2021, 10, 1348 7 of 30 extracts of A. cadamba bark and leaves were examined in both aspirin-induced and pylorus- ligation ulcer models [94]. At oral doses of 200 and 400 mg/kg, both extracts demonstrated the substantial inhibition of gastric lesion in aspirin-induced gastric ulcers and pylorus ligation-induced ulcers. At the same time, the extracts also exhibited a signiﬁcant de- crease in gastric volume, free acidity, pH, total acidity, ulcer inhibition, as well as ulcer index [94]. The gastro-protective efﬁcacy of A. cadamba was investigated in an in vivo mice study showing the ethanolic extract of A. cadamba at two doses of 250 and 500 mg/kg substantially decreased ulceration caused by both HCl and ethanol-dependent doses [95]. Similarly, in another report, the defatted aqueous extract of the leaves of this plant showed substantial analgesic and anti-inﬂammatory activities at different doses at 50, 100, 300, and 500 mg/kg [96,97]. In addition, the methanol extract of the A. cadamba bark has also been successfully assessed for antipyretic, analgesic, and anti-inﬂammatory actions by some researchers [98,99]. 3.8. Asparagus racemosus The A. racemosus of the Liliaceae family is locally known as ‘shatamuli’ in Bangladesh and ‘climbing asparagus’ all over the world [107,108]. Different parts of A. racemosus Plants 2021, 10, 1348 8 of 30 contain different bioactive compounds such as steroidal glycosides, steroidal saponins (e.g., shatavarin I, shatavarin IV); ﬂavonoids (e.g., 8-methoxy-5, 6, 4′- trihydroxyisoﬂavone); alkaloids (e.g., asparagamine A); asparagamine, racemosol, beta-sitosterol, stigmasterol, genistein, and daidzein [108–111]. The plant, particularly its root, has been shown to have anti-ulcerogenic, adaptogenic, anti-oxidant, and anti-dyspepsia activities [112]. The in vivo research indicated the inhibitory effect of A. racemosus on gastric hydrochloric acid secretion that protects gastric mucosal damage [113]. In addition, the reduction in ulcer index using the raw extract of A. racemosus (100 mg/kg/day orally) was comparable to the antiulcer drug ranitidine (30 mg/kg/day orally). The bioactive compound, shatavarin (steroidal saponin), isolated from A. racemosus, acts spontaneously in the treatment of ulcers in a chemically-induced ulcer model by increasing the mucin secretion inside the gastrointestinal tract [114]. 3.9. Beta vulgaris B. vulgaris of the Chenopodiaceae family is generally referred to as ‘beetroot’ or ‘sugar beet’, widely used as a vegetable or as a salad [115]. The root decoction along with a small amount of vinegar is traditionally used for the treatment of ulcers and sores. The alcoholic root extract of B. vulgaris at a concentration of 200–400 mg/kg signiﬁcantly decreased the ulcer index, ulcer score, total acidity, and maintains normal mucosa in pylorus ligation and ethanol-induced ulcer in rat models [116,117]. The active constituents in B. vulgaris are polyphenols, tannins, alkaloids, vitamins (e.g., C, B3, B6, B9), carotenoids, ﬂavonoids, betacyanins, betaxanthins, betanin, and saponins, most likely have inhibitory effects on gastric mucosal injury [116,118,119]. Betalains (molecular formula: C24H26N2O13) (Figure 1), nitrogenous water-soluble compounds, are abundant in B. vulgaris which have strong therapeutic activity against inﬂammatory diseases. This phenolic chromoalkaloid synthesized from the amino acid tyrosine is used as food additives due to its high solubility and lack of toxicity [120]. Pre-treatment with petroleum ether extract of B. vulgaris L. gave signiﬁcant defense against aspirin-induced gastric ulcers [121]. The anti-ulcer effects are likely due to the existence of steroids or phenols in the extract. Moreover, there are signiﬁcant anti-inﬂammatory properties in the aqueous extract of B. vulgaris which was also investigated against the carrageenan-induced edema in rats [122]. 3.11. Caesalpinia pulcherrima C. pulcherrima of the family Fabaceae or Leguminosae is locally known as ‘krishnachura’ in Bangladesh and as ‘peacock ﬂower’ worldwide [130]. Various medicinally active veg- etative sections of this plant including young leaves, bark, fruit, seed, stem, ﬂower, and whole plant are considered as a storehouse of different bioactive compounds such as sterols (e.g., β-sitosterol), ﬂavonoids (e.g., ﬂavones, isoﬂavones, ﬂavanols chalcones, rotenoids), glycosides, organic compounds (e.g., α-phellandrene), essential oils (e.g., β-caryophyllene, γ-Terpinene), and carotenoids (e.g., lutein, zeaxanthin), etc. [130–132]. Traditionally, the extracts of this plant have been used to treat various diseases such as malaria, diarrhea, dysentery, fungal infections, respiratory diseases, inﬂammatory diseases, and microbial diseases [132]. The gastroprotective ability of galactomannan extracted from the seeds of C. pulcherrima L. (GM-CP) was assessed in indomethacin induced acute gastritis model, which showed that GM-CP (10 mg/kg dose) decreased the severity of macroscopic lesion as well as the loss of superﬁcial cells through ameliorating inﬂammatory symptoms including neutrophil inﬁltration, production of TNF-α, thiobarbituric acid, reactive species migration, and adhesion of mesenteric leukocytes [133]. Sharma and Rajani have assessed the anti- inﬂammatory and anti-ulcer efﬁcacy of C. pulcherrima in indomethacin induced cotton pellet granuloma and both aspirin and pylorus-ligation-induced ulcer models respectively [134]. 3.12. Calendula ofﬁcinalis C. ofﬁcinalis L. (family: Asteraceae) is popular with the name ‘gada ful’ to Bangladeshi people. The petals are orally administered in the treatment of stomach pain, inﬂammation, and ulcers [135]. The extract of C. ofﬁcinalis had both antacid property and gastroprotective capacity. Signiﬁcant gastro-curative efﬁcacy of the extract was investigated against absolute ethanol and indomethacin induced ulcerative lesion in rats [136]. Another study also conﬁrmed the presence of gastroprotective properties in the plant extract of C. ofﬁcinalis resulting in the substantial inhibition of the ulcer development caused by chemical and physical agents with 87.15% utmost therapeutic efﬁciency (450 mg/kg bwt) in cold-resistant and stress-induced ulcers [137]. The ﬁndings from lipid peroxidation and enzyme assay clearly exhibited the antioxidant effect of the whole plant of C. ofﬁcinalis on the ulcer condition. Likewise, the ethanolic extract of C. ofﬁcinalis in mice showed substantial anti- inﬂammatory properties against carrageenan as well as dextran–induced paw edema [138]. The extracts substantially inhibited enhanced extents of pro-inﬂammatory cytokines (e.g., IL-6, IL-1β, TNF-α, IFN-γ) and acute-phase protein, C-reactive protein (CRP), in mice generated by LPS injection. The extract also inhibited LPS-induced COX-2 levels in the spleen of mice. The ﬁndings demonstrated that potential anti-inﬂammatory activities of C. ofﬁcinalis extract may be mediated by inhibiting pro-inﬂammatory cytokines as well as COX-2, and subsequent prostaglandin synthesis [138]. 3.10. Bombax ceiba The plant B. ceiba is under the family Bombacaceae and has several medicinal proper- ties against inﬂammatory diseases, such as cancer, dysentery, ulcer, and microbial infec- tions [123]. Bark, latex, leaf, ﬂower, seed, stem, rhizomes, thorn, stem bark, fruit, and heart- wood are the medicinally active parts of B. ceiba which contain common ﬂavonoids (e.g., isovitexin (molecular formula: C21H20O10) (Figure 1), kaempferol 3-O-galactoside (molecu- lar formula: C21H20O11) (Figure 1), gallic acid (molecular formula: C7H6O5) (Figure 1)), quercetin, lupeol, tannic acid, sesquiterpenoids, naphthol, naphthoquinones, polysaccha- rides, anthocyanins, shamimin, lupeol, and alkaloids [123,124]. The young root paste of this plant is traditionally used in the treatment of gynecological disorders, constipation, diarrhea, piles, dysentery, wounds, ulcer, inﬂammation, and urinary diseases [124,125]. Crude plant extracts demonstrated strong analgesic efﬁcacy in acetic acid-induced writhing as well as hot plate tests in mice [126]. Using naloxone, B. ceiba extract-induced analgesia was independent of the opioid receptor; where, mangiferin exhibited signiﬁcant interaction with the receptor at a peripheral site, with a minor contribution at the neuronal stage [126]. This plant is known to have large amounts of gallic and tannic acids that act as astringents precipitating proteins that help to regenerate the damaged epithelial mucosal lining of the ulcerated lesion [127,128]. Similarly, B. ceiba effectively decreased the ulcer index after administration of ﬂower extract (300 mg/kg/day) for seven days, and suppressed gastric inﬂammation by inhibiting TNF-α and interleukin 1β (IL-1β) [129]. Plants 2021, 10, 1348 9 of 30 3.11. Caesalpinia pulcherrima 3.14. Camellia sinensis C. sinensis of the Theaceae family is locally termed as ‘cha’ in Bangladesh and as ‘black tea’ worldwide [146]. The leaves, stems, and twigs contain different active constituents like ﬂavonoids (e.g., thearubigins, theaﬂavins, and catechins), vitamins amino acids, β- carotene, chlorogenic acids, volatile compounds carbohydrates, phenolic acids (e.g., gallic acid, caffeic acid, and cauramic acid), proteins, lipids, and ﬂuoride [147,148]. These components help in the treatment of different diseases with their different protective properties [146]. This plant is traditionally used in the treatment of ﬂatulence, digestion, vomiting, diarrhea, maintaining body temperature, blood sugar, and in the alleviation of stomach discomfort [149,150]. Heteropolysaccharides extracted from C. sinensis exerted gastroprotective properties by reducing ethanol-induced gastric lesions [151]. Moreover, it was also found effective in gastroprotection by gastric mucus maintenance and decreased glutathione levels [151]. Theaﬂavin (molecular formula: C29H24O12) (Figure 1), a major active component of black tea, showed the potency for the healing of indomethacin-mediated gastric ulcers by the synthesis of PGE2, revealing antioxidative aspects and intensiﬁcation of mucin secretion [152]. This polyphelnolic compound also triggers the suppression of several inﬂammatory modulators in the ulcer margin and induces iNOS modulation for gas- tric ulcer healing [153]. Catechin (molecular formula: C15H14O6) (Figure 1), especially epigallocatechin-3-gallate (EGCG) (molecular formula: C22H18O11) (Figure 1), is the major bioactive polyphenol compound of C. sinensis and showed strong antagonistic activity against inﬂammatory respiratory infections in humans caused by Stenotrophomonas mal- tophilia [154]. This compound inhibits the enzyme dihydrofolate reductase of S. maltophilia, which is considered an attractive target for the development of antibacterial molecules [155]. Green tea catechins inhibited TNF synthesis in a murine model of inﬂammatory arthritis and reduced inﬂammation as well as slowed down cartilage breakdown [156]. 3.13. Calotropis procera C. procera of the Asclepiadaecae family is popularly termed ‘akanda’ in Bangladesh and ‘milkweed’ worldwide [139]. The leaf, ﬂower, ﬂower bud, latex, root bark, and root of this plant contain different bioactive compounds such as triterpenoids, lupeol, ﬂavonoids, ﬂavanols, glycosides, resins, cardenolides, mudarine, anthocyanins, α-amyrin, β-amyrin, calactin, calotropin, and β-sitosterol. Bioactive compounds from C. procera show protective properties against different diseases [139,140]. Traditionally, the latex is used in reducing toothache [141], in the treatment of vertigo, paralysis, hair loss, baldness, and rheumatoid/joint swellings. In addition, the leaves have therapeutic values to reduce swelling as well as to treat joint pain. The bark is used in the treatment of eczema, leprosy, and elephantiasis treatment [142]. Different parts of C. procera show anti-inﬂammatory and gastroprotective properties. The leaves and bark of C. procera have curative properties for ulcers and stomach aches. The anti-ulcer efﬁcacy of C. procera extract was determined in white albino rats [143]. The extract signiﬁcantly reduced the ulcer index, ulcer lesion score, leukocyte inﬁltration in Plants 2021, 10, 1348 10 of 30 10 of 30 the gastric epithelial lining, and ameliorated the congestion and necrosis. The anti-ulcer property of the plant was mainly due to the presence of different phyto-constituents like polyphenols, ﬂavonoid and lignin glycoside triterpenoids, tannins, and steroids [144]. In the treatment of ulcerative colitis, colitic rats treated with the methanol extract of dried latex of C. procera (MeDL), demonstrated a signiﬁcant reduction of colonic mucosal damage. This reduction resulted from the inhibition of oxidative stress and pro-inﬂammatory signaling proposes C. procera as a promising therapeutic plant to treat inﬂammatory conditions in the colon [145]. From this study, it was proposed that targeting oxidative stress and NFκB (p65) regulated pro-inﬂammatory signaling could be a potential approach for providing protection in the treatment of colitis. 3.15. Capsicum annuum and C. frutescens C. annuum and C. frutescens from the Solanaceae family are known as ’chili’, ‘pepper’ or simply “capsicum” worldwide, contains different active constituents like solasonine, capsacin, acyclic diterpene glycosides, and capsidiol [130,157]. The fruit is used locally as a spice, which shows potent anti-ulcer and antioxidant properties. It also helps prevent type-2 diabetes [130,157]. The fruit of C. frutescens was taken to treat gastric disorders and ulcers [158]. At doses of 300 and 600 mg/kg body weight, the aqueous extract of the chili pepper (C. frutescens) reduced the gastric ulcer length of aspirin-induced ulcer in experimental rats, which proved the curing properties of the extract of C. frutescen [159]. Capsaicin (molecular formula: C18H27NO3) (Figure 1) is the major bioactive component of these plants, exhibits anti-inﬂammatory characteristics [160,161]. This secondary metabo- lite of Capsicum spp. is also known as capsaicinoid due to their alkaloid nature. Researchers assessed the impact of capsaicin on the mucosa of the stomach, pro-inﬂammatory cy- tokines (e.g., TNF-α, IL-6, IL-1β), and COX-2 in gastric mucosa in two experimental models. Histopathological examinations coupled with molecular studies of stomach sam- Plants 2021, 10, 1348 11 of 30 11 of 30 ples revealed a protective action of gastric mucosa along with a substantial reduction of pro-inﬂammatory cytokines as well as COX-2 in both experimental models [160]. ples revealed a protective action of gastric mucosa along with a substantial reduction of pro-inﬂammatory cytokines as well as COX-2 in both experimental models [160]. 3.17. Cinnamomum cassia C. cassia of the Lauraceae family is commonly known as ‘daruchini’ in Bangladesh and as ‘cinnamon’ worldwide. The bark of cinnamon contains different active constituents such as terpenoids, phenylpropanoids, glycosides, cinnamaldehyde, cinnamic acid, cinnamate, and numerous essential oils (e.g., trans-cinnamaldehyde, cinnamyl acetate, eugenol, L- borneol), etc. [168]. The C. cassia plant is traditionally used as a spice and cures dental problems, prevents colon cancer, and acts as a coagulant to prevent bleeding [169]. It performs different anti-inﬂammatory activities [168–170]. Cinnamaldehyde (molecular formula: C9H8O) (Figure 1), the most abundant phytocomponents of C. cassia, was active as an anti-inﬂammatory agent in gastric inﬂammation. This aromatic aldehyde compound inhibited IL-8 secretion/expression and the nuclear factor kappa B (NF-κB) pathway to treat Helicobacter pylori-induced gastric inﬂammation [171]. 3.18. Chromolaena odorata L. C. odorata, which is commonly known as ‘germany lata’ in Bangladesh, is a member of the Asteraceae family. Several chemical constituents including ﬂavonoids, alkaloids, triterpenes/steroids, monoterpenes, and phenolic acids can play signiﬁcant roles in treat- ing inﬂammatory and ulcer diseases [172]. The plant has been grown in bushy areas, forest zones, and roadsides. The leaf extract of this plant with salt has been used as an oral medicine in the tropical areas of Bangladesh to get rid of ulcers [173]. The ethanol- induced gastric lesion was successfully minimized by applying the extract of C. odorata L. Also, C. odorata extract was effective in arresting internal bleeding due to the presence of polyphenols [174]. 3.16. Carica papaya C. papaya of the Caricaceae family is commonly known as ‘papaya’ [162]. C. papaya contains diverse active constituents like enzymes (e.g., papain, chemopapain, chymopa- pain, peptidase, lysosome, and myrosine), proteins, fats (e.g., myristic, palmitic, stearic, and linoleic), carbohydrates (e.g., glucose, fructose, galactose, and xylitol), minerals, vitamins, volatile compounds, alkaloids (e.g., carpain, pseudocarpain, choline, and caproside), glyco- sides, and carotenoids [163,164]. The most common and signiﬁcant constituent, papain, is a papaya proteinase I (cysteine protease) enzyme which has several therapeutic effects particularly in inﬂammation and gastrointestinal disorders. Traditionally, the decoctions of leaves and dried ﬂowers were used as anti-anemic agents, blood puriﬁers, and in several diseases [164–166]. Fruits are used to treat impotence and ulcer. The crude latex decoction is used to treat anthelmintic, dyspepsia, burns pain, bleeding hemorrhoids, stomachic, and diarrhea [167]. 3.20. Glycyrrhiza glabra G. glabra (family: Fabaceae) has been reported to treat gastric ulcers, oral ulcers, as well as ulcerative colitis [190]. Although the powder form of G. glabra is commonly used to treat cough, gastric pain, and vomiting tendency [191], however, the extract of G. glabra L also has healing properties against gastric ulcer lesion, caused by indomethacin and HCl/Ethanol, in a dose-dependent manner [192]. It has been reported that root of G. glabra, also known as licorice or liquorice, derived compounds stimulate the mucus secretion from the stomach, expand the life span of the surface cell of the stomach, and enhance the prostaglandin level which eventually lead to ulcer healing [193]. According to recent studies, the most important bioactive compounds in licorice are ﬂavonoids, triterpenes, and polysaccharides [194]. Glycyrrhizin (molecular formula: C42H62O16) (Figure 1), the major bioactive compound of G. glabra, is effective against inﬂammation and ulcers [195,196]. This triterpenoid saponin-based compound has two isomers such as 18α-GL & 18β-GL and they have anti-ﬁbrogenic properties [197]. Due to the probable antioxidant effect, extracts of licorice had a healing capacity in gastrointestinal ulceration. In the early 1960s, a succinate derivative of glycerrhetinic acid called carbenoxolone (molecular formula: C34H50O7) (Figure 1) was developed in London as an antiulcer drug and was used to assist in the healing of ulcers as the preferred form of licorice [198]. It is determined that G. glabra can increase superoxide dismutase effectively as an enzymatic defense system to treat ulcerative colitis induced by acetic acid. Moreover, it played a crucial role in diminishing the levels of NO, TNF-α, and IL-6 in a dose-dependent manner [199]. Anti-inﬂammatory mechanisms of licorice and G. glabra include four major factors: prostaglandin E2 (PGE2), TNF, MMPs, and free radicals (including reactive oxygen, and nitrogen species) were reported based on previous several studies [190]. 3.19. Curcuma longa L. C. longa L. of the Zingiberaceae family is locally known as ‘halud’ in Bangladesh and India and commonly termed as ‘turmeric’ worldwide [175]. The rhizome and leaves of C. longa contain many active constituents such as alkaloids, phenolic compounds (e.g., isoﬂavone, diarylheptanoids curcuminoids, diyrenphenate), alcohols, essential oils (e.g., monoterpenes, sesquiterpenes, diterpenes, and triterpenoids), ketones, ß-turmerone, α- turmerone, steroids (e.g., β-sitosterol), and aldehydes [176–178]. Traditionally, this plant has been used to cure dermatitis, cancer, leprosy, hemorrhoids, inﬂammation, and asthma, and shows hepato-protective activity [179,180]. Based on its antioxidant property, curcumin (molecular formula: C21H20O6) (Figure 1), also known as diferuloylmethane, an active component of C. longa, scavenges ROS and regulates matrix metallopeptidases (MMPs) activity to induce antiulcer activity [181,182]. Beside this, curcumin also exerted different pharmacological effects including anti-inﬂammatory activity triggered by suppression of Plants 2021, 10, 1348 12 of 30 12 of 30 PG synthesis [183,184]. It is reported that the substitution group on the methoxy position draws a vital contribution in the anti-inﬂammatory effects of curcumin [185]. In a transgenic mice model, it was shown that phytosomal curcumin exerted strong effects on the activation of anti-inﬂammatory PPARγ (peroxisome proliferator-activated receptor γ) as well as the inhibition of pro-inﬂammatory NF-κB, therefore, it could be used in the treatment of patients with chronic hepatitis B infection [186]. The application of the paste of the rhizome is carried out on injuries, strains, and wounds externally as the primary treatment. The leaves are also used in malaria and jaundice treatment and during pregnancy [187,188]. From the ﬁndings of phase-I clinical trials and toxicology studies conducted by Anand et al., it was concluded that curcumin is safe and tolerated even at very high doses (12 g/day) in humans [189]. A joint report from the World Health Organization (WHO) and the Food and Agriculture Organization (FAO) has recommended that a high intake of curcumin at a concentration of 0-1 mg/kg body weight per day had no adverse health effects on the human body [178]. 3.24. Mangifera indica M. indica of the Anacardiaceae family is commonly known as the ‘mango’ tree [214]. The fruits, stem barks, heartwoods, leaves, and roots of the plant contain active constituents like triterpenoids, polyphenolics (e.g., mangiferin aglycone), sterols (e.g., mangsterol), alkaloids, saponins, tannins and ﬂavonoids (e.g., mangiferin, mangostin, 29-hydroxy mangiferonic), essential oils (e.g., nerol, elemene, linalool, humulene, ocimene), vitamin A, vitamin C, xanthophylls, and β-carotenes [214]. The leaf extract of M. indica, along with rice bran oil, is used traditionally for the treatment of ulcers. The young leaves are also capable of curing dysentery. The seed pulp, along with cornﬂour, can control diabetes. The extracts of the leaves of the mango plant decreased the ulcer index and showed antiulcer properties to ﬁght against in vivo aspirin-induced gastric ulcer [215,216]. The ripe mango juice is used to tackle heat stroke, which is a fatal life-threatening inﬂammatory response [217]. The extract from the bark can treat fever, cold, and vomiting [218]. 3.22. Centella asiatica 3.22. Centella asiatica C. asiatica of the Umbelliferae family is locally known as ‘thankunipata’ in Bangladesh and as ‘Indian pennywort’ worldwide. It originates from the wetlands of Asia [204]. The paste of the leaves of C. asiatica is locally used for ulcers and different gastric disorders. Traditionally, this plant has been used to treat diarrhea, rheumatism, skin diseases, syphilis, and inﬂammation. Several main components i.e., madecassoside, madecassic acid, asi- aticoside, and asiatic acid, present in C. asiatica were extensively studied for therapeutic purposes including ulcers and inﬂammatory diseases [205]. Asiaticoside (molecular for- mula: C48H78O19) (Figure 1), a major active constituent of C. asiatica, plays an important role in healing gastric ulcers [204]. Asiaticoside is a glycoside compound that belongs to the triterpenoid group [206]. Acetic acid-induced gastric ulcers in rats were reduced with a lower lesion score, minimized ulcer size, and reduced or absent leucocytes inﬁltration and submucosal edema, when the plant extract was administered orally. Rats pre-treated with leaf extract exhibited comparatively better protection of the gastric mucosa and had cytoprotective effects [207,208]. 3.23. Lagenaria siceraria L. siceraria of the Cucurbitaceae family is commonly known as ‘bottle gourd’. The fruits, leaves, roots, and seeds of L. siceraria contain different active constituents such as vitamin B, amino acids, and ascorbic acid (vitamin C) [157,209]. The leaves are cooked and taken by women as a potherb and to relieve the pain during menstruation. The syrups from the fruits are also used to treat bronchial abnormalities like pectoral cough, asthma, etc. [210,211]. The extract of L. siceraria has both strong anti-ulcer and antioxidant activities, though the molecular mechanisms of both anti-ulcer and anti-oxidant activities were not investigated [212]. However, the doses of 250 and 400 mg/kg were safe as there was no indications of signs of toxicity or mortality [212]. From another study, a dose up to 1000 mg/kg could be safe after the repeated administration of methanolic extract of L. siceraria fruit for 28 days [213]. 3.21. Hibiscus rosa-sinensis H. rosa-sinensis (family: Malvaceae) is locally known as ‘joba’ in Bangladesh and commonly known as ‘China rose’ worldwide [200]. The root, leaf, and ﬂower of H. rosa- sinensis contains different active constituents such as tannins, steroids, anthraquinones, essential oils, quinines, mucilage, phenols, reducing sugars, ﬂavonoids, carbohydrates, free amino acids, alkaloids, proteins, terpenoids, cardiac glycosides, and saponins [200]. Traditionally, the root of the plant is used for treating ulcers. The administration of aqueous ﬂower extract from this plant (250 mg/kg) revealed gastric ulcer suppressive activity against pylorus-ligation, aspirin-induced, and ethanol-induced ulcers in vivo and this protective activity occurred due to the presence of ﬂavonoids and tannins as free radical quenchers [201]. Fruits are used externally to cure sprains, wounds, and ulcers [200,202,203]. Plants 2021, 10, 1348 13 of 30 13 of 30 3.25. Mimosa pudica M. pudica of the Fabaceae family is locally known as ‘lajjaboti’ in Bangladesh and is commonly known as ‘zombie’ or ‘shy plant’ worldwide. The leaf juice or whole plant decoction helps in treating urinary tract infection, dysentery, pain in the body or head, tooth pain, and snakebite injury [218,219]. The fresh leaf and seed decoctions are effectively used in curing intestinal ulcers traditionally [218]. It has been shown that the methanolic extract of M. pudica exhibited more gastroprotective properties than chloroform extract after administrating two different doses (100 and 200 mg/kg) for a duration of eight days [220]. It has enhanced gastroprotective properties because of the presence of phyto-constituents (e.g., ﬂavonoids, alkaloids, and tannins) and free radical scavenging activity [220]. The ethanolic extract of M. pudica, at the dose of 400 mg/kg, signiﬁcantly attenuated ulcerative Plants 2021, 10, 1348 14 of 30 14 of 30 colitis induced by 4% acetic acid and potentially reduced both myeloperoxidase and malondialdehyde in rats when compared to the reference drug prednisolone [221]. 3.28. Psidium guajava P. guajava of the Myrtaceae family is commonly known as ‘guava’, which contains different active chemical constituents like tannin, resin, quercetin, crystals of calcium oxalate, guaijaverin, fat, galactose-speciﬁc lecithins, cellulose, volatile oil, mineral salts, and ﬂavonoids [228,232]. The decoction prepared from the leaves of P. guajava is useful in treating ulcers. The extract of P. guajava leaves is also effective in the problems associated with diabetes and stomachache [218,228,233]. 3.27. Moringa oleifera M. oleifera of the Moringaceae family is locally known as ‘shajna’ in Bangladesh and as ‘drum-stick tree’ worldwide [223]. This plant contains active constituents like alkaloids, beta carotene, tannins, beta sitosterol, zeatin, quercetin, ﬂavonoids, kaempferom, protein, vitamins, minerals, amino acids, phenolic acids, natural sugars, phytosterols, saponin, and terpenoids [227,228]. Quercetin (molecular formula: C15H10O7) (Figure 1), a ﬂavonoid compound in M. oleifera, showed strong anti-inﬂammatory activity [229]. The leaf of M. oleifera helps in the treatment of ulcers, indigestion, asthma, and sinus pain [219,227,228]. The ethanolic root extract signiﬁcantly decreased ulcer index, total acidity, and enhanced gastric pH at both doses of 350 and 500 mg/kg. The extract also has anti-secretory and cytoprotective potentiality [230]. The alcoholic leaf extract also resulted in the reduction of acid pepsin secretion and ulcer lesion [38]. The acetone, methanol, chloroform, and petroleum ether extracts of the leaves and fruits of M. oleifera were investigated on both gastric and duodenal ulcers using acetic acid, pylorus ligation, indomethacin, ethanol, and cold-restraint stress-induced gastric ulcer and cysteamine-induced duodenal ulcer model [231]. The methanol and acetone extract of the leaves exerted signiﬁcant gastric antisecretory and gastric cytoprotective effects in pylorus-ligated rats and both ethanol- and indomethacin-induced gastric ulcers, respectively. Compared to the insigniﬁcant antiulcer effect of fruit, the leaf extract reduced both cysteamine-induced duodenal ulcers and the stress-induced gastric ulcers remarkably [231]. 3.26. Momordica charantia M. charantia is a climbing plant of the Cucurbitaceae family. It is often called ‘bitter gourd’ worldwide and as ‘corolla’ in Bangladesh [222,223]. Powder prepared from the whole plant is locally used in treating diversiﬁed ulcers [218]. The local people use the unripe fruits as vegetables and cook them. The highest percentage of gastric ulcer inhibi- tion was shown to be 62.85% in the ethanol-induced ulcer model at a dose of 100 mg/kg compared to the standard ranitidine [224]. There was signiﬁcant healing activity with this plant extract to treat acetic acid-induced gastric ulcers [225]. The extract successfully reduced the ulcer index and inhibited the development of gastric ulcers in all the experi- mental ulcer models including indomethacin-induced, pylorus-ligated, ethanol-induced, cysteamine-induced duodenal ulcers, and stress-induced ulcer models [226]. 3.31. Solanum nigrum S. nigrum of the Solanaceae family is commonly known as ‘black nightshade’ and locally known as ‘kakmachi’ [209]. The active constituents in this plant are phytosterols, glycopro- teins, glycoalkaloids, saponins, polysaccharides, ﬂavonoids, gallic acid, alkaloids, catechin, naringenin, protocatechuic acid, rutin, caffeic acid, and epicatechin [130,202,238,239]. Peo- ple consume the fresh leaves of S. nigrum traditionally in the treatment of intestinal ulcers including hepatotoxicity, body pain, cancer, abdominal problems, and in improving the functions of CNS and the brain [130,202,238]. 3.32. Syzygium aromaticum S. aromaticum of the Myrtaceae family is commonly known as ‘clove’ and locally known as ‘lobongo’ in Bangladesh [240]. S. aromaticum contains active volatile and non-volatile constituents like ﬂavonoids, essential oils (e.g., phenyl propanoids, carvacrol, eugenol, thymol, cinnamaldehyde), triterpenes, hydroxycinnamic acid, sterol (e.g., campesterol, stigmasterol) hydroxyphenylpropene, tannins, and hydroxybenzoic acids [130,241–243]. The ﬂower bud of the plant helps to prevent ulcers. Traditionally, it is used in dental care for tooth aches, oral ulcers, as a disinfectant in wounds and insect bites, for purifying blood, for maintaining cardiovascular health, as a platelet inhibitor, and in boosting the immune system [236,241]. 3.33. Terminalia chebula T. chebula, of the Combretaceae family, is commonly known as ‘chebulic myrobalan’. In Bangladesh and India, it is well known as ‘haritoki’ [202]. This plant contains diverse bioactive chemical compounds such as chebulic acid, sorbitol, chebulinic acid, tannic acid, chebulagic acid, lucilage, gallic acid, tannin, corilagin, ﬁxed oils, ellagic acid, resin, ﬂavonoids, fructose, amino acids, and sterols [130,202,244]. T. chebula mixed with triphala and sindhu salt, is used in treating ulcers and ulcerated wounds. The fruit helps in the treatment of blood dysentery and stomachache. The maceration of the bark of T. chebula, in addition to other medicinal plants, helps in waist pain and pain in bones [202,244,245]. Chebulinic acid (molecular formula: C41H32O27), is also called 1,3,6-tri-O-galloyl-2,4- chebuloyl-β-D-glucopyranoside, is an ellagitannin abundant in the fruits of T. chebula had strong therapeutic efﬁcacy on gastric ulcers [246]. 3.30. Shorea robusta S. robusta of the Dipterocarpaceae family and is commonly known as ‘sal tree’ and locally known as ‘shaal’, in Bangladesh [236]. This plant contains different active constituents like mangiferonic acid, uvaol, ursolic acid, αand β amyrin, asiatic acid, tri, and tetrehydrox- yursenoic acid [236]. Ointments prepared from S. robusta are traditionally used in curing different ailments such as ulcers, wounds, hemorrhoids, burns, dermatitis, pain, swelling, ear problems, and eye problems. The resin or oleoresin (gum) of the plant is used in curing diarrhea, gonorrhea, and dysentery [236]. Rats received S. robusta resin at doses of 150 and 300 mg/kg for treating ethanol and pyloric ligation-induced gastric ulcer, prevented gastric mucosal damage, decreased gastric juice volume, total acidity, and pepsin secretion [237]. 3.29. Scindapsus ofﬁcinalis S. ofﬁcinalis (family: Araceae), which is locally known as ‘guruchi lota’, is extensively used by folk medicinal herbalists in Bangladesh in treating ulcers, indigestion, anti-emetic, and helminthiasis [234]. When an in vivo study was conducted to ﬁgure out the efﬁciency of the fruit extract of S. ofﬁcinalis to treat pyloric-ligation-induced gastric ulcers in Wistar rats, the high dose of the extract (500 mg/kg) showed the potential to minimize the ulcer index. The anti-ulcer activity might be ascribed to the free radical scavenging activity [235]. Plants 2021, 10, 1348 15 of 30 15 of 30 3.30. Shorea robusta 3.35. Zingiber ofﬁcinale 3.35. Zingiber ofﬁcinale Z. ofﬁcinale of the Zingiberaceae family is commonly known as ‘ginger’, which is tra- ditionally used as a medicinal preparation in the treatment of peptic ulcers, diarrhea, allergy, smallpox, asthma, urticaria, fever, impotence, and bronchitis [216,250,251]. Sev- eral previous studies in animal models showed that gastrointestinal ulcers were induced by hypothermic restraint stress and NSAIDs [252–254]. Zingerone (molecular formula: C11H14O3) (Figure 1), a phenolic active constituent of ginger (9.25%) [255], showed the anti- ulcer efﬁcacy in vivo when Wistar rats were fed with zingeron doses (50, 100, 200 mg/kg), by lowering the number and length of ethanol-induced ulcers in zingerone uptaken groups. The effect of zingeron is protective on the artiﬁcially induced ulcer because of its free radi- cal quenching potentiality [256]. Research suggested that ginger constituents ameliorate low-dose aspirin-induced gastric ulceration in the gastrointestinal tract. Therefore, a novel prodrug of aspirin designed as 6-gingerol aspirinate reduced acute stomach injury induced by aspirin in mice [257]. It is proposed that the anti-ulcer and healing capacities of zinger, are achieved due to its strong thromboxane synthetase feature, inhibition of K+-ATPase, gastric H+, and H. pylori growth by phenolic antioxidants [252]. All the medicinal plants discussed above show anti-ulcer activity and their uses are in practice among people since ancient time. These plants not only cure ulcers but also show potency against other diseases. The modes of action of bioactive compounds isolated from medicinal plants is summarized in Figure 2. Moreover, other similar medicinal plants with antiulcer and anti-inﬂammatory activity are summarized in Table 1, with their therapeutic uses and medicinal parts to be used, for easy identiﬁcation. Table 1. Common medicinal plants of Bangladesh used in the treatment of ulcer, inﬂammatory, and other related diseases. 3.34. Tinospora cordifolia T. cordifolia (family: Menispermaceae), locally known as ‘pipolti’, is traditionally used as a treatment for gastric trouble and ulcers [247]. Moreover, the anti-ulcer efﬁciency of T. cordifolia extracts was assessed in ethanol and the pylorus ligation-induced ulcer model whereas a remarkable reduction of ulcer index, gastric volume, and total acidity sup- ported the efﬁcacy of the extract as an anti-ulcer agent [248]. The major phytocomponents which were isolated from the T. cordifolia, sesquiterpene tinocordifolin, tinocordifolioside, sesquiterpene glycoside, arabinogalactan, tinocordiside, makisterone, magnoﬂorine, and palmatine could play a vital role in reducing the illness [249]. Plants 2021, 10, 1348 16 of 30 16 of 30 3.35. Zingiber ofﬁcinale Name (Family) Local Name in Bangladesh Commonly Used Medicinal Parts Therapeutic Uses References Acacia senegal (Leguminosae) Babla Roots, bark, wood leaves, ﬂowers, gums, and seeds Used to treat ulcers, inﬂammation, to cure stomach and throat pain [228,258] Acacia farnesiana (Leguminosae) Belatibabul Leaves, bark, and ﬂowers Anti-inﬂammatory, anti-hepatotoxic, anti-pyritic and anti-ulcerogenic [259,260] Achyranthes aspera (Amaranthaseae) Apang Whole plant, roots, and seeds Used to treat ulcers and inﬂammation [261,262] Albizia procera (Mimosaceae) Silkorai Bark and leaves Used to treat ulcers, threadworms, skin scabies, and tooth pain [233] Allophylus serratus (Sapindaceae) Tippani Leaves, roots, ﬂowers and seeds Used to treat ulcers, inﬂammation, and gastrointestinal disorders [130,263] Alstonia scholaris (Apocynaceae) Chatim Bark and latex Used to treat ulcer, dysentery, and rheumatism [36,264] Amaranthus viridis (Amaranthaceae) Noteyshak Leaves and seeds Used to treat ulcer and inﬂammation [265] Amberboa moschata (Asteraceae) Jam Roots Used to treat ulcers, malignancy, and menstrual disorders [66] edicinal plants of Bangladesh used in the treatment of ulcer, inﬂammatory, and other related diseases. Table 1. Common medicinal plants of Bangladesh used in the treatment of ulcer, inﬂammatory, and other Plants 2021, 10, 1348 17 of 30 Table 1. Cont. 3.35. Zingiber ofﬁcinale Name (Family) Local Name in Bangladesh Commonly Used Medicinal Parts Therapeutic Uses References Angelica sinensis (Apiaceae) Not known Aerial parts Used to treat ulcerative colitis [266] Anogeissus latifolia (Combretacea) Dhai Roots, bark, leaves and fruits Used to treat inﬂammation, ulcer, dysentery, hemorrhoids, and liver diseases [261,267] Azadirachta indica (Meliaceae) Neem Leaves, roots, seeds, and bark Anti-inﬂammatory, anti-pyretic, anti-ulcer, anti-arrhythmic, anti-protozoal, and gastrointestinal disease [268–270] Basella alba Linn (Basellaceae) Puishak Leaves Used to treat ulcers and constipation [259] Butea frondosa (Leguminosae) Palash Flower, roots, gums, stem, bark and seeds Anti-ulcerogenic, anti-hemorrhagic activity, and septic sore throats [36] Carissa congesta (Apocynaceae) Karamcha Roots and leaves Used to treat diabetes and ulcers [157] Cissus quadrangularis (Vitaceae) Harjora or Harbhanga Stems and rhizomes Used to treat stomach trouble and ulcers [135] Colocasia esculenta (Araceae) Mukhikachu Whole plant Used to treat tumor, ulcers, cancer, constipation, and indigestion [157,219,233,271] Commiphora mukul (Burseraceae) Guggul Guggul gum Used to treat ulcers, atherosclerosis, rheumatism, and hypercholesterolemia [228,272] Desmostachya bipinnata (Gramineae) Kush or Durva Roots Used to treat ulcers, cancers, and diarrheal disease [130,273] Dyospyros perigrina (Ebenaceae) Gaab Fruits and seeds Used to treat ulcer, diarrhea, dysentery, and wounds [233] Dyospyros philippensis (Ebenaceae) Boniok Fruits and seeds Used to treat ulcer, diarrhea, dysentery, and wounds [233] Euphorbia hirta (Euphorbiaceae) Bara or Dudhia Whole plant Used to treat ulcers, inﬂammation, and bronchitis [219,233,273] Euphorbia neriifolia (Eurphorbiaceae) Mansaij or Patasij Leaves, ﬂowers, fruits, and seeds Used to treat ulcers, scabies, and schizophrenia [228,274] Excoecaria agallocha (Euphorbiaceae) Gewa Leaves, bark, roots Used to treat microbial infections, cancers, wound and ulcers. [130,275] Ficus religiosa (Urticaceae) Aswatha or Panbot Leaves, stem bark, seeds, and roots Used to treat ulcers, asthma, epilepsy, and inﬂammatory gastric problems [276,277] Table 1. Cont. Plants 2021, 10, 1348 18 of 30 Table 1. Cont. 3.35. Zingiber ofﬁcinale Name (Family) Local Name in Bangladesh Commonly Used Medicinal Parts Therapeutic Uses References Ginkgo biloba (Ginkgoaceae) Adel or Ginkgo Root Applied to cure colitis [278] Heliotropium indicum (Boraginaceae) Hatisur Whole plant Used to treat ulcers, sores, and rheumatism [233,244] Lannea coromandelica (Anacardiaceae) Jiga Bark Anti-eruptions, anti-leprous, anti-ulcer, and ulcerative dyspepsia [158,233] Lawsonia inermis (Lythraceae) Mehedi Leaves Cures wounds and ulcers [228,244] Lens esculenta (Leguminosae) Masur Seeds Used to treat ulcers [233] Lippia nodiﬂora (Verbenaceae) Bhui Okar Leaves and whole plant Used to treat bronchial problem and ulcers [259] Ludwigia adscendens (Onagraceae) Keshardam Whole plant Used to treat ulcers [233] Mikania micrantha (Asteraceae) Toopainna Lata Leaves Used to treat gastric ulcers and discomfort of digestive tract [66] Nerium indicum (Apocynaceae) Karabi Roots, leaves, and whole plant Anti-ulcer, diuretic, and reduce swellings [279] Oxystelma esculentum (Asclepiadaceae) Dudhi or Dudhia Lata Leaves, petiole, stem, roots, and rhizomes Anti-ulcer, diuretic, and anti-bronchitis activity [261,280] Piper betel (Piperaceae) Paan Leaves Used as digestive aid, anti-oxidant, anti-inﬂammatory, and analgesic property [130,281] Plumeria alba (Apocynaceae) Kathgolap Bark, leaves, ﬂower buds, and latex Used to treat syphilitic ulcers [279] Polyalthia longifolia (Annonaceae) Debdaru Whole plant Anti-ulcerogenic, hepatoprotective, anti-inﬂammatory, blood pressure, fever, and moisturizing activity [282,283] Scoparia dulcis (Scrophulariaceae) Michri Dana Leaves Used in the treatment of inﬂammation and nerve problems [244] Sesbania grandiﬂora (Leguminosae) Bokful Leaves Used to treat ulcers [284,285] Smilax ovalifolia ( Smilacaceae) Kumarilata Leaves and stems Used to treat ulcers [66] Vernonia patula (Asteraceae) Kuksim Aerial parts Anti-ulcer, anti-inﬂammatory, and anti-dropsy [219] Xanthium indicum (Asteraceae) Ghagra Stems, roots, fruits, leaves, and whole plant Used to treat ulcers, tumors, and smallpox [219] Zizyphus mauritiana (Rhamnaceae) Ber or Kool Roots and bark Used to treat wounds, fever, ulcers, and diarrhea [233] Table 1. Cont. 19 of 30 Plants 2021, 10, 1348 Figure 2. Modes of action of bioactive compounds of medicinal plants to treat ulcers and inﬂammatory diseases. Figure 2. Modes of action of bioactive compounds of medicinal plants to treat ulcers and inﬂammatory diseases. odes of action of bioactive compounds of medicinal plants to treat ulcers and inﬂammatory diseases. Figure 2. Modes of action of bioactive compounds of medicinal plants to treat ulcers and inﬂammatory diseases. References 1. Bishayee, A.; Sethi, G. Bioactive natural products in cancer prevention and therapy: Progress and 2016, 40–41, 1–3. [CrossRef] 2. Mintah, S.O.; Asafo-Agyei, T.; Archer, M.-A.; Junior, P.A.-A.; Boamah, D.; Kumadoh, D.; Appiah, A.; Ocloo, A.; Boakye, Y.D.; Agyare, C. Medicinal plants for treatment of prevalent diseases. In Pharmacognosy-Medicinal Plants; IntechOpen: London, UK, 2019. 3. Sumner, J. The Natural History of Medicinal Plants; Timber Press: London, UK, 2000. 4. Arif, T.; Bhosale, J.D.; Kumar, N.; Mandal, T.K.; Bendre, R.S.; Lavekar, G.S.; Dabur, R. Natural products—Antifungal agents derived from plants. J. Asian Nat. Prod. Res. 2009, 11, 621–638. [CrossRef] 4. Arif, T.; Bhosale, J.D.; Kumar, N.; Mandal, T.K.; Bendre, R.S.; Lavekar, G.S.; Dabur, R. Na derived from plants. J. Asian Nat. Prod. Res. 2009, 11, 621–638. [CrossRef] 5. Cragg, G.M.; Newman, D.J. Natural products: A continuing source of novel drug leads. Biochim. Biophys. Acta 2013, 1830, 3670–3695. [CrossRef] 6. Imadi, S.R.; Mahmood, I.; Gul, A. Medicinal Plants Against Cancer. In Plant and Human Health, Volume 1: Ethnobotany and Physiology; Ozturk, M., Hakeem, K.R., Eds.; Springer: Cham, Switzerland, 2018; pp. 139–196. 6. Imadi, S.R.; Mahmood, I.; Gul, A. Medicinal Plants Against Cancer. In Plant and Human Health, Volume 1: Ethnobotany and Physiology; Ozturk, M., Hakeem, K.R., Eds.; Springer: Cham, Switzerland, 2018; pp. 139–196. 7 Laila U ; Akram M ; Shariati M A ; Hashmi A M ; Akhtar N ; Tahir I M ; Ghauri A O ; Munir N ; Riaz M ; Akhter N Role of 7. Laila, U.; Akram, M.; Shariati, M.A.; Hashmi, A.M.; Akhtar, N.; Tahir, I.M.; Ghauri, A.O.; Munir, N.; Ri medicinal plants in HIV/AIDS therapy. Clin. Exp. Pharm. Physiol. 2019, 46, 1063–1073. [CrossRef] 8 Siddi i M H Al i S A Al Wh ibi M H H i Z Ali H M El Z id M E A i i i p py p y 8. Siddiqui, M.H.; Alamri, S.A.; Al-Whaibi, M.H.; Hussain, Z.; Ali, H.M.; El-Zaidy, M.E. A mini-review of anti-hepatitis B virus activity of medicinal plants. Biotechnol. Biotechnol. Equip. 2017, 31, 9–15. [CrossRef] 8. Siddiqui, M.H.; Alamri, S.A.; Al-Whaibi, M.H.; Hussain, Z.; Ali, H.M.; El-Zaidy, M.E. A mini-rev activity of medicinal plants. Biotechnol. Biotechnol. Equip. 2017, 31, 9–15. [CrossRef] y p q p 9. Dias, D.A.; Urban, S.; Roessner, U. A historical overview of natural products in drug discovery. Metabolites 2012, 2, 303–336. [CrossRef] 10. 4. Conclusions and Future Directions All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by Rural Development Administration (RDA), grant number PJ015726, Republic of Korea. Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Data Availability Statement: Not applicable. Acknowledgments: This work was carried out with the support of the Cooperative Research Pro- gram for Agriculture Science and Technology Development (grant number PJ015726), RDA, Republic of Korea. The ﬁgures were created with Biorender.com (accessed on 21 May 2021). Conﬂicts of Interest: The authors declare no conﬂict of interest. 4. Conclusions and Future Directions There is a belief that the solutions to all health problems can be found in nature. Currently, humans are victimized by various life-threatening diseases, and in the form of Plants 2021, 10, 1348 20 of 30 20 of 30 medicinal plant, nature has proven its potency in preventing and curing these diseases. Between 70% and 80% of people throughout the world are solely dependent on primitive or herbal medicines [286]. The worldwide demand for herbal medicine is great and is growing. However, the trials and experiments in this sector are very limited until recently, and this reﬂects our lack of knowledge about nature. All around the world, there are huge numbers of medicinal plants but the medicinal value of less than one-third of them have been discovered and identiﬁed. Still now, more comprehensive, and reliable scientiﬁc studies are needed to evaluate and ensure the safety and efﬁciency of medicinal plants, as well as their potential metabolites in ulcers and inﬂammatory disease treatment. The quality of the targeted active compounds should be tested repeatedly or batch-to-batch for ensuring reproducibility as well as uniformity of the active components. The implementation of randomized clinical trials overcoming exiting challenges like study design, selection of controls, and active ingredients standardization is urgently needed. International and local regulatory bodies should come forward to facilitate and monitor the clinical trials for achieving a breakthrough outcome. Author Contributions: Conceptualization, S.R.A., M.F.R. and M.M.H.C.; investigation, S.R.A., M.F.R., A.R., R.C. and A.B.; writing: S.R.A., M.F.R., A.R., R.C., K.K. and M.M.H.C.; visualization, R.C. and M.F.R.; project administration M.M.H.C. and K.-H.B., supervision, M.M.H.C. and K.-H.B., reviewing, M.M.H.C., K.-H.B. and K.K., editing: K.-H.B., M.M.H.C. and K.K.; funding acquisition, K.-H.B. All authors have read and agreed to the published version of the manuscript. Author Contributions: Conceptualization, S.R.A., M.F.R. and M.M.H.C.; investigation, S.R.A., M.F.R., A.R., R.C. and A.B.; writing: S.R.A., M.F.R., A.R., R.C., K.K. and M.M.H.C.; visualization, R.C. and M.F.R.; project administration M.M.H.C. and K.-H.B., supervision, M.M.H.C. and K.-H.B., reviewing, M.M.H.C., K.-H.B. and K.K., editing: K.-H.B., M.M.H.C. and K.K.; funding acquisition, K.-H.B. All authors have read and agreed to the published version of the manuscript. Author Contributions: Conceptualization, S.R.A., M.F.R. and M.M.H.C.; investigation, S.R.A., M.F.R., A.R., R.C. and A.B.; writing: S.R.A., M.F.R., A.R., R.C., K.K. and M.M.H.C.; visualization, R.C. and M.F.R.; project administration M.M.H.C. and K.-H.B., supervision, M.M.H.C. and K.-H.B., reviewing, M.M.H.C., K.-H.B. and K.K., editing: K.-H.B., M.M.H.C. and K.K.; funding acquisition, K.-H.B. y herjee, P.K. Quality Control of Herbal Drugs: An Approach to Evaluation of Botanicals; Business Horizons: New De References Ethnobotanical informations on the species of selected areas in Nilgiri Biosphere Reserve, the Western Ghats, India. J. Res. Biol. 2015, 5, 43–57. 17. Curtis, P.; Gaylord, S. Safety Issues in the Interaction of Conventional, Complementary, and Alternative Health Care. Complement. Health Pr. Rev. 2005, 10, 3–31. [CrossRef] 18. Kalra, P.; Sharma, S.; Kumar, S. Antiulcer effect of the methanolic extract of Tamarindus indica seeds in different experimental models. J. Pharm. 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https://openalex.org/W4240927038	https://zenodo.org/record/4999644/files/eqr0551%20kevin%20.pdf	English	null	Emergency Remote Teaching during COVID-19: A Comparison of Student Perceptions	null	2,021	cc-by	4,533	Education Quarterly Reviews Fuchs, Kevin. (2021), Emergency Remote Teaching during COVID-19: A Comparison of Student Perceptions. In: Education Quarterly Reviews, Vol.4, No.2, 593-599. ISSN 2621-5799 DOI: 10.31014/aior.1993.04.02.303 The online version of this article can be found at: https://www.asianinstituteofresearch.org/ Published by: The Asian Institute of Research The Education Quarterly Reviews is an Open Access publication. It may be read, copied, and distributed free of charge according to the conditions of the Creative Commons Attribution 4.0 International license. The Asian Institute of Research Education Quarterly Reviews is a peer-reviewed International Journal. The journal covers scholarly articles in the fields of education, linguistics, literature, educational theory, research, and methodologies, curriculum, elementary and secondary education, higher education, foreign language d i hi d l i h d i d i f i l d h fi ld f d l d Education Quarterly Reviews Fuchs, Kevin. (2021), Emergency Remote Teaching during COVID-19: A Comparison of Student Perceptions. In: Education Quarterly Reviews, Vol.4, No.2, 593-599. ISSN 2621-5799 DOI: 10.31014/aior.1993.04.02.303 The online version of this article can be found at: https://www.asianinstituteofresearch.org/ Published by: The Asian Institute of Research The Education Quarterly Reviews is an Open Access publication. It may be read, copied, and distributed free of charge according to the conditions of the Creative Commons Attribution 4.0 International license. The Asian Institute of Research Education Quarterly Reviews is a peer-reviewed International Journal. The journal covers scholarly articles in the fields of education, linguistics, literature, educational theory, research, and methodologies, curriculum, elementary and secondary education, higher education, foreign language d i hi d l i h d i d i f i l d h fi ld f d l d Education Quarterly Reviews Education Quarterly Reviews Fuchs, Kevin. (2021), Emergency Remote Teaching during COVID-19: A Comparison of Student Perceptions. In: Education Quarterly Reviews, Vol.4, No.2, 593-599. Fuchs, Kevin. (2021), Emergency Remote Teaching during COVID-19: A Comparison of Student Perceptions. In: Education Quarterly Reviews, Vol.4, No.2, 593-599. ISSN 2621-5799 DOI: 10.31014/aior.1993.04.02.303 Abstract The pandemic has shaken up the higher education landscape around the world, with responses from institutions falling into three categories: retaining in-class teaching with social distancing, adopting hybrid models (blended learning, limiting the number of students on campus), or transitioning to fully online teaching. However, there is a significant difference between emergency remote teaching and a genuine shift to online/hybrid learning, with the key distinguishing term being “emergency.” In response to the global pandemic, the higher education community is now working on the continuous development of action plans in a quest to identify means to manage the crisis more efficiently. The purpose of this paper is to analyze the perceived performance of ERT from the perspective of undergraduate students. For that purpose, samples (n=332) were taken in two different geographical settings, i.e., Thailand and Sweden. Moreover, it is the objective to compare both samples and identify similarities and inadequacies which help stakeholders to manage ERT more efficiently in the future. Keywords: Emergency Remote Teaching, Higher Education, Virtual Classroom, ERT, Thailand, Swede Kevin Fuchs1 ulty of Hospitality and Tourism, Prince of Songkla University, Thailand. Email: kevin.f@phuket.psu.ac.th 1 Faculty of Hospitality and Tourism, Prince of Songkla University, Thailand. Email: kevin.f@phuket.psu.ac.th DOI: 10.31014/aior.1993.04.02.303 DOI: 10.31014/aior.1993.04.02.303 The online version of this article can be found at: https://www.asianinstituteofresearch.org/ Published by: The Asian Institute of Research The Education Quarterly Reviews is an Open Access publication. It may be read, copied, and distributed free of charge according to the conditions of the Creative Commons Attribution 4.0 International license. The Asian Institute of Research Education Quarterly Reviews is a peer-reviewed International Journal. The journal covers scholarly articles in the fields of education, linguistics, literature, educational theory, research, and methodologies, curriculum, elementary and secondary education, higher education, foreign language education, teaching and learning, teacher education, education of special groups, and other fields of study related to education. As the journal is Open Access, it ensures high visibility and the increase of citations for all research articles published. The Education Quarterly Reviews aims to facilitate scholarly work on recent theoretical and practical aspects of education. The Asian Institute of Research Education Quarterly Reviews Vol.4, No.2, 2021: 593-599 ISSN 2621-5799 Copyright © The Author(s). All Rights Reserved DOI: 10.31014/aior.1993.04.02.303 The Asian Institute of Research Education Quarterly Reviews Vol.4, No.2, 2021: 593-599 ISSN 2621-5799 Copyright © The Author(s). All Rights Reserved DOI: 10.31014/aior.1993.04.02.303 Emergency Remote Teaching during COVID-19: A Comparison of Student Perceptions Kevin Fuchs1 1. Introduction All research undertaken during the pandemic represents a transient response from instructors and institutions. The sustainability of university education is one of the concerns expressed during the global COVID-19 epidemic. According to Muftahu (2020), the pandemic generated extremely difficult economic conditions that jeopardize university education's long-term viability. He further said that uncertainty and insufficient funding is likely to lead to a lack of support for university courses and potentially jeopardize the long-term viability of higher education. The global COVID-19 pandemic has also brought attention to the need for higher education institutions to develop more adaptable teaching methods (Schlesselman, 2020). The pandemic led all universities around the world to cancel classes, affecting the vast majority of the student population. Some institutions were able to offer remote learning or online education, but the majority of institutions were unprepared to respond quickly and mitigate the effects of COVID-19's absolute lockdown (Hodges et al., 2020; Schlesselman, 2020; Muftahu, 2020). It is the purpose of this paper to share the latest work-in-progress research concerning the perceived performance by undergraduate students concerning emergency remote teaching in two different geographical settings, i.e., Thailand and Sweden, and compare their perceptions. 593 Vol.4, No.2, 2021 Education Quarterly Reviews Vol.4, No.2, 2021 Asian Institute of Research 2.1. Secondary Data 2.1. Secondary Data The sample representing the data collected in Thailand was taken from an earlier study (Fuchs & Karrila, 2021). Henceforth, the data will be referred to as “secondary data” for this paper. The design of the questionnaire, sampling procedure, and data analysis were adopted from Fuchs and Karrila (2021). However, the data was further modified to allow for easier comparison with the empirical data collected in Sweden. These modifications included the removal of responses from students in their fourth and fifth years. The included sample accounts for 174 responses (n1=174) from Thailand (Table 1). 2. Methodology 2.1. Secondary Data 2.2. Empirical Data 2.2. Empirical Data The sample representing the data collected in Sweden was collected from undergraduate students in a full-time business degree program in the second quarter of 2021 during a nationwide lockdown as the result of COVID- 19. The collected data will be referred to as “empirical data” for this paper. The design of the questionnaire, sampling procedure, and data analysis were adopted from Fuchs and Karrila (2021) to allow for easier comparison with the secondary data collected in Thailand. The included sample accounts for 158 responses (n2=158) from Sweden, as shown in Table 1 below. 3.1. Student perceptions about ERT by geography 3.1. Student perceptions about ERT by geography The results from both surveys can be seen in Table 2, wherein the Thai sample indicates mean ratings ranging from 3.47 (No. 1) to 4.07 (No. 4). In comparison, the results from the Swedish sample range from 2.87 (No. 1) to 4.23 No. 3). Similarly, the distribution of responses from the sample taken in Thailand is narrower, wherein the gap increases with the Swedish sample. The same observation is confirmed by the higher standard deviation (SD) noted in the empirical data compared to the secondary data from Thailand. Furthermore, independent T- tests were performed for each attribute on the assumption that « Mean2 ≠ Mean3 », wherein a violation of the assumption was observed for four attributes (No. 1; No. 4; No. 6; No. 10) between both samples. Based on the ten attributes that were surveyed when students were asked to evaluate their perception of the performance of emergency remote teaching, six items ranked higher in Thailand, wherein the remaining four items ranked higher in Sweden. Table 2: Perceived performance ratings given by students (summarized from surveys) No. Attribute Description1 Mean2 SD2 Mean3 SD3 t-value p-value 1 The teacher begins the class with a review of the previous class 3.47 1.01 2.87 0.91 -5.714 < .001a 2 The teacher presents the material in an interesting and engaging way 3.55 1.06 3.99 1.19 3.573 < .001 3 The teacher presents the material in an organized and coherent way 3.71 1.03 4.23 1.12 4.407 < .001 4 The teacher is knowledgeable about the content of the course 4.07 1.02 3.77 1.23 -2.452 0.015a 5 The teacher is friendly and patient with the students 3.97 0.99 3.89 1.08 -0.643 0.520 6 The course material is well and professionally prepared 3.75 1.00 3.59 1.20 -1.363 0.174a 7 The course material is easy to access in the LMS 3.84 1.01 3.82 1.11 -0.140 0.888 8 Students are engaged to actively participate in the discussion 3.76 1.00 3.89 1.06 1.127 0.261 9 I am learning something which I consider valuable 3.68 1.05 3.87 1.16 1.557 0.120 10 I am finding the course challenging and stimulating 3.52 1.22 3.44 1.38 -0.522 0.602a 1 Ratings obtained from a Likert-type five points scale ranging from lowest rating (1) to highest rating (5), i.e. 2.3 Profile of the participants The aggregate of both samples yielded 332 eligible responses (n1= 174; n2= 158) that were included for further analysis in this paper. The corresponding socio-demographic profile of the participants can be found in Table 1. The aggregate of both samples yielded 332 eligible responses (n1= 174; n2= 158) that were included for further analysis in this paper. The corresponding socio-demographic profile of the participants can be found in Table 1. Table 1: Socio-demographic profile of respondents (summarized from surveys) Table 1: Socio-demographic profile of respondents (summarized from surveys) Characteristics Thailand (n1=174) Sweden (n2=158) Absolute Percent Absolute Percent Gender Female 122 70% 76 48% Male 52 30% 82 52% Year of study First-year 49 28% 83 53% Second-year 83 48% 49 31% Third-year 42 24% 26 16% Age range 20 years or below 126 72% 90 57% 21 years or above 48 28% 68 43% Nationality Local 149 86% 93 59% Foreign 25 14% 65 41% Preferred mode of study Traditional Classroom 138 79% 69 44% Virtual Classroom 36 21% 89 56% It can be stated that in the Thai sample, seven out of ten students were female, while the sample from Sweden contained an almost balanced distribution of gender (male=52%; female=48%). Additionally, it is noteworthy that the Thai students were younger than their peers in Sweden. In Thailand, 72% were 20 years or younger, while that number was at 57% for the Swedish students. Another notable characteristic is the preferred mode of study, wherein 79% of the students in Thailand prefer an on-site study arrangement with the traditional classroom, while 56% of students in Sweden prefer emergency remote teaching that was conducted fully virtually. Lastly, the ratio of local students to foreign students is higher in Thailand than in Sweden. 14% of the 594 Education Quarterly Reviews Vol.4, No.2, 2021 Asian Institute of Research Thai sample were foreign degree students, whereas three times as many (41%) from the Swedish sample were foreign. 3.1. Student perceptions about ERT by geography Not At All Satisfied (1), Not Very Satisfied (2), Somewhat Satisfied (3), Very Satisfied (4), and Extremely Satisfied (5) 2 Mean rating of responses (mean) and standard deviation (SD) calculated from the sample taken in Thailand 3 Mean rating of responses (mean) and standard deviation (SD) calculated from the sample taken in Sweden a Levene’s test is significant (p < .05), suggesting a violation of the assumption of equal variances The largest disagreement between both samples was noted with attribute No. 3, which asked the students to rate the performance about “the teacher presents the material in an organized and coherent way.” Students in Sweden gave a relatively high score of 4.23 and the students in Thailand rated this attribute 0.55 points lower, at 3.71. Another noteworthy result is the low mean rating for attribute No. 1, which asked the students if “the teacher The largest disagreement between both samples was noted with attribute No. 3, which asked the students to rate the performance about “the teacher presents the material in an organized and coherent way.” Students in Sweden gave a relatively high score of 4.23 and the students in Thailand rated this attribute 0.55 points lower, at 3.71. Another noteworthy result is the low mean rating for attribute No. 1, which asked the students if “the teacher 595 Education Quarterly Reviews Vol.4, No.2, 2021 Asian Institute of Research begins the class with a review of the previous class.” The responses from the Swedish sample indicate a rating of 2.87 compared to a rating of 3.47 from the Thai sample. Amongst both samples, there is an agreement that this attribute was perceived with the lowest performance in both countries (Table 2). begins the class with a review of the previous class.” The responses from the Swedish sample indicate a rating of 2.87 compared to a rating of 3.47 from the Thai sample. Amongst both samples, there is an agreement that this attribute was perceived with the lowest performance in both countries (Table 2). 3.2. Student perceptions about ERT by preferred mode of study Figure 1: Comparison of responses by preferred mode of study (summarized from surveys) 3.24 3.59 3.82 3.91 3.91 3.60 3.80 3.73 3.69 3.43 3.10 4.04 4.18 3.95 3.97 3.80 3.88 3.97 3.90 3.57 2.75 2.85 2.95 3.05 3.15 3.25 3.35 3.45 3.55 3.65 3.75 3.85 3.95 4.05 4.15 4.25 4.35 4.45 1 2 3 4 5 6 7 8 9 10 Traditional Classroom (Total; n=207) Virtual Classroom (Total; n=125) Figure 1: Comparison of responses by preferred mode of study (summarized from surveys) It can be conclusively stated that both groups of students, regardless of their preferred mode of study, were generally satisfied with the performance of their ERT classes during the COVID-19 pandemic. A similar hypothesis was observed through a related case study, which attested “that students achieved better results under emergency remote teaching” (Iglesias-Pradas, Hernández-García, Chaparro-Peláez, & Prieto, 2021). Iglesias- Pradas et al. (2021) further claim that “the choice of delivery mode did not seem to affect students’ academic performance,” which is something this paper did not investigate. However, it could serve as a possible explanation for the relatively high approval rating for the virtual classroom by the sampled undergraduate It can be conclusively stated that both groups of students, regardless of their preferred mode of study, were generally satisfied with the performance of their ERT classes during the COVID-19 pandemic. A similar hypothesis was observed through a related case study, which attested “that students achieved better results under emergency remote teaching” (Iglesias-Pradas, Hernández-García, Chaparro-Peláez, & Prieto, 2021). Iglesias- Pradas et al. (2021) further claim that “the choice of delivery mode did not seem to affect students’ academic performance,” which is something this paper did not investigate. However, it could serve as a possible explanation for the relatively high approval rating for the virtual classroom by the sampled undergraduate It can be conclusively stated that both groups of students, regardless of their preferred mode of study, were generally satisfied with the performance of their ERT classes during the COVID-19 pandemic. A similar hypothesis was observed through a related case study, which attested “that students achieved better results under emergency remote teaching” (Iglesias-Pradas, Hernández-García, Chaparro-Peláez, & Prieto, 2021). Iglesias- Pradas et al. (2021) further claim that “the choice of delivery mode did not seem to affect students’ academic performance,” which is something this paper did not investigate. 3.2. Student perceptions about ERT by preferred mode of study 3.2. Student perceptions about ERT by preferred mode of study Next, the following graphics visualize the distribution of total responses (n=332) based on the preferred mode of study (Figure 1), as well as the year of study (Figure 2). The same analysis was conducted for gender and age range. However, the former did not reveal any noteworthy results, wherein the latter is correlated to the characteristic year of study and displays similar results. Based on the preferred mode of study, it can be noted that students who prefer the virtual classroom (Figure 1; green line) gave a higher performance rating compared to students that prefer the traditional classroom (Figure 1; blue line). In particular, the attributes “the teacher presents the material interestingly and engagingly” (No. 2) and “the teacher presents the material in an organized and coherent way” (No. 3) received the largest deviations in responses. The students that preferred the virtual classroom expressed that they were very satisfied (4.04 and 4.18 respectively) with how the teacher presented the course material and that the material was presented interestingly and engagingly. Irrespective of their preferred mode of study, both groups of students had a relative agreement with items No. 4, No. 5, and No. 7, where the variance was less or equal to 0.10 (Figure 1). Moreover, it should be mentioned that none of the ten attributes received a mean rating below the neutral threshold, i.e., 3.00, for either group of students. Both groups of students gave the lowest performance rating for item No. 1, which asked about a review of the previous class at the beginning of each lecture. It could either be that the lecturer did not place much emphasis on this aspect, or that this action was merely neglected in both locations by the course instructor. 3.3. Student perceptions about ERT by year of study Also, there is an agreement amongst multiple attributes when grouping the responses by year of study, as shown in Figure 2. Notably, the perceived performance amongst first-year students ranks the lowest (Figure 2; red line) and increases with the year of study. Based on this observation, it can be hypothesized that students have higher satisfaction with ERT classes as they progress to the next year in their studies. A similar occurrence was already mentioned in a case study that reported first-year students’ dissatisfaction with emergency remote teaching due to a lack of socialization and peer interaction (Fuchs, 2021). An Albanian case study (Xhelili et al., 2021) concluded with a noteworthy finding that suggested, “online learning cannot replace the classroom. […] students’ are not familiar enough with technology-based education”. Based on this finding, paired with the results shown by the year of study, it could be theorized that the students become more proficient with digital technologies as they progress in their studies and, therefore, their satisfaction increases with online-based education as seen in Figure 2. Students in the third year of study rated five out of ten items higher than 4.00, which demonstrates their high satisfaction. In opposition to this sentiment, students in the first year of their study did not rate any attribute above 4.00, while 3.88 (No. 5) was the highest-rated item corresponding to the statement “the teacher is friendly and patient with the students.” Another noteworthy observation is that students in the first, second, and third year of study agreed that “the teacher begins the class with a review of the previous class” (No. 1) was perceived with their lowest satisfaction rating amongst all ten attributes. Moreover, all three groups agreed that the teacher presented the class material in an organized and coherent way (No. 3), which is reflected in their respective satisfaction ratings (Year 1 = 3.87; Year 2 = 4.00; Year 3 = 4.03). 3.2. Student perceptions about ERT by preferred mode of study However, it could serve as a possible explanation for the relatively high approval rating for the virtual classroom by the sampled undergraduate 596 Education Quarterly Reviews Asian Institute of Research Vol.4, No.2, 2021 students in this study. The reasonably high satisfaction amongst both groups of students – those that prefer the virtual classroom as well as those that prefer the traditional classroom –validates similar case studies on emergency remote teaching (Agarwal & Kaushik, 2020) that attest to the high satisfaction of students with the alternative mode of studying. students in this study. The reasonably high satisfaction amongst both groups of students – those that prefer the virtual classroom as well as those that prefer the traditional classroom –validates similar case studies on emergency remote teaching (Agarwal & Kaushik, 2020) that attest to the high satisfaction of students with the alternative mode of studying. 4. Conclusion and Future Works The outcomes that were revealed as the result of this study were threefold: Firstly, students in Thailand and Sweden are generally satisfied with the perceived performance of their respective emergency remote teaching. Furthermore, students that prefer the virtual classroom during ERT gave higher satisfaction ratings compared to students that prefer a traditional classroom arrangement before COVID-19. Finally, first-year students appear to be less satisfied with emergency remote teaching than their older peers. The study revealed that as the year of study progresses, the perceived satisfaction with emergency remote teaching increases. As noted by Barbour et al. (2020) in their technical review about emergency remote teaching, “the threat of COVID-19 has presented some unique challenges for institutions of higher education”. Therefore, it is not too farfetched to assume that educators and students wish to move beyond the current impacts of this global pandemic. With little doubt, this is a stressful circumstance and, when it is over, universities will be able to determine how well they were able to apply ERT to maintain instructional continuity. Plenty of research has been conducted over the last months since the initial outbreak of COVID-19 in January 2019. Even though emergency remote teaching does not occur regularly, it is safe to assume that some regions in the world will be confronted with it again at some point in time. This research has raised more questions than it has provided answers. Thus, more research into this issue of student perceptions related to emergency remote teaching should be carried out with the implication to increase the preparedness and quality in the near future. 3.3. Student perceptions about ERT by year of study Figure 2: Comparison of responses by year of study (summarized from surveys) 3.03 3.71 3.87 3.84 3.88 3.51 3.76 3.73 3.70 3.31 3.26 3.85 4.00 3.89 3.87 3.74 3.81 3.78 3.85 3.56 3.34 3.69 4.03 4.16 4.15 3.87 4.01 4.06 3.75 3.66 2.75 2.85 2.95 3.05 3.15 3.25 3.35 3.45 3.55 3.65 3.75 3.85 3.95 4.05 4.15 4.25 4.35 4.45 1 2 3 4 5 6 7 8 9 10 Year 1 (Total; n=132) Year 2 (Total; n=132) Year 3 (Total; n=68) Figure 2: Comparison of responses by year of study (summarized from surveys) 3.03 3.71 3.87 3.84 3.88 3.51 3.76 3.73 3.70 3.31 3.26 3.85 4.00 3.89 3.87 3.74 3.81 3.78 3.85 3.56 3.34 3.69 4.03 4.16 4.15 3.87 4.01 4.06 3.75 3.66 2.75 2.85 2.95 3.05 3.15 3.25 3.35 3.45 3.55 3.65 3.75 3.85 3.95 4.05 4.15 4.25 4.35 4.45 1 2 3 4 5 6 7 8 9 10 Year 1 (Total; n=132) Year 2 (Total; n=132) Year 3 (Total; n=68) Figure 2: Comparison of responses by year of study (summarized from surveys) Van Nuland, Hall, and Langley (2020) discovered that “universities are looking to e-learning tools to facilitate what used to be face-to-face laboratory experiences in an online environment” more rapidly due to the ongoing Van Nuland, Hall, and Langley (2020) discovered that “universities are looking to e-learning tools to facilitate what used to be face-to-face laboratory experiences in an online environment” more rapidly due to the ongoing 597 Education Quarterly Reviews Asian Institute of Research Vol.4, No.2, 2021 pandemic. This observation could offer a possible explanation concerning the lower satisfaction ratings amongst students in their earlier studies. Most of the practice-based classes and laboratory exercises are usually held in the first and second year of study, wherein the third year is more theory-based with the development of applied concepts. Notably, none of the student groups gave a very high satisfaction rating for finding the course challenging and stimulating (Year 1 = 3.31; Year 2 = 3.56; Year 3 = 3.66). Acknowledgments The author would like to thank the participants that contributed to the research by answering the survey. Moreover, the author would like to declare no potential conflicts of interest concerning the research, authorship, or publication of this article. The data that support the findings of this study are available from the corresponding author upon reasonable request. Schlesselman, L. S. (2020). Perspective from a Teaching and Learning Center during Emergency Remote Teaching. American Journal of Pharmaceutical Education, 84(8). https://doi.org/10.5688/ajpe8142 Van Nuland, S. E., Hall, E., & Langley, N. R. (2020). STEM crisis teaching: Curriculum design with e‐ learning tools. FASEB BioAdvances, 2(11), 631-637. https://doi.org/10.1096/fba.2020-00049 Xhelili, P., Ibrahimi, E., Rruci, E., & Sheme, K. (2021). Adaptation and perception of online learning during COVID-19 pandemic by Albanian university students. International Journal on Studies in Education, 3(2), 103-111. https://doi.org/10.46328/ijonse.49 Xhelili, P., Ibrahimi, E., Rruci, E., & Sheme, K. (2021). Adaptation and perception of online learning during COVID-19 pandemic by Albanian university students. International Journal on Studies in Education, 3(2), 103-111. https://doi.org/10.46328/ijonse.49 g f , ( ) p g jp Van Nuland, S. E., Hall, E., & Langley, N. R. (2020). STEM crisis teaching: Curriculum design with e‐ learning tools. FASEB BioAdvances, 2(11), 631-637. https://doi.org/10.1096/fba.2020-00049 Schlesselman, L. S. (2020). Perspective from a Teaching and Learning Center during Emergency Remot Teaching. American Journal of Pharmaceutical Education, 84(8). https://doi.org/10.5688/ajpe8142 , ( ) p g g g g y Teaching. American Journal of Pharmaceutical Education, 84(8). https://doi.org/10.5688/ajpe8142 Van Nuland, S. E., Hall, E., & Langley, N. R. (2020). STEM crisis teaching: Curriculum design with e‐ learning tools. FASEB BioAdvances, 2(11), 631-637. https://doi.org/10.1096/fba.2020-00049 Xhelili, P., Ibrahimi, E., Rruci, E., & Sheme, K. (2021). Adaptation and perception of online learning during COVID 19 pandemic by Albanian university students International Journal on Studies in Education 3(2) References Agarwal, S., & Kaushik, J. S. (2020). Student’s perception of online learning during COVID pandemic. The Indian Journal of Pediatrics, 87, 554-554. https://doi.org/10.1007/s12098-020-03327-7 Agarwal, S., & Kaushik, J. S. (2020). Student’s perception of online learning during COVID pandemic. The Indian Journal of Pediatrics, 87, 554-554. https://doi.org/10.1007/s12098-020-03327-7 Barbour, M. K., LaBonte, R., Hodges, C., Moore, S., Lockee, B., Trust, T. & Kelly, K. (2020). Understanding pandemic pedagogy: Differences between emergency remote, remote, and online teaching. State of the Nation: K-12 e-Learning in Canada. https://doi.org/10.13140/RG.2.2.31848.70401 Á Iglesias-Pradas, S., Hernández-García, Á., Chaparro-Peláez, J., & Prieto, J. L. (2021). Emergency remote teaching and students’ academic performance in higher education during the COVID-19 pandemic: A case study. Computers in Human Behavior, 119, 106713. https://doi.org/10.1016/j.chb.2021.106713 Fuchs, K. (2021). Advances in Tourism Education: A Qualitative Inquiry about Emergency Remote Teaching in Higher Education. Journal of Environmental Management and Tourism, 12(2), 538-543. https://doi.org/10.14505//jemt.v12.2(50).23 Fuchs, K., & Karrila, S. (2021). The Perceived Satisfaction with Emergency Remote Teaching (ERT) amidst COVID-19: An Exploratory Case Study in Higher Education. The Education and Science Journal, 23(5), 116-130. https://doi.org/10.17853/1994-5639-2021-5-116-130 Hodges, C., Moore, S., Lockee, B., Trust, T., & Bond, A. (2020). The difference between emergency remote teaching and online learning. Educause Review, 27, 1-12. Muftahu, M. (2020). Higher education and covid-19 pandemic: Matters arising and the challenges of sustaining academic programs in developing African universities. International Journal of Educational Research Review, 5(4), 417-423. https://doi.org/10.24331/ijere.776470 598 Education Quarterly Reviews Asian Institute of Research Vol.4, No.2, 2021 599
https://openalex.org/W4392840541	https://bsj.uobaghdad.edu.iq/index.php/BSJ/article/download/816/747	Arabic	null	Evaluation of the one electron expectation values for different wave function of Be atom	Mağallaẗ baġdād li-l-ʿulūm	2,021	cc-by	2,365	مجلة أم سلمة للعلوم مجلة أم سلمة للعلوم مجلد4 ( 3 ) 7002 مجلد4 ( 3 ) 7002 حساب القيمة المتوقعة لتواجد اال لكترون المفرد للدوال الموحية المختلفة لذرة البيريليوم Be بان حسن عادل األسعد * خليل هادي البياتي* صالح عبد هللا حسون* تاريخ قبول النشر8/4/ 7002 الخال ة حساب القيمة المتوقعة لتواجد اال لكترون المفرد للدوال الموحية المختلفة لذرة البيريليوم Be بان حسن عادل األسعد * خليل هادي البياتي* صالح عبد هللا حسون* تاريخ قبول النشر8/4/ 7002 الخال ة خليل هادي البياتي* ا ال: ان الهدف من البحث هو حساب ت واجد االلكترون حول النواة من خالل دراسة دالة توزيع الكثافة القطرية لجسيم واحدD(r1) لذرة البيريليوم Beوبأستخدام مجموعة من الدوال الموجية لهارتري- فوك (1960,1974,1993) وقد تمت الدراسة باستخدام طريقة التجز ئة لذرة البيريليوم في الحالة1s22s2 ،حيث تم تحليل ذر ة البيريليومBe الى ستة ازواج من الدوال الموجية زوج في الغالفK وزوج في الغالف L والبقيه في األغلفة الوسطية KL والنتائج المستحصلة تم حسابها عدديا بأستخدام برامج حاسوبيه بأستخدام برنامج الماثكاد(Mathcad) الدالة الموجية وجي ان نظرية هارتري فوك تمتاز باستخدا مها الدوال الموجية ذات التماثل العكسي والتي يمكن كتابتها بشكل محددة سليترحيث يمكن كتابة محدد سليترSlatre determinant لألنظمة الذرية بالمعادلة االتية[1] :- حيث انSnl(r) يمثل اوربيتال سليترSlater type- orbitals ،اماξ تمثل دليل المدارorbital exponent . ا (1) … ) N ( ).... 2 ( ) 1 ( ) ! N ( ) N ,.... 2,1 ( N 2 1 2 / 1      النظرية ان احتمالية تواجد االلكترونات في كل غالف الكتروني يعرف بدالة توزيع الكثافة القطرية لجسيم واحد وبصورة عامة يعّرف بالمعادلة االتية[1] :- (7) … حيث ان          0 2 0 1 2 1 1 2 1 1 ) r( r 4 d ) r( r ) r( D N 3 2 1 N 2 1 N 2 1 1 dx .... dX dX d ) X .... X , X ( ) X .... X , X ( N ) r (        النظرية ان احتمالية تواجد االلكترونات في كل غالف الكتروني يعرف بدالة توزيع الكثافة القطرية لجسيم واحد وبصورة عامة يعّرف بالمعادلة االتية[1] :-   2 النظرية ان احتمالية تواجد االلكترونات في كل غالف الكتروني يعرف بدالة توزيع الكثافة القطرية لجسيم واحد وبصورة عامة يعّرف بالمعادلة االتية[1] :- (7) … حيث ان          0 2 0 1 2 1 1 2 1 1 ) r( r 4 d ) r( r ) r( D N 3 2 1 N 2 1 N 2 1 1 dx .... dX dX d ) X .... X , X ( ) X .... الدالة الموجية القيمة المتوقعة للغالف KαLα= KβLα 1993[4] 1974[3] 1960[2] n 14.40451 14.40597 14.55654 -2 2.10220 2.10216 2.12579 -1 1 1 1 0 1.53220 1.53234 1.52864 1 4.32955 4.33237 4.32159 2 من الجداول السابقة تم حساب معدل القيمة المتوقعة :من المعادلة االتية- (11) ...               1993 n 1 1974 n 1 1960 n 1 Total n 1 r r r 3 1 r   n 1r القيمة المتوقعة للغالف KαLα= KβLα 1993[4] 1974[3] 1960[2] n 14.40451 14.40597 14.55654 -2 2.10220 2.10216 2.12579 -1 1 1 1 0 1.53220 1.53234 1.52864 1 4.32955 4.33237 4.32159 2   n 1r ( جدول1) القيمة المتوقعة لذرة البيريليوم(Be) للغالف KαKβ لعدد من الدوال الموجية المنشورة في السنوات 1993,1974,1960 . ا ( جدول1) القيمة المتوقعة لذرة البيريليوم(Be) للغالف KαKβ لعدد من الدوال الموجية المنشورة في السنوات 1993,1974,1960 . القيمة المتوقعة للغالفKαKβ 1993[4] 1974[3] 1960[2] n 27.75338 27.75583 27.75345 -2 3.68188 3.68183 3.68188 -1 1 1 1 0 0.41499 0.41499 0.41499 1 0.23295 0.23295 0.16532 2   n 1r 1993,1974,1960 . القيمة المتوقعة للغالفKαKβ 1993[4] 1974[3] 1960[2] n 27.75338 27.75583 27.75345 -2 3.68188 3.68183 3.68188 -1 1 1 1 0 0.41499 0.41499 0.41499 1 0.23295 0.23295 0.16532 2 ( جدول2) القيمة المتوقعة لذرة البيريليوم(Be) للغالف LαLβ ل عدد من الدوال الموجية المنشورة في السنوات 1993,1974,1960 . القيمة المتوقعة للغالف LαLβ 1993[4] 1974[3] 1960[2] n 1.05564 1.05611 1.35915 -2 0.52252 0.52248 0.56969 -1 1 1 1 0 2.64852 2.64865 2.64136 1 8.41665 8.42051 8.40033 2 ( جدول3) ال قيمة المتوقعة لذرة البيريليوم(Be) للغالف KαLα= KβLα لعدد من الدوال الموجية المنشورة في السنوات1993,1974,1960 . 1.53220 1.53234 1.52864 1 4.32955 4.33237 4.32159 2 من الجداول السابقة تم حساب معدل القيمة المتوقعة :من المعادلة االتية- (11) ... الدالة الموجية X , X ( N ) r (        N يمثل عدد االلكترونات الدالة1  تمثل دالة االلكترو ن االول وتسمى هذه الدالة ببرم المدار وتعتمد على متجه البرم ومتجه :الفضاء وتعرف بالمعادلة االتية- j ا (2) l … استخدمتj=12 للمصدر[2] j=6 للمصدر[3] j=7 للمصدر[4]    j 1 i i χ i c  (8) … حيث ان ويمكن االستفادة من حساب توزيع الكثافة القطرية لجسيم واحد في ايجاد القيم المتوقعة لجسيم واحد كما في المعادلة[5] (9) … اذ ان2 - ≥ n ≥ 2 وعندما تكون قيمةn تساو ي (صفر) فأن القيمة المتوقعة يجب ان تساوي (واحد) ألن الدالة متعايره اي ان ...(10) وللمزيد من المعلومات يمكن مراجعة المصدر رقم [6] K K K K d d d    sin       0 1 n 1 1 n 1 dr r ) r ( D r       0 0 1 1 1 0 1 1 1 dr ) r ( D dr r ) r ( D ويمكن كتابةnlm x : والمتمثلة بالصيغة االتية- (3) … ) s ( ) , ( m Y ) r ( n R ) , , (r, m n χ           ويمكن كتابةnlm x : والمتمثلة بالصيغة االتية- (3) … ) s ( ) , ( m Y ) r ( n R ) , , (r, m n χ           ويمكن كتابةnlm x : والمتمثلة بالصيغة االتية- (3) … ) s ( ) , ( m Y ) r ( n R ) , , (r, m n χ           حيث انRnl(r) يمثل الجزء القطري من الدالة : الموجية ويعرف بالمعادلة االتية- (4) … ) (r n S m n N (r) n R     الحسابات والنتائج بأستخدام المعادلة(9) تم حساب القيمة المتوقعة لجسيم واحد للغالفKαKβ لذرة البيريليوم(Be) لدوال موجية مختلفة والنتائج موضحة في الجدول1 ،   n 1r (6) 393 مجلد4 ( 3 ) 7002 مجلة أم سلمة للعلوم وكذلك للغالف LαLβ وا لنتائج موضحة في الجدول 2 اما بالنسبة للغالف KαLα=KβLαفأن النتائج موضحةفي الجدول3. وكذلك للغالف LαLβ وا لنتائج موضحة في الجدول 2 اما بالنسبة للغالف KαLα=KβLαفأن النتائج موضحةفي الجدول3. الدالة الموجية جدول ( 4 ) معدل القيمة لذرة البريليومBe وحسب االغلفة KαLα= KβLα LαLβ KαKβ n 14.40506 1.05581 27.75432 -2 2.10219 0.5225 3.68186 -1 1 1 1 0 1.53225 2.64856 0.41499 1 4.33855 8.41792 0.23295 2 2 1 0 1 2 0 10 20 30 27.754 0.2 33 ex1s1T s ( ) 3 ex2s1T s ( ) 3 ex3T s ( ) 3 2 2  s شكل ( 1 ) معدل القيمة لذرة البريليومBe وحسب االغلفة   n 1r   n 1r n K-Shell KL-Shell L-Shell               1993 n 1 1974 n 1 1960 n 1 Total n 1 r r r 3 1 r   n 1r   n 1r   n 1r القيمة المتوقعة للغالفKαKβ 1993[4] 1974[3] 1960[2] n 27.75338 27.75583 27.75345 -2 3.68188 3.68183 3.68188 -1 1 1 1 0 0.41499 0.41499 0.41499 1 0.23295 0.23295 0.16532 2   n 1r من الجداول السابقة تم حساب معدل القيمة المتوقعة :من المعادلة االتية- (11) ...               1993 n 1 1974 n 1 1960 n 1 Total n 1 r r r 3 1 r ( جدول2) القيمة المتوقعة لذرة البيريليوم(Be) للغالف LαLβ ل عدد من الدوال الموجية المنشورة في السنوات 1993,1974,1960 . ا جدول ( 4 ) معدل القيمة لذرة البريليومBe وحسب االغلفة KαLα= KβLα LαLβ KαKβ n 14.40506 1.05581 27.75432 -2 2.10219 0.5225 3.68186 -1 1 1 1 0 1.53225 2.64856 0.41499 1 4.33855 8.41792 0.23295 2   n 1r جدول ( 4 ) معدل القيمة لذرة البريليومBe وحسب االغلفة KαLα= KβLα LαLβ KαKβ n 14.40506 1.05581 27.75432 -2 2.10219 0.5225 3.68186 -1 1 1 1 0 1.53225 2.64856 0.41499 1 4.33855 8.41792 0.23295 2   n 1r ( جدول3) ال قيمة المتوقعة لذرة البيريليوم(Be) للغالف KαLα= KβLα لعدد من الدوال الموجية المنشورة في السنوات1993,1974,1960 . اما القيم التوقعة عندما تكون1 n  :كما يأتي- n=-2 as HF(1974)>HF(1993)>HF(1960) 3-Clementi, E. and Roetti.1974.Atomic Data and Nuclear Data Tables, 14:177- 478.  الغالفKαLα= KβLα القيم المتوقعة لمجموعة الدوال الموجية لهارتري فوك :كاالتي-  الغالفKαLα= KβLα القيم المتوقعة لمجموعة الدوال الموجية لهارتري فوك :كاالتي-  الغالفKαLα= KβLα القيم المتوقعة لمجموعة الدوال الموجية لهارتري فوك :كاالتي- 4-Bunge, C.F, J.A.Barrientos, and A.V. Bunge .1993.Roothaan-Hartree Fock ground State atomic Wave function, Atomic Data Nucl.Data Table, 53:113- 124. يا n=-2 as HF(1974)>HF(1960)>HF(1993) n=-1 as HF(1993) =HF(1960)>HF(1974) 5-Al-bayati, K.H., A.K.Ahamd and N. CH.Al-Tamimei 2006. Calculation of the one –particle expectation Values to some atoms and ions. Um-Salama Science Journal, College of Science for Women, Baghdad University,3:246-253. 5-Al-bayati, K.H., A.K.Ahamd and N. CH.Al-Tamimei 2006. Calculation of the one –particle expectation Values to some atoms and ions. Um-Salama Science Journal, College of Science for Women, Baghdad University,3:246-253. 6-Al-Asaad, B.H, A.2005. Study of the physical properties for the electrons outer shells for some atoms, M.Sc Thesis college of Scince for Women, Baghdad University,Baghdad, Iraq. اما القيم التوقعة عندما تكون1 n  :كما يأتي- n=-2 as HF(1974)> HF(1960)>HF(1993) اما القيم التوقعة عندما تكون1 n  :كما يأتي- n=-2 as HF(1974)> HF(1960)>HF(1993)  تكون القيمة المتوقعة للغالف KαKβ عندما قيمn سالبةاكبر مما ع ليه عند القيم الموجبة لـn اي ان احتمالية تواجد االلكترونات تكون في ال م ناطق .القريبة من النواة  تكون القيمة المتوقعة للغالف KαKβ عندما قيمn سالبةاكبر مما ع ليه عند القيم الموجبة لـn اي ان احتمالية تواجد االلكترونات تكون في ال م ناطق .القريبة من النواة  L n 1 K n 1 r r      عند القيم الموجبة لـn وذلك ألن النوى الثقيلة قوة التجاذب بين الدالة الموجية 1 1 1 0 1.53225 2.64856 0.41499 1 4.33855 8.41792 0.23295 2 2 1 0 1 2 0 10 20 30 27.754 0.2 33 ex1s1T s ( ) 3 ex2s1T s ( ) 3 ex3T s ( ) 3 2 2  s شكل ( 1 ) معدل القيمة لذرة البريليومBe وحسب االغلفة   n 1r n K-Shell KL-Shell L-Shell   n 1r ( جدول3) ال قيمة المتوقعة لذرة البيريليوم(Be) للغالف KαLα= KβLα لعدد من الدوال الموجية المنشورة في السنوات1993,1974,1960 .   n 1r ( جدول3) ال قيمة المتوقعة لذرة البيريليوم(Be) للغالف KαLα= KβLα لعدد من الدوال الموجية المنشورة في السنوات1993,1974,1960 .   n 1r 2 1 0 1 2 0 10 20 30 27.754 0.2 33 ex1s1T s ( ) 3 ex2s1T s ( ) 3 ex3T s ( ) 3 2 2  s   n 1r K-Shell KL-Shell L-Shell L-Shell 394 مجلة أم سلمة للعلوم مجلد4 ( 3 ) 7002 مناقشة النتائج عند مقارنة القيم ال م توقعة لمجموعة الدوال : الموجية المختلفة نجد ان-  الغالفKαKβ اغلب القيم متساوية باستثناءالقيم عندما تكونn=-2,-1 تكون حسب العالقة n=-2 as HF(1974) >HF(1960)>HF(1993) n=-1as HF(1993)=HF(1960) >HF(1974) مناقشة النتائج عند مقارنة القيم ال م توقعة لمجموعة الدوال : الموجية المختلفة نجد ان-  الغالفKαKβ اغلب القيم متساوية باستثناءالقيم عندما تكونn=-2,-1 تكون حسب العالقة n=-2 as HF(1974) >HF(1960)>HF(1993) n=-1as HF(1993)=HF(1960) >HF(1974) الكتروناتها كبيرة الت سمح بتواجد الكترونات بعيدا عنها .والعكس صحيح الكتروناتها كبيرة الت سمح بتواجد الكترونات بعيدا عنها .والعكس صحيح المصادر: 1- King, F.W.1991.Radial electronic density function for selected low-lying excited 2s state of Li isoelectronic series Phys.Rev, 44: 3350-3353. 2-Roothaan, C.C, L.Sachs,and A.W.Weiss.1960.Analytical self- consistent field function for the atomic configurations 1s2,1s22s, and 1s22s2 ,Reviews of modern Physics, 32:186- 193 3-Clementi, E. and Roetti.1974.Atomic Data and Nuclear Data Tables, 14:177- 478. 4-Bunge, C.F, J.A.Barrientos, and A.V. Bunge .1993.Roothaan-Hartree Fock ground State atomic Wave function, Atomic Data Nucl.Data Table, 53:113- 124. 5-Al-bayati, K.H., A.K.Ahamd and N. CH.Al-Tamimei 2006. Calculation of the one –particle expectation Values to some atoms and ions. Um-Salama Science Journal, College of Science for Women, Baghdad University,3:246-253. 6-Al-Asaad, B.H, A.2005. Study of the physical properties for the electrons outer shells for some atoms, M.Sc Thesis college of Scince for Women, Baghdad University,Baghdad, Iraq. المصادر: 1- King, F.W.1991.Radial electronic density function for selected low-lying excited 2s state of Li isoelectronic series Phys.Rev, 44: 3350-3353. ,ا n=-2 as HF(1974) >HF(1960)>HF(1993) n=-1as HF(1993)=HF(1960) >HF(1974)  الغالف LαLβ القيم المتوقعة لمجموعة الدوال :الموجية لهارتري فوك كاالتي- , , , A.W.Weiss.1960.Analytical self- consistent field function for the atomic configurations 1s2,1s22s, and 1s22s2 ,Reviews of modern Physics, 32:186- 193 يا ي n=-2 as HF(1974)>HF(1960)>HF(1993) n=-1 as HF(1993) >HF(1960)>HF(1974) اما القيم التوقعة عندما تكون1 n  :كما يأتي- n=-2 as HF(1974)>HF(1993)>HF(1960)  الغالفKαLα= KβLα القيم المتوقعة لمجموعة الدوال الموجية لهارتري فوك :كاالتي- n=-2 as HF(1974)>HF(1960)>HF(1993) n=-1 as HF(1993) =HF(1960)>HF(1974) اما القيم التوقعة عندما تكون1 n  :كما يأتي- n=-2 as HF(1974)>HF(1993)>HF(1960) 393 Evaluation of the one electron expectation values for different wave function of Be atom مجلد4 ( 3 ) 7002 مجلة أم سلمة للعلوم Physics department Baghdad University - College of Science for Women Physics department Baghdad University - College of Science for Women * Khalil H.Al-bayati* Salaah A.Hasson* Salaah A.Hasson* Abstract: The aim of this work is to evaluate the one- electron expectation value from the radial electronic density function D(r1) for different wave function for the 2S state of Be atom . The wave function used were published in 1960,1974and 1993, respectavily.   n 1r Using Hartree-Fock wave function as a Slater determinant has used the partitioning technique for the analysis open shell system of Be (1s22s2) state, the analyze Be atom for six-pairs electronic wave function , tow of these are for intra-shells (K,L) and the rest for inter-shells(KL) . The results are obtained numerically by using computer programs (Mathcad). 393
https://openalex.org/W2963647620	https://epublications.marquette.edu/cgi/viewcontent.cgi?article=1246&context=civengin_fac	English	null	Evaluating the Variability of Urban Land Surface Temperatures Using Drone Observations	Remote sensing	2,019	cc-by	13,152	Marquette University Marquette University e-Publications@Marquette e-Publications@Marquette Civil and Environmental Engineering Faculty Research and Publications Civil, Construction, and Environmenta Engineering, Department o 7-20-2019 Evaluating the Variability of Urban Land Surface Temperatures Evaluating the Variability of Urban Land Surface Temperatures Using Drone Observations Using Drone Observations Joseph Naughton Walter M. McDonald Civil, Construction, and Environmental Engineering, Department of Received: 30 May 2019; Accepted: 16 July 2019; Published: 20 July 2019 Received: 30 May 2019; Accepted: 16 July 2019; Published: 20 July 2019 Abstract: Urbanization and climate change are driving increases in urban land surface temperatures that pose a threat to human and environmental health. To address this challenge, we must be able to observe land surface temperatures within spatially complex urban environments. However, many existing remote sensing studies are based upon satellite or aerial imagery that capture temperature at coarse resolutions that fail to capture the spatial complexities of urban land surfaces that can change at a sub-meter resolution. This study seeks to ﬁll this gap by evaluating the spatial variability of land surface temperatures through drone thermal imagery captured at high-resolutions (13 cm). In this study, ﬂights were conducted using a quadcopter drone and thermal camera at two case study locations in Milwaukee, Wisconsin and El Paso, Texas. Results indicate that land use types exhibit signiﬁcant variability in their surface temperatures (3.9–15.8 ◦C) and that this variability is inﬂuenced by surface material properties, traﬃc, weather and urban geometry. Air temperature and solar radiation were statistically signiﬁcant predictors of land surface temperature (R2 0.37–0.84) but the predictive power of the models was lower for land use types that were heavily impacted by pedestrian or vehicular traﬃc. The ﬁndings from this study ultimately elucidate factors that contribute to land surface temperature variability in the urban environment, which can be applied to develop better temperature mitigation practices to protect human and environmental health. Keywords: land surface temperature; drones; unmanned aerial vehicles; thermal remote sensing www.mdpi.com/journal/remotesensing Evaluating the Variability of Urban Land Surface Temperatures Evaluating the Variability of Urban Land Surface Temperatures Using Drone Observations Using Drone Observations Joseph Naughton Walter M. McDonald Follow this and additional works at: https://epublications.marquette.edu/civengin_fac Part of the Civil Engineering Commons Follow this and additional works at: https://epublications.marquette.edu/civengin_fac remote sensing Article Evaluating the Variability of Urban Land Surface Temperatures Using Drone Observations Joseph Naughton and Walter McDonald * Department of Civil, Construction & Environmental Engineering, Marquette University, P.O. Box 1881, Milwaukee, WI 53211, USA * Correspondence: walter.mcdonald@marquette.edu; Tel.: +1-414-288-2117 Evaluating the Variability of Urban Land Surface Temperatures Using Drone Observations oseph Naughton and Walter McDonald * Department of Civil, Construction & Environmental Engineering, Marquette University, P.O. Box 1881, Milwaukee, WI 53211, USA Joseph Naughton and Walter McDonald * Joseph Naughton and Walter McDonald * * Correspondence: walter.mcdonald@marquette.edu; Tel.: +1-414-288-2117 * Correspondence: walter.mcdonald@marquette.edu; Tel.: +1-414-288-2117 Received: 30 May 2019; Accepted: 16 July 2019; Published: 20 July 2019 www.mdpi.com/journal/remotesensing 1. Introduction Urban areas across the world are subject to thermal stresses caused by the surface urban heat island (SUHI) eﬀect where urban land surfaces experience higher temperatures than their surrounding rural areas. This is in large part due to the replacement of undeveloped vegetated land with anthropogenic materials that absorb more solar radiation and have diﬀerent heat capacity and surface radiative properties [1]. This results in higher surface temperatures that pose a signiﬁcant threat to human health [2], as well as higher storm runoﬀtemperatures that can harm aquatic life [3–5]. These stresses are only expected to grow with increases in global temperatures and urban populations; therefore, it is critical that we understand the fundamental processes that drive land surface temperature (LST) to develop solutions that can protect human and environmental health. To that end, thermal remote sensing is an important tool for evaluating urban land surface temperatures. This includes satellite sensors such as ASTER, MODIS and Landsat that can capture land surface temperatures at 30 m–1 km resolutions [6]. Data from these satellites have been used to extensively study urban land surface temperatures and their eﬀects [7–14]. However, while satellite remote sensing is valuable for evaluating LST across a city scale, the spatial resolution precludes its applications to smaller spatial scales that better reﬂect the spatial complexity of the urban environment. To acquire higher resolution thermal data, studies have used aerial reconnaissance or downscaling Remote Sens. 2019, 11, 1722; doi:10.3390/rs11141722 2 of 18 Remote Sens. 2019, 11, 1722 techniques [15,16]; however, these are still at resolutions (4–10 m) that cannot capture changes that occur on a sub meter resolution. Furthermore, satellite remote sensing is temporally constrained to intervals between 1–14 days. Aerial ﬂights do not have the same temporal constraints; however, doing so at on-demand temporal resolutions would not be economically practical. Therefore, these methods are inadequate for evaluating changes in urban LST that occur throughout the day or capturing the spatial heterogeneity of urban LST at small scales. This challenge is important to overcome as urban land surfaces are spatially complex and signiﬁcant variations in land cover can occur on a sub meter spatial resolution [17]. While existing research has demonstrated that the spatial conﬁguration of land use classiﬁcations at a city scale are important (i.e. 1. Introduction industrial, residential, forest) [18,19], less is known about the importance of the spatial conﬁguration and variations in LST at smaller scales (i.e., sidewalks, grass medians, ﬂowerbeds, etc.). In addition, the urban environment is dynamic and land surface temperatures can be signiﬁcantly inﬂuenced by other factors besides land cover material properties [20]. Land surface temperature may therefore vary signiﬁcantly across small spatial scales; however, the factors that control this variation are not well deﬁned. Doing so requires direct measurements of surface temperatures across wide spatial and temporal scales, yet little research to date has evaluated the spatial variability in temperature among urban land use types in sub-meter resolutions. This may be due to measurement limitations, as satellite data is too coarse and in-situ temperature probes are too expensive to densely distribute across an urban landscape. Therefore, new and innovative approaches to measuring land surface temperatures at small spatial and temporal scales are needed to assess thermal variability across land use types in the urban environment. Unmanned Aerial Vehicles (UAVs) or drones, are a technology that can meet this challenge. Recent advances in UAVs and radiometric thermal cameras have made it possible to capture land surface temperatures on-demand and at sub-meter spatial resolutions that accurately reﬂect the spatial complexity and detail of land surface temperatures in the urban environment [21]. UAVs also have advantages in that they can be ﬂown on demand to capture LST at temporal resolutions unmatched by satellite or aerial imagery. While the limited battery life of around 30 minutes for quad-copter UAVs constrains the area that can be captured in a single ﬂight, their spatial and temporal resolutions oﬀer signiﬁcant advantages for evaluating the variability of LST in the urban environment at ﬁne spatial and temporal scales. p We therefore present a study to evaluate the variability of temperatures across urban land surfaces using a UAV. In this study, we apply a UAV and radiometric thermal camera to capture land surface temperatures at high-resolutions (13 cm) in two case study locations: Milwaukee, Wisconsin and El Paso, Texas. Using data collected throughout a calendar year, we evaluate the variability in land surface temperatures, develop models to predict mean land surface temperature based upon weather parameters and evaluate the diurnal trends in urban land surface temperature. 1. Introduction To do so, we (1) quantify land surface temperature variability across diﬀerent surface types, (2) evaluate variance in temperature across diﬀerent surface types based upon meteorological and/or other derived parameters (e.g., albedo, normalized diﬀerence vegetation index, apparent thermal inertia, etc.), (3) predict land surface temperature based upon meteorological parameters and (4) assess diurnal variability in land surface temperature magnitude and uncertainty. Ultimately, this study helps to elucidate factors that contribute to land surface temperature variability in the urban environment at small spatial scales, which can then be applied to develop better temperature mitigation strategies. 2.1. Case Study Locations Two case study locations were chosen for this project: (1) a portion of Marquette University’s campus in Milwaukee, WI and (2) a portion University of Texas El Paso’s (UTEP) campus in El Paso, Texas (Figure 1). The Marquette and UTEP case study areas were roughly 21,300 m2 and 27,300 m2, 3 of 18 Remote Sens. 2019, 11, 1722 respectively and included a balance of both natural landscape and impervious gray surfaces. Surface types within each case study location were manually delineated using ESRI’s ArcMap software. The nine surfaces types identiﬁed at Marquette and UTEP and their respective surface areas are listed in Table 1. The speciﬁc locations on each campus were chosen for their variety of surface types, similarities in land use between the two locations and suitability for drone takeoﬀ/landing and ﬂying. In addition, these locations provide a contrast in geography, climate and weather that are helpful in testing the generalizability of our ﬁndings. For example, Milwaukee’s climate is classiﬁed by Koppen and Geiger as Dfa (Humid Continental Hot Summers With Year Around Precipitation) and receives 870 mm of precipitation annually, while El Paso is classiﬁed as BWk (Cold Desert Climate) and receives 221 mm of precipitation annually [22,23]. emote Sens. 2019, 11, x FOR PEER REVIEW 3 of 19 ine surfaces types identified at Marquette and UTEP and their respective surface areas are listed in able 1. The specific locations on each campus were chosen for their variety of surface types, imilarities in land use between the two locations and suitability for drone takeoff/landing and flying. n addition, these locations provide a contrast in geography, climate and weather that are helpful in esting the generalizability of our findings. For example, Milwaukee’s climate is classified by Koppen nd Geiger as Dfa (Humid Continental Hot Summers With Year Around Precipitation) and receives 70 mm of precipitation annually, while El Paso is classified as BWk (Cold Desert Climate) and eceives 221 mm of precipitation annually [22,23]. Figure 1. Visual imagery of the case study locations: Marquette University (a) and University of Texas El Paso (UTEP) (b). Visual imagery of Marquette was captured from a drone on 11 August 2018. Visual imagery of UTEP was pulled from Google Maps on 13 March 2019. (a) (b) Figure 1. Visual imagery of the case study locations: Marquette University (a) and University of Texas El Paso (UTEP) (b). 2.1. Case Study Locations Visual imagery of Marquette was captured from a drone on 11 August 2018. Visual imagery of UTEP was pulled from Google Maps on 13 March 2019. (a) (b) (a) igure 1. Visual imagery of the case study locations: Marquette University (a) and University of Texas l Paso (UTEP) (b). Visual imagery of Marquette was captured from a drone on 11 August 2018. isual imagery of UTEP was pulled from Google Maps on 13 March 2019. Figure 1. Visual imagery of the case study locations: Marquette University (a) and University of Texas El Paso (UTEP) (b). Visual imagery of Marquette was captured from a drone on 11 August 2018. Visual imagery of UTEP was pulled from Google Maps on 13 March 2019. Table 1 Su fa e ty e a d u fa e a ea ithi ea h a e tudy lo atio Table 1. Surface types and surface areas within each case study location. T bl 1 S f t d f ithi h t d l ti Table 1. Surface types and surface areas within each case study location. Table 1. Surface types and surface areas within each case study location. MARQUETTE UTEP Surface Type Surface Area (m2) Surface Type Surface Area (m2) Grass 2,738 Rooftop (rammed earth) 503 Sidewalk 904 Desert Shrub 173 Rooftop (composite) 336 Rooftop (composite) 2,047 Road (asphalt) 3,299 Parking Lot (asphalt) 1,350 Parking Lot (concrete) 908 Sidewalk (concrete) 9,808 Rooftop (rubber) 6,057 Road (asphalt) 4,253 Canopy Cover 4,758 Parking Lot (concrete) 4,081 Shrub/mulch 2,272 Grass 1,270 MARQUETTE UTEP Surface Type Surface Area (m2) Surface Type Surface Area (m2) Grass 2738 Rooftop (rammed earth) 503 Sidewalk 904 Desert Shrub 173 Rooftop (composite) 336 Rooftop (composite) 2047 Road (asphalt) 3299 Parking Lot (asphalt) 1350 Parking Lot (concrete) 908 Sidewalk (concrete) 9808 Rooftop (rubber) 6,057 Road (asphalt) 4253 Canopy Cover 4758 Parking Lot (concrete) 4081 Shrub/mulch 2272 Grass 1270 Solar 65 Canopy Cover 3782 2.3. Data Collection Methods Two datasets were collected during the 2018 calendar year: (1) surface temperature measured at 12:00 PM across the entire year and (2) surface temperature measured on a diurnal cycle. To evaluate surface temperature across the entire year, fourteen ﬂights in Milwaukee and one in El Paso were recorded between 26 February and 13 September 2018 (Table 2). To evaluate the diurnal cycle of temperature, three ﬂights in Milwaukee and one in El Paso measured temperature throughout the day at 9:00 AM, 12:00 PM, 3:00 PM and 5:00 PM (Table 3). Weather data was collected at Marquette from a station on top of Engineering Hall and weather data at UTEP was collected from a weather station 10.5 km away at El Paso International Airport. Each station recorded air temperature, relative humidity, wind speed, wind direction, relative humidity, solar radiation and atmospheric pressure. Drone imagery was captured autonomously using a third-party photogrammetry software called Pix4Dcapture. Using this software, autonomous ﬂight paths were programmed to the drone prior to each mission. Programmed ﬂight path information included drone speed, altitude and image overlap. Drone speed was set at 54 km/h for visual and multispectral ﬂights but set at a lower threshold of 30.6 km/h for thermal ﬂights due to the diﬀerence in image capture speed between the two camera technologies. The ﬂight altitude for each mission was set to the United States Federal Aviation Administration (FAA) maximum allowable limit of 120 m, which resulted in thermal imagery at a 13 cm pixel size. Finally, the image overlap was set to 85%, which provided reliable overlap for stitching an orthomosaic during data processing. Table 2. Flight log and summary of meteorological variables recorded for Marquette and UTEP during ﬁfteen noon ﬂights. Solar 2.2. Equipment .2. Equipment Remote sensing data was collected using a DJI Matrice 100 (M100) quadcopter UAV. The M100 was deployed at our case study locations with three types of camera payloads—visual, multispectral nd infrared. These cameras include the DJI Zenmuse X3 visual (12 MP), Zenmuse X3 multispectral Blue-Green-NIR 680-800nm at 12 MP) and DJI Zenmuse XTR radiometric thermal (13 mm, 30 hz and Remote sensing data was collected using a DJI Matrice 100 (M100) quadcopter UAV. The M100 was deployed at our case study locations with three types of camera payloads—visual, multispectral and infrared. These cameras include the DJI Zenmuse X3 visual (12 MP), Zenmuse X3 multispectral (Blue-Green-NIR 680–800 nm at 12 MP) and DJI Zenmuse XTR radiometric thermal (13 mm, 30 Hz and spectral bandwidth of 7–13 µm). Additionally, ground temperatures were validated using a Nubee NUB8380 Digital Infrared Thermometer. 4 of 18 Remote Sens. 2019, 11, 1722 2.3. Data Collection Methods Flight Number Flight Date Flight Time Air Temp (◦C) Relative Humidity (%) Wind Speed (m/s) Wind Dir (Degrees) Solar Rad (kW/m2) Pressure (kPa) MU 1 26 February2018 12:00 PM −1.7 54 4.0 225 0.00 102.2 MU 2 12 April 2018 12:00 PM 12.4 65.8 6.8 285 0.41 100.2 MU 3 8 May 2018 12:00 PM 26.4 22.1 4.2 218 0.81 101.7 UTEP 1 20 May 2018 12:00 PM 27.8 26 5.8 120 0.96 101.7 MU 4 13 June 2018 12:00 PM 25.3 33.3 3.4 321 0.89 101.2 MU 5 29 June 2018 12:00 PM 31.5 54.7 5.4 193 0.80 101.0 MU 6 11 July 2018 12:00 PM 25.9 44.1 2.0 91 0.78 101.9 MU 7 12 July 2018 12:00 PM 27.4 43.2 5.9 204 0.60 101.8 MU 8 17 July 2018 12:00 PM 25 38.9 3.0 38 0.74 101.6 MU 9 18 July 2018 12:00 PM 22.5 56.3 3.1 101 0.83 101.8 MU 10 25 July 2018 12:00 PM 28.6 31.9 2.5 271 0.83 101.4 MU 11 10 August 2018 12:00 PM 25.8 58.8 2.3 84 0.77 101.3 MU 12 31 August 2018 12:00 PM 25.8 49.9 4.0 158 0.09 101.6 MU 13 12 September 2018 12:00 PM 26.1 55.3 3.1 168 0.56 101.9 MU 14 13 September 2018 12:00 PM 22.8 64.9 4.2 127 0.68 102.0 Table 3. Flight log and summary of meteorological variables recorded for Marquette and UTEP during four diurnal ﬂights. Flight Number Flight Date Flight Time Air Temp (◦C) Relative Humidity (%) Wind Speed (m/s) Wind Dir (Degrees) Solar Rad (kW/m2) Pressure (kPa) MU1 13 June 2018 9:00 AM 22.5 42.8 4.4 320 0.73 101.1 MU1 13 June 2018 12:00 PM 25.3 33.3 3.4 321 0.89 101.2 MU1 13 June 2018 3:00 PM 27.5 20.1 3.0 328 0.80 101.2 MU1 13 June 2018 5:00 PM 27.9 19.8 2.0 285 0.51 101.2 MU2 17 July 2018 9:00 AM 23.8 37.7 3.1 8 0.52 101.6 MU2 17 July 2018 12:00 PM 25 38.9 3.0 38 0.74 101.6 MU2 17 J l 2018 3 00 PM 25 41 2 3 0 38 0 76 101 7 Table 2. Flight log and summary of meteorological variables recorded for Marquette and UTEP during ﬁfteen noon ﬂights. light log and summary of meteorological variables recorded for Marquette and UTEP during ﬂi ht 3. 2.4. Thermal Data Processing After data collection in the ﬁeld, a series of post-processing steps were performed using Pix4D and ESRI’s ArcMap to stitch the drone thermal imagery into orthomosaics, correct temperature values for emissivity and extract surface temperature data for analysis. First, Pix4D was used to stitch the captured thermal images into orthomosaics, export the orthomosaics as a 32-bit TIFF and georeference them for application within ArcMap. Once in ArcMap, an emissivity correction was applied to each thermal orthomosaic. Emissivity is a measure of how well a material can emit energy as thermal radiation and diﬀerent materials have diﬀerent values of emissivity depending on their surface properties [24]. Land use classiﬁcations that were previously delineated for each case study area were used to apply emissivity values to the target surfaces. The emissivity values for each land use classiﬁcation used in this study are listed in Table 4 and are based upon a review of emissivity studies. These emissivity values were then applied in the following emissivity correction equation derived from Stefan-Boltzmann Law: Ttarget = 4 s T4sensor −(1 −ε) ∗T4 background ε (1) (1) where Ttarget is the actual temperature of the target surface [K], Tsensor is the temperature measured by the infrared camera [K], Tbackground is the recorded air temperature [K] and ε is the emissivity value of the target surface [25]. This equation was used to correct each surface type for their respective emissivity before performing spatial data analysis. where Ttarget is the actual temperature of the target surface [K], Tsensor is the temperature measured by the infrared camera [K], Tbackground is the recorded air temperature [K] and ε is the emissivity value of the target surface [25]. This equation was used to correct each surface type for their respective emissivity before performing spatial data analysis. Table 4. Emissivity values for each surface type. Table 4. Emissivity values for each surface type. Land Use Type Emissivity Value Reference Grass 0.979 [26] Shrub/mulch 0.928 [27] Road (asphalt) 0.95 [28,29] Parking Lot (concrete) 0.91 [29–31] Sidewalk (concrete) 0.91 [24,29–31] Rooftop (tar and stone) 0.973 [24] Rooftop (black rubber) 0.859 [24] Solar Panel 0.85 [32] Canopy Cover 0.977 [33] Once the thermal data were corrected for emissivity, spatial data analysis was performed in ArcMap. First, a land use feature map was created that categorized the surface types in each case study location. 2.3. Data Collection Methods Flight log and summary of meteorological variables recorded for Marquette and UTEP during i l ﬂi ht Table 3. Flight log and summary of meteorological variables recorded for Marquette and UTEP during four diurnal ﬂights. four diurnal ﬂights. Flight Number Flight Date Flight Time Air Temp (◦C) Relative Humidity (%) Wind Speed (m/s) Wind Dir (Degrees) Solar Rad (kW/m2) Pressure (kPa) MU1 13 June 2018 9:00 AM 22.5 42.8 4.4 320 0.73 101.1 MU1 13 June 2018 12:00 PM 25.3 33.3 3.4 321 0.89 101.2 MU1 13 June 2018 3:00 PM 27.5 20.1 3.0 328 0.80 101.2 MU1 13 June 2018 5:00 PM 27.9 19.8 2.0 285 0.51 101.2 MU2 17 July 2018 9:00 AM 23.8 37.7 3.1 8 0.52 101.6 MU2 17 July 2018 12:00 PM 25 38.9 3.0 38 0.74 101.6 MU2 17 July 2018 3:00 PM 25 41.2 3.0 38 0.76 101.7 MU2 17 July 2018 5:00 PM 22.8 57.9 3.4 34 0.50 101.7 MU3 10 August 2018 9:00 AM 27.4 46.1 2.4 33 0.70 101.3 MU3 10 August 2018 12:00 PM 25.7 58.8 2.3 84 0.77 101.3 MU3 10 August 2018 3:00 PM 27.4 46.1 2.4 33 0.70 101.3 MU3 10 August 2018 5:00 PM 27.4 33.4 2.4 37 0.43 101.2 UTEP1 20 May 2018 9:00 AM 25 32 5.8 90 0.66 101.8 UTEP1 20 May 2018 12:00 PM 27.8 26 5.8 120 0.96 101.7 UTEP1 20 May 2018 3:00 PM 31.1 17 4.0 120 0.83 101.4 UTEP1 20 May 2018 5:00 PM 31.1 21 4.9 90 0.50 101.3 5 of 18 Remote Sens. 2019, 11, 1722 2.5. Surface Parameters In addition to surface temperature, three other material properties were derived from visual and multispectral imagery, converted into spatial distribution rasters and averaged for each surface type. These include albedo (S), normalized diﬀerence vegetation index (NDVI) and apparent thermal inertia (ATI). Albedo, a measure of solar reﬂectance of a material, was derived from blue, green, red and near-IR image bands as shown in the following equation: (2) S = cbbk + cggk + crrk + ciik (2) where cb = 0.17, cg = −0.13, cr = 0.33 and ci = 0.54 are derived constants and bk, gk, rk and ik are the band reﬂectance’s for—blue, bk (420–492 nm); green, gk (533–587 nm); red, rk (604–664 nm); and near-IR, ik (833–920 nm) [34]. where cb = 0.17, cg = −0.13, cr = 0.33 and ci = 0.54 are derived constants and bk, gk, rk and ik are the band reﬂectance’s for—blue, bk (420–492 nm); green, gk (533–587 nm); red, rk (604–664 nm); and near-IR, ik (833–920 nm) [34]. Visual and multispectral imagery were also used to derive NDVI, which is a measure of the degree of live vegetation and is commonly used to evaluate soil moisture dynamics, erosion potential and plant and crop health. As shown in Equation (3), NDVI is a function of near-IR and red band reﬂectance and is estimated on a scale of −1 to +1, with higher values indicating higher vegetative cover and greater plant health [35]. NDVI = (NIR −Red) (NIR + Red) (3) (3) Finally, ATI was derived for each surface type from albedo (S), solar correction (SCR) and the diurnal temperature amplitude (DTA) (Equation (4)). ATI is an estimation of thermal inertia from remotely sensed observations and can be estimated from diurnal changes in temperature. Speciﬁcally, ATI is derived from solar correction (SCR), albedo (S) and the diurnal temperature amplitude (DTA), where DTA is the diﬀerence between the maximum and minimum surface temperature recorded at the time the remote images were captured and SCR is the solar correction factor (Equation (5)), which is dependent on geographic location, the local latitude (θ) and the solar declination (ϕ) [36]. ATI = SCR(1 −S) DTA (4) (4) SCR = sinθ sinϕ(1 − tanθ tanϕ)2 + cosθ cosϕ arccos(−tanθ tanϕ) (5) 2.4. Thermal Data Processing Then inconsistencies within these areas, such as a parked car within a parking lot, human traﬃc on a sidewalk or construction materials on the street, were clipped and removed for each ﬂight. Once these inconsistencies were removed, zonal statistics was applied to compute summary statistics of each surface type such as mean and standard deviation of the land surface temperature. A complete ﬂow-chart of the process from ﬂight programming to developing summary statistics is shown in Figure 2. In total this process took about 3 h to complete for each ﬂight. Figure 2. Flow chart of data collection and processing. Figure 2. Flow chart of data collection and processing. Remote Sens. 2019, 11, 1722 6 of 18 2.6. Model Development Drone observations were applied to develop empirical models of land surface temperature. These include (1) a regression model to predict spatially averaged surface temperatures at 12:00 PM based upon meteorological variables and (2) a model to assess diurnal variability and predict surface temperatures throughout a given day. Multi-variable regression models were developed to predict spatially averaged surface temperature of the fourteen Milwaukee and single El Paso 12:00 PM flights using MATLAB and the statistical software package JMP 13 [37]. Response screening was performed for each of the respective datasets to identify the strength of relationship between surface temperature (response) and meteorological parameters (predictors). Between the two case study locations, six surface types that were common to both locations were used as response variables: grass, canopy cover, concrete parking lot, concrete sidewalk, composite rooftop and road surface. Meteorological predictor variables included air temperature, relative humidity, preceding 24 h rainfall, wind speed, wind direction, barometric pressure and solar radiation. After response screening, stepwise linear regression was then performed to predict land surface temperature based upon meteorological parameters as represented in following equation: y = β0 + β1x1 + β2x2 + · · · + βkxk (6) (6) y = β0 + β1x1 + β2x2 + · · · + βkxk 7 of 18 Remote Sens. 2019, 11, 1722 where y is the response variable, β0, β1, . . . , βk are the regression coeﬃcients and x1, x2, . . . , xk are the predictor variables for k predictors [38]. These models were developed using data from both the Milwaukee and El Paso ﬂights; therefore, to evaluate the inﬂuence and leverage of the El Paso dataset we computed Cook’s D inﬂuence and hat matrix leverage statistics [38]. Finally, we explored the variation in surface temperatures as they change throughout the day (9 AM, 12 PM, 3 PM and 5 PM) and evaluated if this variation could be explained by any meteorological parameters. In addition to exploring diurnal changes in variability, we applied the data to develop a model to predict land surface temperatures throughout the day for the six land use types common to each location. 2.6. Model Development To do so, we applied the drone data collected on the four diurnal ﬂight missions to estimate land surface temperatures based upon the solar radiation and the diﬀerence between the air and land surface temperatures, which have been found to be statistically signiﬁcant predictors for diurnal estimates of pavement temperatures [39]. First, we computed a parameter (g) based upon the drone-derived mean land surface temperature and measured air temperature and solar radiation: g = Ts −Ta ∗S (7) (7) where Ts is the mean surface temperature of the land use, Ta is the measured air temperature and S is the measured solar radiation (kW). Next, g at a given hour i was estimated using a Gaussian peak model given by the following: gi = a ∗e−0.5∗( i−b c ) 2 (8) (8) where gi, is the parameter g at hour i, a is the peak value, b is the critical point and c is the growth rate [40]. Using this model, the mean land surface temperature can be predicted based upon air temperature and solar radiation for any time of day using the following: where gi, is the parameter g at hour i, a is the peak value, b is the critical point and c is the growth rate [40]. Using this model, the mean land surface temperature can be predicted based upon air temperature and solar radiation for any time of day using the following: Ts,i = Ta,i + (gi/Si) (9) (9) where Ts,i is the estimated surface temperature at hour i and Ta,i and Si are the air temperature and solar radiation at hour i. Taken as a whole, these models test both the suitability of predicting drone-derived mean land surface temperatures based upon meteorological variables, as well as the generalizability of our ﬁndings by including data from sites in two diﬀerent geomorphologic and climatic regions. 3.1. Surface Temperature Variability Note GRS = grass; SM = shrub/mulch; CPY = canopy; PL = parking lot; SW = sidewalk; RTC = composite rooftop; RTR = rubber rooftop; RD = road; SLR = solar. p g ; ; p p; p; ; We then summarized the average temperature, standard deviation and coefficient of variation of each surface type for the fourteen recorded flights in Milwaukee, WI and the single flight in El Paso, TX (Table 5). Generally, gray surfaces exhibited higher temperatures throughout the year than green surfaces. In El Paso, the asphalt parking lot exhibited the highest average temperature (51.7 °C) and grass exhibited the lowest (41.6 °C), while in Milwaukee the black rubber rooftop exhibited the highest average temperature (57.4 °C) and canopy cover exhibited the lowest (30.4 °C). In terms of variation, the lowest degrees of variation typically occurred in the parking lots and grass. However, We then summarized the average temperature, standard deviation and coefficient of variation of each surface type for the fourteen recorded flights in Milwaukee, WI and the single flight in El Paso, TX (Table 5). Generally, gray surfaces exhibited higher temperatures throughout the year than green surfaces. In El Paso, the asphalt parking lot exhibited the highest average temperature (51.7 ◦C) and grass exhibited the lowest (41.6 ◦C), while in Milwaukee the black rubber rooftop exhibited the highest average temperature (57.4 ◦C) and canopy cover exhibited the lowest (30.4 ◦C). In terms of variation, the lowest degrees of variation typically occurred in the parking lots and grass. However, there is a noted difference in the variation between the two locations; the road in Milwaukee had the highest coefficient of variation of 0.32, while the road in El Paso had the lowest at 0.04. This may be due to a difference in traffic on the days that flights were conducted. The location in Milwaukee is located near the city center and is subject to heavy and constant vehicular traffic, while the location in El Paso is in a restricted traffic area and experienced very low vehicle activity on the weekend that the flight was conducted. Table 5. Average temperature, standard deviation and coeﬃcient of variation of nine surface types From 14 recorded ﬂights on Marquette University campus and from one ﬂight recorded on UTEP’s campus. Table 5. 3.1. Surface Temperature Variability We evaluated the land surface temperature variability of each ﬂight across common land use types and generally found that green surfaces had a greater degree of variability than gray surfaces, with the exception being the rubber rooftop. As an example, the distribution of surface temperature data (1,986,543 total data points) is shown in Figure 3 for a ﬂight recorded on July 11, 2018. The six gray surfaces recorded a smaller distribution of temperature on average but had more extreme values than green surfaces (Figure 3a). Gray surfaces retain more heat from the sun because of their high emissivity and ATI and therefore typically have higher surface temperatures. Additionally, non-normal behavior was identiﬁed for both canopy cover and rubber rooftop (Figure 3b). Canopy cover exhibits a left skew while the rubber rooftop exhibits a right skew. The canopy cover had a variation of tree types and therefore a variation of leaf area indices (LAI), which may be a reason for the skew in the temperature data. The rubber rooftop also exhibited a strong right skew, which may be due to small materials on the roof surface, such as ventilation pipes and drainage grates, that were diﬃcult to detect and may not have been removed from the dataset. Therefore, this caused a distribution of lower temperatures to be recorded. Remote Sens. 2019, 11, 1722 to detect and may not h lower temperatures to b 8 of 18 of (b) Figure 3. Boxplot distribution of surface temperature (a); and histogram of surface temperature (b). Data from flight recorded on July 11, 2018. Note GRS = grass; SM = shrub/mulch; CPY = canopy; PL = ki l SW id lk RTC i f RTR bb f RD d SLR l (a) (b) Figure 3. Boxplot distribution of surface temperature (a); and histogram of surface temperature (b). Data from ﬂight recorded on July 11, 2018. Note GRS = grass; SM = shrub/mulch; CPY = canopy; PL = parking lot; SW = sidewalk; RTC = composite rooftop; RTR = rubber rooftop; RD = road; SLR = solar. (b) (a) (b) Figure 3. Boxplot distribution of surface temperature (a); and histogram of surface temperature (b). Data from flight recorded on July 11, 2018. Note GRS = grass; SM = shrub/mulch; CPY = canopy; PL = Figure 3. Boxplot distribution of surface temperature (a); and histogram of surface temperature (b). Data from ﬂight recorded on July 11, 2018. 3.1. Surface Temperature Variability Average temperature, standard deviation and coeﬃcient of variation of nine surface types From 14 recorded ﬂights on Marquette University campus and from one ﬂight recorded on UTEP’s campus. Location Surface Type Temp (◦C) Standard Dev (◦C) Coeﬀ. of Variation MU Grass 34.7 7.9 0.15 Shrub/mulch 40.7 6.2 0.12 Canopy 30.4 7.2 0.16 Parking Lot 38.7 4.2 0.08 Sidewalk 36.3 11.2 0.21 Rooftop—Composite 47.6 6.2 0.1 Rooftop—Rubber 57.4 15.8 0.22 Road 32.5 15.4 0.32 Solar Panels 47.0 7.2 0.13 UTEP Grass 41.6 6.1 0.1 Canopy 46.6 6.3 10 Desert Shrub 46.2 6.7 0.11 Parking Lot (asphalt) 51.7 4.9 0.07 Parking (concrete) 45.3 7.4 0.12 Sidewalk 43.1 8.2 0.13 Rooftop—Composite 47.3 7.2 0.11 Rooftop—Dzong 46.2 6.3 0.1 Road 48.4 2.8 0.04 9 of 18 Remote Sens. 2019, 11, 1722 The distribution of the average temperature, standard deviation and coeﬃcient of variation for the fourteen Milwaukee, WI ﬂights is further illustrated in Figure 4. The shrub/mulch, composite rooftop and solar panels have the most consistent variability among the land use types as shown in the boxplot distribution of their coeﬃcient of variation, while the greatest spread in variation occurred in the road and sidewalk. This may indicate that areas that are not subject to human traﬃc (e.g., shrub/mulch ﬂower beds, rooftops and solar panels) have more consistent variability in their temperatures, while other areas that are subject to intermittent human traﬃc (e.g., roads and sidewalks) have inconsistent temperature variabilities. We also evaluated if the variability in land surface temperature correlated with any meteorological parameters but found no statistically signiﬁcant predictors. Remote Sens. 2019, 11, x FOR PEER REVIEW 10 of 19 Fi 4 B l di ib i f d d d i i d ffi i f i i Grass Shrub/mulch Canopy Parking Lot Sidewalk RT Composite RT Rubber Road Solar 20 40 60 Average Temperature Grass Shrub/mulch Canopy Parking Lot Sidewalk RT Composite RT Rubber Road Solar 0 10 20 30 Standard Deviation Grass Shrub/mulch Canopy Parking Lot Sidewalk RT Composite RT Rubber Road Solar 0.2 0.4 0.6 0.8 Coefficient of Variation Figure 4. Boxplot distribution of average temperature, standard deviation and coeﬃcient of variation from 14 recorded ﬂights in Milwaukee, WI. Figure 4. Boxplot distribution of average temperature, standard deviation and coeﬃcient of variation from 14 recorded ﬂights in Milwaukee, WI. from 14 recorded flights in Milwaukee, WI. 3.1. Surface Temperature Variability We evaluated the variation in surface temperatures throughout the day and found that the highest degree of variation occurred at noon. This is demonstrated in the Figure 5, which shows box plots of the standard deviation for six land use types: grass, canopy, parking lot, sidewalk, composite roof and road for data from both MU and UTEP. As illustrated, all land use types have the greatest standard deviation in temperatures during 12:00 PM, with lower levels of deviation in the morning and late afternoon. This trend suggests that as surfaces heat up, they do so at different rates, which We evaluated the variation in surface temperatures throughout the day and found that the highest degree of variation occurred at noon. This is demonstrated in the Figure 5, which shows box plots of the standard deviation for six land use types: grass, canopy, parking lot, sidewalk, composite roof and road for data from both MU and UTEP. As illustrated, all land use types have the greatest standard deviation in temperatures during 12:00 PM, with lower levels of deviation in the morning and late afternoon. This trend suggests that as surfaces heat up, they do so at diﬀerent rates, which contributes to more variability during mid-day. contributes to more variability 3.2. Impact of the Built Environment 5 10 15 20 Standard Deviation (°C) Road Sidewalk Rooftop Parking Lot Canopy Grass We also evaluated the spatial distribution of surface temperature to locate and identify factors of the built environment that contribute to temperature variability. Figure 5 illustrates the spatial distribution of surface temperatures for a ﬂight on July 8th, 2018. One factor of variability is the reﬂectance and shaded cover from nearby buildings. For example, sidewalks in close proximity to Engineering Hall exhibited higher temperatures, most likely due to the sun’s reﬂectance oﬀits glass paneling. Two similarly sized sidewalk areas were compared and results show the average temperature was 4.7 ◦C hotter for the location closer to the building than the one farther away. In comparison to the sidewalk, parking lot land uses had more consistent variability, perhaps because there were fewer nearby buildings or large trees to exacerbate (glass reﬂectance) or reduce (shaded cover) their temperature. This indicates proximity to nearby buildings or other structures can be a signiﬁcant factor of uncertainty in predicting surface temperatures. 10 of 18 which Remote Sens. 2019, 11, 1722 and late afternoo contributes to m 9 12 15 17 9 12 15 17 9 12 15 17 9 12 15 17 9 12 15 17 9 12 15 17 Time (hr) 0 5 10 15 20 Standard Deviation (°C) Road Sidewalk Rooftop Parking Lot Canopy Grass Figure 5. Standard deviation distributions for six land use types at hour 9, 12, 15 and 17 at both the MU and UTEP locations. Remote Sens. 2019, 11, x FOR PEER REVIEW 11 of 19 Figure 5. Standard deviation distributions for six land use types at hour 9, 12, 15 and 17 at both the MU and UTEP locations. 3.2 Impact of the Built Environment We also evaluated the spatial distribution of surface temperature to locate and identify factors of the built environment that contribute to temperature variability. Figure 5 illustrates the spatial distribution of surface temperatures for a flight on July 8th, 2018. One factor of variability is the reflectance and shaded cover from nearby buildings. For example, sidewalks in close proximity to Engineering Hall exhibited higher temperatures, most likely due to the sun’s reflectance off its glass paneling. Two similarly sized sidewalk areas were compared and results show the average temperature was 4.7 °C hotter for the location closer to the building than the one farther away. contributes to more variability 3.2. Impact of the Built Environment In comparison to the sidewalk, parking lot land uses had more consistent variability, perhaps because Figure 5. Standard deviation distributions for six land use types at hour 9, 12, 15 and 17 at both the MU and UTEP locations. paneling. Two similarly sized sidewalk areas were compared and results show the average temperature was 4.7 °C hotter for the location closer to the building than the one farther away. In comparison to the sidewalk, parking lot land uses had more consistent variability, perhaps because Other sources of land surface temperature uncertainty are traﬃc and parked cars. Traﬃc ﬂow along a roadway intermittently blocks the suns radiation, thereby impacting the surface temperatures of the roadway pavement below. This creates a concentrated pocket of cooler surface temperatures called a heat shadow, which results in variations in surface temperatures across the pavement. This is especially pronounced in pavement lots with parked cars as illustrated in Figure 6b, which shows the distribution of surface temperatures within a parking lot. In this ﬁgure a parked car rooftop, pavement surface and heat shadow recorded temperatures of 69.6 ◦C, 47.8 ◦C and 49.0 ◦C, respectively, all within a space of ~50 m2. there were fewer nearby buildings or large trees to exacerbate (glass reflectance) or reduce (shaded cover) their temperature. This indicates proximity to nearby buildings or other structures can be a significant factor of uncertainty in predicting surface temperatures. Other sources of land surface temperature uncertainty are traffic and parked cars. Traffic flow along a roadway intermittently blocks the suns radiation, thereby impacting the surface temperatures of the roadway pavement below. This creates a concentrated pocket of cooler surface temperatures called a heat shadow, which results in variations in surface temperatures across the pavement. This is especially pronounced in pavement lots with parked cars as illustrated in Figure 6b, which shows the distribution of surface temperatures within a parking lot. In this figure a parked car rooftop, pavement surface and heat shadow recorded temperatures of 69.6 °C, 47.8 °C and 49.0 °C, respectively all within a space of ~ 50 m2 p y p Figure 6. Spatial distribution of temperature from a flight recorded on 11 July 2018 (a) and zoomed in spatial distribution of temperature for the concrete parking lot from a flight recorded on 11 July 2018 (b). 3.3. Surface Properties and Land Surface Temperature 3.3 Surface Properties and Land Surface Temperature Surface Type Albedo NDVI ATI Grass 0.317 0.369 0.198 Shrub/mulch 0.502 0.402 0.183 Canopy 0.378 0.490 0.209 Parking Lot 0.673 0.091 0.121 Sidewalk 0.472 0.144 0.195 Rooftop–Composite 0.580 0.101 0.156 Rooftop–Rubber 0.406 0.096 0.219 Road 0.518 0.117 0.179 Solar 0.333 0.143 0.217 g g ( ) apparent thermal inertia (ATI) values for each surface type. Surface Type Albedo NDVI ATI Grass 0.317 0.369 0.198 Shrub/mulch 0.502 0.402 0.183 Canopy 0.378 0.490 0.209 Parking Lot 0.673 0.091 0.121 Sidewalk 0.472 0.144 0.195 Rooftop – Composite 0.580 0.101 0.156 Rooftop – Rubber 0.406 0.096 0.219 Road 0.518 0.117 0.179 Solar 0.333 0.143 0.217 Figure 7. Spatial distribution of tempearture (a), albedo (b), NDVI (c) and ATI (d) for a flight recorded on 11 August 2018. High: 89 °C Low: -2.8°C (a) (b) (c) (d) High: 0.91 Low: 0 High: 1 Low: -1 High: 0.22 Low: 0.02 Figure 7. Spatial distribution of tempearture (a), albedo (b), NDVI (c) and ATI (d) for a ﬂight recorded on 11 August 2018. Table 6. Average albedo, normalized diﬀerence vegetation index (NDVI) and apparent thermal inertia (ATI) values for each surface type. apparent thermal inertia (ATI) values for each surface type. Surface Type Albedo NDVI ATI Figure 7. Spatial distribution of tempearture (a), albedo (b), NDVI (c) and ATI (d) for a flight recorded on 11 August 2018. High: 89 °C Low: -2.8°C (a) (b) (c) (d) High: 0.91 Low: 0 High: 1 Low: -1 High: 0.22 Low: 0.02 Figure 7. Spatial distribution of tempearture (a), albedo (b), NDVI (c) and ATI (d) for a ﬂight recorded on 11 August 2018. Figure 7. Spatial distribution of tempearture (a), albedo (b), NDVI (c) and ATI (d) for a flight recorded on 11 August 2018. High: 89 °C Low: -2.8°C (a) (b) (c) (d) High: 0.91 Low: 0 High: 1 Low: -1 High: 0.22 Low: 0.02 Figure 7. Spatial distribution of tempearture (a), albedo (b), NDVI (c) and ATI (d) for a ﬂight recorded on 11 August 2018. Figure 7. Spatial distribution of tempearture (a), albedo (b), NDVI (c) and ATI (d) for a flight recorded on 11 August 2018. Figure 7. Spatial distribution of tempearture (a), albedo (b), NDVI (c) and ATI (d) for a ﬂight recorded on 11 August 2018. To further explore this variability and assess its impact on surface temperatures, we plotted these surface properties against land surface temperature. 3.3. Surface Properties and Land Surface Temperature 3.3 Surface Properties and Land Surface Temperature 3.3. Surface Properties and Land Surface Temperature 3.3 Surface Properties and Land Surface Temperature Drone data was applied to derive surface properties including albedo, NDVI and ATI, of the surface types in the case study (Table 6). The light concrete parking lot exhibited the highest albedo (0.673) while grass exhibited the lowest (0.317). The spatial distribution of temperature, albedo, NDVI and ATI at the Milwaukee, WI case study location is shown in Figure 7. As illustrated, these surface material properties have a large degree of variation across the case study area. Drone data was applied to derive surface properties including albedo, NDVI and ATI, of the surface types in the case study (Table 6). The light concrete parking lot exhibited the highest albedo (0.673) while grass exhibited the lowest (0.317). The spatial distribution of temperature, albedo, NDVI and ATI at the Milwaukee, WI case study location is shown in Figure 7. As illustrated, these surface material properties have a large degree of variation across the case study area. T bl 6 A lb d li d diff t ti i d (NDVI) d Table 6. Average albedo, normalized diﬀerence vegetation index (NDVI) and apparent thermal inertia (ATI) values for each surface type. Surface Type Albedo NDVI ATI Grass 0.317 0.369 0.198 Shrub/mulch 0.502 0.402 0.183 Canopy 0.378 0.490 0.209 Parking Lot 0.673 0.091 0.121 Sidewalk 0.472 0.144 0.195 Rooftop–Composite 0.580 0.101 0.156 Rooftop–Rubber 0.406 0.096 0.219 Road 0.518 0.117 0.179 Solar 0.333 0.143 0.217 g , g ( ) apparent thermal inertia (ATI) values for each surface type. Surface Type Albedo NDVI ATI Grass 0.317 0.369 0.198 Shrub/mulch 0.502 0.402 0.183 Canopy 0.378 0.490 0.209 Parking Lot 0.673 0.091 0.121 Sidewalk 0.472 0.144 0.195 Rooftop – Composite 0.580 0.101 0.156 Rooftop – Rubber 0.406 0.096 0.219 Road 0.518 0.117 0.179 Solar 0.333 0.143 0.217 Figure 7. Spatial distribution of tempearture (a), albedo (b), NDVI (c) and ATI (d) for a flight recorded on 11 August 2018. High: 89 °C Low: -2.8°C (a) (b) (c) (d) High: 0.91 Low: 0 High: 1 Low: -1 High: 0.22 Low: 0.02 Figure 7. Spatial distribution of tempearture (a), albedo (b), NDVI (c) and ATI (d) for a ﬂight recorded on 11 August 2018. Table 6. Average albedo, normalized diﬀerence vegetation index (NDVI) and apparent thermal inertia (ATI) values for each surface type. contributes to more variability 3.2. Impact of the Built Environment The hotter surfaces (red) in the right image are parked cars and the cooler surfaces (blue) are heat shadows visible after parked cars leave. (a) (b) High: 70 °C Low: 2.8 °C High: 89 °C Low: -2.8 °C Figure 6. Spatial distribution of temperature from a ﬂight recorded on 11 July 2018 (a) and zoomed in spatial distribution of temperature for the concrete parking lot from a ﬂight recorded on 11 July 2018 (b). The hotter surfaces (red) in the right image are parked cars and the cooler surfaces (blue) are heat shadows visible after parked cars leave. Figure 6. Spatial distribution of temperature from a flight recorded on 11 July 2018 (a) and zoomed in spatial distribution of temperature for the concrete parking lot from a flight recorded on 11 July 2018 (b). The hotter surfaces (red) in the right image are parked cars and the cooler surfaces (blue) are heat shadows visible after parked cars leave. Figure 6. Spatial distribution of temperature from a ﬂight recorded on 11 July 2018 (a) and zoomed in spatial distribution of temperature for the concrete parking lot from a ﬂight recorded on 11 July 2018 (b). The hotter surfaces (red) in the right image are parked cars and the cooler surfaces (blue) are heat shadows visible after parked cars leave. 11 of 18 11 of 18 Remote Sens. 2019, 11, 1722 Remote Sens 2019 11 x FOR PEE 3.4.1 Spatially Averaged Surface Temperature Regression Model 3.4.1. Spatially Averaged Surface Temperature Regression Model Multi-variable linear regression models were developed to predict spatially averaged surface temperature and it was found that air temperature and solar radiation are significant predictors (Figure 9). Standard least squares regression was applied to develop models that predict the surface temperature of six land use types: grass, canopy cover, parking lot (concrete), sidewalk, rooftop (composite) and road. The models had an average R2 of 0.71 with the parking lot having the greatest of (0.89) and the road the lowest (0.37). The parked cars and heat shadows were clipped out as inconsistencies before analysis occurred and therefore the parking lot surface had the most homogenous distribution of temperatures. The grass model had the second greatest R2 (0.84) and had a similarly homogenous distribution. Contrarily, the roadway surface had a much less homogenous distribution of temperatures and thus the road model had a low predictive power and statistical significance. This may be due in large part due to the difficulty of clipping out inconsistencies related to nonstationary objects (e.g. moving cars) combined with their impact on pavement temperatures. Multi-variable linear regression models were developed to predict spatially averaged surface temperature and it was found that air temperature and solar radiation are significant predictors (Figure 9). Standard least squares regression was applied to develop models that predict the surface temperature of six land use types: grass, canopy cover, parking lot (concrete), sidewalk, rooftop (composite) and road. The models had an average R2 of 0.71 with the parking lot having the greatest of (0.89) and the road the lowest (0.37). The parked cars and heat shadows were clipped out as inconsistencies before analysis occurred and therefore the parking lot surface had the most homogenous distribution of temperatures. The grass model had the second greatest R2 (0.84) and had a similarly homogenous distribution. Contrarily, the roadway surface had a much less homogenous distribution of temperatures and thus the road model had a low predictive power and statistical significance. This may be due in large part due to the difficulty of clipping out inconsistencies related to nonstationary objects (e.g., moving cars) combined with their impact on pavement temperatures. The data collected in El Paso, TX was evaluated for inﬂuence and leverage and it was found that it did not have high inﬂuence or leverage in any of the six models. 3.3. Surface Properties and Land Surface Temperature 3.3 Surface Properties and Land Surface Temperature Figure 8 illustrates temperature plotted against its respective albedo for the 611,460 total data points captured by the drone imagery and results show clusters that form for different surface types. Some of these clusters exhibit either a (1) low range in albedo and high range in temperature or (2) high range in albedo and low range in temperature. For example, the road exhibits a low range in albedo and high range in temperature, implying the variability in roadway temperatures are more dependent on meteorological (e.g. exposure to solar radiation) and human (e.g. traffic) variables than physical properties (e.g. albedo). On the other hand, the parking lot has a higher but similar range in albedo, yet it has a much lower variability in temperature. This could be due to the fact that the parking lot has a range of materials from asphalt to concrete coupled with a much lower level of traffic as compared to the roadway, which is more To further explore this variability and assess its impact on surface temperatures, we plotted these surface properties against land surface temperature. Figure 8 illustrates temperature plotted against its respective albedo for the 611,460 total data points captured by the drone imagery and results show clusters that form for diﬀerent surface types. Some of these clusters exhibit either a (1) low range in albedo and high range in temperature or (2) high range in albedo and low range in temperature. For example, the road exhibits a low range in albedo and high range in temperature, implying the variability in roadway temperatures are more dependent on meteorological (e.g., exposure to solar radiation) and human (e.g., traﬃc) variables than physical properties (e.g., albedo). On the other hand, the parking lot has a higher but similar range in albedo, yet it has a much lower variability in temperature. This could be due to the fact that the parking lot has a range of materials from asphalt to concrete coupled with a much lower level of traﬃc as compared to the roadway, which is more homogenous and experiences constant vehicular traﬃc that intercepts land surface exposure to solar radiation. Therefore, this graphic may support the previous statement that there are anthropogenic variables, such as intermittent human foot or vehicular traﬃc, that are signiﬁcant to land surface 12 of 18 olar enic Remote Sens. 2019, 11, 1722 homogenous and radiation. Theref temperature processes. 3 4 Temperature Prediction Mod 3.4. Temperature Prediction Models 3.4. Temperature Prediction Models Drone observations were applied to develop empirical models of land surface temperature. These include (1) a regression model to predict spatially averaged surface temperatures at 12:00 PM based upon environmental variables and (2) a diurnal model to predict surface temperatures throughout a given day. Drone observations were applied to develop empirical models of land surface temperature. These include (1) a regression model to predict spatially averaged surface temperatures at 12:00 PM based upon environmental variables and (2) a diurnal model to predict surface temperatures throughout a given day. 3.3. Surface Properties and Land Surface Temperature 3.3 Surface Properties and Land Surface Temperature Overall these results suggest that patterns in the physical properties of urban materials may provide insight into surface temperature variability. temperature processes. Overall these results suggest that patterns in the physical properties of urban materials may provide insight into surface temperature variability. Figure 8. Surface temperature data plotted against albedo from a flight recorded on 11 August 2018. 30 40 50 60 70 80 Temperature (°C) 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 Albedo Canopy Cover Shrub/mulch Parking Lot Sidewalk Road RT Composite RT Rubber Grass Figure 8. Surface temperature data plotted against albedo from a ﬂight recorded on 11 August 2018. 4 T P di i M d l Figure 8. Surface temperature data plotted against albedo from a flight recorded on 11 August 2018. Figure 8. Surface temperature data plotted against albedo from a ﬂight recorded on 11 August 2018. 3.4.1 Spatially Averaged Surface Temperature Regression Model 3.4.1. Spatially Averaged Surface Temperature Regression Model To evaluate inﬂuence we used Cook’s D and found that the El Paso data points all fell below the threshold of 2.4 (max 0.19) to be considered high-inﬂuence points [38]. In addition, we used the hat matrix to evaluate leverage and found that no El Paso data points exhibited high leverage in the model. The agreeability of the data across the two case study areas indicates that the ﬁndings in this study may have generalizability beyond the case study locations. Remote Sens. 2019, 11, 1722 beyond the case study 13 of 18 13 of 18 Figure 9. Temperature prediction models of six surface types: grass (a), canopy cover (b), parking lot (c), sidewalk (d), composite rooftop (e) and road (f). UTEP datapoint is fitted in green. Note the 95% confidence intervals are in blue Recorded Temperature (°C) Recorded Temperature (°C) Recorded Temperature (°C) Recorded Temperature (°C) Recorded Temperature (°C) Recorded Temperature (°C) 1.85+0.62AT+8.05SR R2=0.37 p-value=0.06 20.6+1.13AT+0.67SR R2=0.78 p-value=0.0001 14.87+0.61AT+11.53SR R2=0.70 p-value=0.0007 8.81+1.17AT+4.31SR R2=0.89 p-value<0.0001 11.71+0.40AT+15.85SR R2=0.68 p-value=0.0011 17.73+0.64AT+8.29SR R2=0.84 p-value<0.0001 (a) (b) (c) (d) (e) (f) Figure 9. Temperature prediction models of six surface types: grass (a), canopy cover (b), parking lot (c), sidewalk (d), composite rooftop (e) and road (f). UTEP datapoint is ﬁtted in green. Note the 95% conﬁdence intervals are in blue. Recorded Temperature (°C) 17.73+0.64AT+8.29SR R2=0.84 p-value<0.0001 (a) Recorded Temperature (°C) 11.71+0.40AT+15.85SR R2=0.68 p-value=0.0011 (b) Recorded Temperature (°C) 14.87+0.61AT+11.53SR R2=0.70 p-value=0.0007 (d) Recorded Temperature (°C) 8.81+1.17AT+4.31SR R2=0.89 p-value<0.0001 (c) Recorded Temperature (°C) 20.6+1.13AT+0.67SR R2=0.78 p-value=0.0001 (e) Recorded Temperature (°C) 1.85+0.62AT+8.05SR R2=0.37 p-value=0.06 (f) Figure 9. Temperature prediction models of six surface types: grass (a), canopy cover (b), parking lot (c), sidewalk (d), composite rooftop (e) and road (f). UTEP datapoint is fitted in green. Note the 95% Figure 9. Temperature prediction models of six surface types: grass (a), canopy cover (b), parking lot (c), sidewalk (d), composite rooftop (e) and road (f). UTEP datapoint is ﬁtted in green. Note the 95% conﬁdence intervals are in blue. confidence intervals are in blue. 3.4.2. Diurnal Prediction Model 3.4.2 Diurnal Prediction Model Finally, models were developed to predict land surface temperature throughout the day based upon the air temperature and solar radiation (Equations 7-9). The diurnal data was fit with a Gaussian peak distribution and it was found that the parking lot and composite rooftop had the best model fit with an R2 of 0.83 and 0.78, respectively, while all other models had an R2 value of 0.53 or below Finally, models were developed to predict land surface temperature throughout the day based upon the air temperature and solar radiation (Equations (7)–(9)). The diurnal data was ﬁt with a Gaussian peak distribution and it was found that the parking lot and composite rooftop had the best model ﬁt with an R2 of 0.83 and 0.78, respectively, while all other models had an R2 value of 0.53 or below (Figure 10). While this approach is constrained by a limited number of data points from four ﬂights and only four numerical x-axis variables, there are a few insights we can gain from these results. The ﬁrst is that these models conﬁrm what was found in the previous regression models: it is much easier to predict the land surface temperature of homogenous materials, such as pavements and rooftops, than it is to predict land surfaces that have a greater distribution in texture and material, such as canopy. The second is that anthropogenic variables, such as pedestrians and vehicular traﬃc that are diﬃcult to quantify, may inﬂuence the ability to predict surface temperatures based upon meteorological variables. This was shown by the lower model ﬁt in the high-traﬃc roadways and sidewalks as compared to the low-traﬃc parking lot. Remote Sens. 2019, 11, 1722 sidewalks as compared 14 of 18 14 of 18 Figure 10. Gaussian peak models of six surface types: grass (a), canopy cover (b), parking lot (c), sidewalk (d), composite rooftop (e) and road (f). confidence intervals are in blue. 3.4.2. Diurnal Prediction Model Note that GRS = grass; CPY = canopy; PL = parking lot; SW = sidewalk; RTC = composite rooftop; AT = air temperature; SR = solar radiation; t = time 8 10 12 14 16 18 Time (hr) 0 5 10 15 (GRS-AT)SR Grass 8 10 12 14 16 18 Time (hr) 0 5 10 15 20 (CPY-AT)SR Canopy Cover 8 10 12 14 16 18 Time (hr) 5 10 15 20 (PL-AT)SR Parking Lot 8 10 12 14 16 18 Time (hr) 0 5 10 15 (SW-AT)SR Sidewalk 8 10 12 14 16 18 Time (hr) 0 5 10 15 20 25 (RTC-AT)SR RT Composite 8 10 12 14 16 18 Time (hr) -20 -10 0 10 20 (RD-AT)SR Road 10.20𝑒ି଴.ହ(௧ିଵଶ.ଽ଻) ଶ.଼଺ ଶ R2 = 0.50 7.69𝑒ି଴.ହ(௧ିଵଶ.ହଶ) ଶ.଺ହ ଶ R2 = 0.33 16.26𝑒ି଴.ହ(௧ିଵଷ.଼ଽ) ଷ.ସହ ଶ R2 = 0.83 13.18𝑒ି଴.ହ(௧ିଵସ.଴ଵ) ଷ.ଵ଻ ଶ R2 = 0.53 17.12𝑒ି଴.ହ(௧ିଵସ.଼଴) ଶ.ସ଴ ଶ R2 = 0.41 20.61𝑒ି଴.ହ(௧ିଵଷ.଻଼) ଷ.ଶ଴ ଶ R2 = 0.78 Figure 10. Gaussian peak models of six surface types: grass (a), canopy cover (b), parking lot (c), sidewalk (d), composite rooftop (e) and road (f). Note that GRS = grass; CPY = canopy; PL = parking lot; SW = sidewalk; RTC = composite rooftop; AT = air temperature; SR = solar radiation; t = time. 8 10 12 14 16 18 Time (hr) 0 5 10 15 (GRS-AT)SR Grass 10.20𝑒ି଴.ହ(௧ିଵଶ.ଽ଻) ଶ.଼଺ ଶ R2 = 0.50 8 10 12 14 16 18 Time (hr) 0 5 10 15 20 (CPY-AT)SR Canopy Cover 7.69𝑒ି଴.ହ(௧ିଵଶ.ହଶ) ଶ.଺ହ ଶ R2 = 0.33 Canopy Cover Time (hr) 8 10 12 14 16 18 Time (hr) 0 5 10 15 (SW-AT)SR Sidewalk 13.18𝑒ି଴.ହ(௧ିଵସ.଴ଵ) ଷ.ଵ଻ ଶ R2 = 0.53 8 10 12 14 16 18 Time (hr) 5 10 15 20 (PL-AT)SR Parking Lot 16.26𝑒ି଴.ହ(௧ିଵଷ.଼ଽ) ଷ.ସହ ଶ R2 = 0.83 Parking Lot Sidewalk Sidewalk 8 10 12 14 16 18 Time (hr) 0 5 10 15 20 25 (RTC-AT)SR RT Composite 20.61𝑒ି଴.ହ(௧ିଵଷ.଻଼) ଷ.ଶ଴ ଶ R2 = 0.78 8 10 12 14 16 18 Time (hr) -20 -10 0 10 20 (RD-AT)SR Road 17.12𝑒ି଴.ହ(௧ିଵସ.଼଴) ଶ.ସ଴ ଶ R2 = 0.41 Figure 10. Gaussian peak models of six surface types: grass (a), canopy cover (b), parking lot (c), sidewalk (d), composite rooftop (e) and road (f). Note that GRS = grass; CPY = canopy; PL = parking Figure 10. confidence intervals are in blue. 3.4.2. Diurnal Prediction Model Gaussian peak models of six surface types: grass (a), canopy cover (b), parking lot (c), sidewalk (d), composite rooftop (e) and road (f). Note that GRS = grass; CPY = canopy; PL = parking lot; SW = sidewalk; RTC = composite rooftop; AT = air temperature; SR = solar radiation; t = time. lot; SW = sid 4. Discussion 4. Discussion We have presented a case study that applied high resolution drone measurements (13 cm) to evaluate urban surface temperatures and results indicate that there is a wide variability in surface temperature behavior across urban land use types. Some of the uncertainty in land surface temperature variability may be attributable to human movement patterns, land surface properties or urban geometry. Results indicate that mean land surface temperatures can be predicted based upon solar radiation and air temperature. By elucidating some of the factors that inﬂuence land surface temperature variability, we hope to contribute to the growing body of knowledge centered around land surface temperature in the urban environment. To this end, our ﬁndings suggest that when parameterizing models, it is important to understand the unique relationship between surface material properties, urban geometry, weather and human movement. For example, the results indicate that pedestrian or vehicular traﬃc may have an impact on land surface temperature variability across sidewalks, parking lots and streets. Depending on the volume of cars, either parked or moving, this can greatly impact the temperature proﬁle of paved Remote Sens. 2019, 11, 1722 15 of 18 15 of 18 surfaces. Parked cars can create heat shadows which cool the surface below and our study demonstrates that when a car moves it can reveal temperatures as low as 8.3 ◦C cooler than the exposed surface. In addition, results have identiﬁed several factors of urban geometry that aﬀect land surface temperatures. Urban factors such as building reﬂectivity and surface altitude can impact solar radiation, which then inﬂuences surface temperatures in locations impacted by these eﬀects. For example, sidewalks often lie near buildings and depending on a buildings reﬂectance or shadows this can make sidewalk temperatures more vulnerable to temperature ﬂuctuations. In this study, sidewalk temperatures impacted by glass reﬂectance were on average 4.7 ◦C hotter that sidewalks not impacted by reﬂectance. Therefore, knowledge of the spatial distribution of urban geometry is important for predicting and evaluating land surface temperatures in the built environment. While addressing urban geometry or pedestrian and vehicular traﬃc within our prediction models is outside of the scope of this project, future work should evaluate how to incorporate these important parameters into land surface temperature predictions. lot; SW = sid 4. Discussion Results indicate air temperature and solar radiation are signiﬁcant predictors of mean land surface temperature in both of our models and it was found this relationship holds true in both Milwaukee, WI and El Paso, TX. Because the model holds true across two diﬀerent climatic regions, the models developed in this project may be generalizable beyond their case study regions. In addition, these models can also be easily applied as air temperature and solar radiation are commonly measured across the world. The generalizability of these ﬁndings also has important implications for engineering applications that use predictions of land surface temperatures. Urban land surface temperatures are often used by public health oﬃcials to mitigate the impact of the urban heat island eﬀect on human health [2], in developing binders and mixers of pavement in roadway designs [41] or to estimate the impact of land surface temperatures on receiving stream temperatures [42–44]. This study also demonstrates several advantages and disadvantages of using drones as compared to satellite or in-situ imagery. The case studies we evaluated were restricted to the size of a city block around 46,000 m2 and even though battery life would have allowed us to collect an area ten times this size, we were restricted by United States Federal Aviation Administration UAV pilot rules that restrict the ﬂight of UAVs to within line of sight of the pilot. In an urban environment with tall buildings the line of sight may be the primary constraint on coverage area. Therefore, a disadvantage of UAVs is that ﬂight time and legal restrictions may constrain the ﬂight areas to small portions of a city. However, this could be overcome with ﬁxed-wing drones that are able cover a greater area, in addition to relaxed regulations that allow ﬂights beyond the line of sight [45]. Despite the restriction on the spatial extent of the study area, advantages of UAVs over satellites or in-situ methods are their ability to collect distributed temperature data at spatial resolutions (13 cm) that reﬂect small scale changes in the urban environment. In addition, satellite data is restricted to daily to weekly observations while drones can be ﬂown on-demand, which allows them to capture temperature changes throughout the day. Overall, this study highlights the utility of using drone observations to capture the variability of urban land surface temperatures at small spatial scales. lot; SW = sid 4. Discussion Urban environments are spatially complex, making it diﬃcult to capture the spatial distribution of observable phenomena outside of high-resolution remote sensing techniques. Our ﬁndings suggest that drones could also be good tools for evaluating the variability of other parameters of the urban environment that are important for environmental studies such as soil moisture, leaf area index or impervious cover. Therefore, it is important for studies such as this one that evaluate the spatial complexities of the urban environment in order to improve the methods that we use to model and understand urban systems. 5. Conclusions • Data were consistent in the models between Milwaukee, WI and El Paso, TX, suggesting that the ﬁndings in this study may be generalizable beyond the case study locations. Overall, our findings suggest that land surface temperature variability in the urban environment can come from several sources including surface material properties, urban geometry, weather and pedestrian and vehicular traffic. This has direct implications for land surface temperature models that are used for urban environmental studies. As climate change and urbanization continue to exacerbate the SUHI, studies such as this are important for gaining a better understanding of the complexities of land surface temperatures. Ultimately this improved understanding will help to develop better methods and procedures to mitigate the impact of land surface temperatures on human and environmental health. Author Contributions: J.N. provided investigation, data collection, data analysis, and writing of the original draft. W.M. contributed conceptualization, supervision, data collection and draft editing. Funding: This project was funded by the Marquette OPUS College of Engineering Earl B. and Charlotte Nelson Award. Funding: This project was funded by the Marquette OPUS College of Engineering Earl B. and Charlotte Nelson Award. Acknowledgments: The authors would like to acknowledge and sincerely thank Saurav Kumar and Wissam Atwah at the University of Texas El Paso for their help in collecting data in El Paso, Tx. Acknowledgments: The authors would like to acknowledge and sincerely thank Saurav Kumar and Wissam Atwah at the University of Texas El Paso for their help in collecting data in El Paso, Tx. Conﬂicts of Interest: The authors declare no conﬂict of interest. 5. Beitinger, T.L.; Bennett, W.A.; McCauley, R.W. Temperature Tolerances of North American Freshwater Fishes Exposed to Dynamic Changes in Temperature. Environ. Biol. Fishes 2000, 58, 237–275. [CrossRef] 5. Conclusions The main objectives of this work were to apply drone imagery to capture land surface temperature variability and develop models to predict mean land surface temperatures. This was done through the application of high-resolution thermal imagery as a parameterizing tool for model development. The results revealed that land surface temperature variability is extensive and inﬂuenced by numerous Remote Sens. 2019, 11, 1722 16 of 18 variables related to urban environments and that air temperature and solar radiation are signiﬁcant predictors of mean land surface temperature. Conclusions from this study hold true in both Milwaukee, WI and El Paso, TX, indicating they could be generalizable to regions beyond these two case study locations. y The key ﬁndings from this study were: The key ﬁndings from this study were: • Land surface variability was signiﬁcant and ranged between (3.9–15.8 ◦C) for common land use types. • Areas that experienced pedestrian or vehicular traﬃc exhibited higher variabilities than comparable surfaces that did not. In Milwaukee, the high-traﬃc road had a coeﬃcient of variation of 0.32 as compared to 0.08 for the low-traﬃc parking lot. This indicates that human traﬃc may impact land surface temperatures due to the heat-shadow eﬀect. • Urban geometry has an inﬂuence on land surface temperatures; shadows and reﬂectance from buildings showed a signiﬁcant inﬂuence on the temperatures of nearby land surfaces throughout the day. Sidewalk temperatures impacted by glass reﬂectance were on average 4.7 ◦C hotter that sidewalks not impacted by reﬂectance. • Land surface temperature variability is low in the morning, peaks at noon and goes back down in the evening. This may indicate that as surfaces heat up, they do so at diﬀerent rates, which contributes to more variability during mid-day. • Land surface temperature variability is low in the morning, peaks at noon and goes back down in the evening. 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https://openalex.org/W2926665458	https://ctajournal.biomedcentral.com/track/pdf/10.1186/s13601-018-0239-2	English	null	Genetic associations of the response to inhaled corticosteroids in asthma: a systematic review	Clinical and translational allergy	2,019	cc-by	14,223	© The Author(s) 2019. 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 There is wide variability in the response to inhaled corticosteroids (ICS) in asthma. While some of this heterogeneity of response is due to adherence and environmental causes, genetic variation also influences response to treatment and genetic markers may help guide treatment. Over the past years, researchers have investigated the relationship between a large number of genetic variations and response to ICS by performing pharmacogenomic studies. In this systematic review we will provide a summary of recent pharmacogenomic studies on ICS and discuss the latest insight into the potential functional role of identified genetic variants. To date, seven genome wide association stud‑ ies (GWAS) examining ICS response have been published. There is little overlap between identified variants and meth‑ odologies vary largely. However, in vitro and/or in silico analyses provide additional evidence that genes discovered in these GWAS (e.g. GLCCI1, FBXL7, T gene, ALLC, CMTR1) might play a direct or indirect role in asthma/treatment response pathways. Furthermore, more than 30 candidate-gene studies have been performed, mainly attempting to replicate variants discovered in GWAS or candidate genes likely involved in the corticosteroid drug pathway. Single nucleotide polymorphisms located in GLCCI1, NR3C1 and the 17q21 locus were positively replicated in independent populations. Although none of the genetic markers has currently reached clinical practise, these studies might provide novel insights in the complex pathways underlying corticosteroids response in asthmatic patients. Keywords: Inhaled corticosteroids, Asthma, Genetics, Genomics, GWAS noxious environmental exposures, misdiagnosis, and truly steroid refractory disease, genetic variation might also be an important factor influencing treatment response in patients with asthma [4, 6–8]. Approximately 70% of the variance in ICS response is suggested to be due to genetic factors [4, 9]. Genetic associations of the response to inhaled corticosteroids in asthma: a systematic review Ozlem Keskin1, Niloufar Farzan2, Esra Birben3, Hayriye Akel4, Cagatay Karaaslan4, Anke H. Maitland‑van der Zee2,5, Michael E. Wechsler6, Susanne J. Vijverberg2 and Omer Kalayci3* Clinical and Translational Allergy Clinical and Translational Allergy Keskin et al. Clin Transl Allergy (2019) 9:2 https://doi.org/10.1186/s13601-018-0239-2 Open Access Introductionf Affecting up to 18% of the world’s population, asthma is a chronic airway disease and inhaled corticosteroids (ICS) are the preferred first-line treatment for persis- tent asthma [1]. However, there is a wide variability in response to ICS [2–4] such that up to 35–40% of the patients receiving ICS for 8–12 weeks do not show signif- icant improvement in lung function [2, 3]. Furthermore, 10% of the asthmatic patients on maintenance ICS treat- ment may remain symptomatic or at high risk of asthma attacks in spite of the regular use of this medication [5]. In addition to poor adherence to medication, continuing Over the past three decades, researchers have investi- gated the relationship between a large number of genetic variations and response to ICS by performing pharma- cogenomic studies. The main aim of these studies was to identify genetic markers that could help physicians opti- mize asthma treatment. The high number of publications has enabled systematic literature reviews that summarize the findings of the studies and evaluate the potential clin- ical relevance of the findings. One of these systematic lit- erature reviews was published by Farzan et al. [10]. This © The Author(s) 2019. 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. the findings of the studies and evaluate the potential clin ical relevance of the findings. One of these systematic lit erature reviews was published by Farzan et al. [10]. Thi Correspondence: okalayci63@gmail.com 3 Pediatric Allergy and Asthma Unit, Hacettepe University School of Medicine, 06100 Ankara, Turkey Full list of author information is available at the end of the article Correspondence: okalayci63@gmail.com 3 Pediatric Allergy and Asthma Unit, Hacettepe University School of Medicine, 06100 Ankara, Turkey Full list of author information is available at the end of the article *Correspondence: okalayci63@gmail.com 3 Pediatric Allergy and Asthma Unit, Hacettepe University School of Medicine, 06100 Ankara, Turkey Full list of author information is available at the end of the article Keskin et al. Clin Transl Allergy (2019) 9:2 Keskin et al. GWAS (Genome‑wide association studies) GWAS (Genome‑wide association studies) GWAS have provided the opportunity to detect novel pharmacogenetic variants related to ICS response by scanning a high number of genetic variants across the entire genome. In the systematic review by Farzan et al. [10], the results of the four GWAS that had been con- ducted prior to 2015 have been described in detail. These GWAS had identified four new loci to be associated with ICS response. These loci harbored the GLCCI1 (gluco- corticoid-induced transcript 1 protein), T, FBXL7 (F-box and leucine-rich repeat protein 7) and ALLC (Allantoi- case) genes [12–15]. SNPs within GLCCI1, T gene, and ALLC were associated with changes in the lung function and rs10044254 SNP within FBXL7 was associated with changes in the asthma symptom scores. From the iden- tified SNPs within these genes, rs10044254 was the only SNP that reached the genome-widesignificance thresh- old. SNPs within the GLCCI1, T gene, and FBXL7, were associated with ICS response in pediatric asthma popu- lations and were conducted by the same research group [12–14]. These studies included Caucasians from the Single-Nucleotide Polymorphism Health Association- Asthma Resource Project (SHARP). Although the three GWAS all studied the SHARP population, the methods of the GWAS differed quite a lot with regard to sample size, study design, outcome measurements, and genotyp- ing platforms. Introductionf Clin Transl Allergy (2019) 9:2 Page 2 of 25 study reviewed pharmacogenomics studies of ICS and leukotriene modifiers that were published between 1999 and 2015. Furthermore, SNPs that were positively rep- licated at least once in an independent population were discussed in detail. Despite the large number of pharma- cogenomics studies, there is still no genetic marker used in clinical practice to optimize asthma treatment with ICS. However, due to the rapid decrease in the costs of genotyping and emergence of advanced genotyping tech- nologies, efforts are still ongoing to replicate previously identified markers and/or identify new genetic mark- ers by performing Genome-wide association studies (GWAS). One of the important issues of single nucleo- tide polymorphisms (SNPs) that are identified by GWAS is that their function and relation to the disease/trait of interest is often not clear and therefore might inhibit fur- ther progression towards clinical implementation. This has encouraged researchers to perform in vitro and/or in silico studies to unravel the potential role of the SNPs/ genes in the asthma-treatment response pathway. Since the field is rapidly evolving, this follow-up systematic review aims to (1) provide a summary of the pharma- cogenomics studies on ICS published after 2015 and (2) provide the latest insight into the potential functional role of the identified genes/variants identified in GWAS, in order to provide an overview of the latest promising clinical markers. This systematic review is reported according to PRISMA guidance [11]. This systematic review is reported according to PRISMA guidance [11]. Novel pharmacogenomics studies From 2015 to 2018, nine additional studies were iden- tified through PubMed. Of these studies, three were GWAS and six were candidate gene studies. Methods Clin Transl Allergy Asthma and/or Lung funcon Corcosteroids Genecs (n=1106) Asthma and/or Lung funcon Corcosteroids Pharmacogenecs (n=93) Asthma and/or Lung funcon Corcosteroids Pharmacogenomics (n=122) Asthma and/or Lung funcon Corcosteroids Genecs (n=126) Asthma and/or Lung funcon Corcosteroids Pharmacogenecs (n=66) Asthma and/or Lung function Corcosteroids Pharmacogenomics (n=77) Aer general key word based search, arcles was selected according to criteria Selected arcles for review (n=46) Aer reading the full-text, duplicated and unrelated arcles were excluded and ﬁnal list was formed Fig. 1 Flowchart for the inclusion of ICS pharmacogenomic studies Asthma and/or Lung funcon Corcosteroids Genecs (n=1106) Asthma and/or Lung funcon Corcosteroids Pharmacogenecs (n=93) Asthma and/or Lung funcon Corcosteroids Pharmacogenomics (n=122) Aer general key word based search, arcles was selected according to criteria Asthma and/or Lung funcon Corcosteroids Genecs (n=1106) Asthma and/or Lung funcon Corcosteroids Pharmacogenecs (n=93) Asthma and/or Lung funcon Corcosteroids Pharmacogenomics (n=122) Aer general key word based search, arcles was selected according to criteria Asthma and/or Lung funcon Corcosteroids Genecs (n=126) Asthma and/or Lung funcon Corcosteroids Pharmacogenecs (n=66) Asthma and/or Lung function Corcosteroids Pharmacogenomics (n=77) Selected arcles for review (n=46) Aer reading the full-text, duplicated and unrelated arcles were excluded and ﬁnal list was formed Fig. 1 Flowchart for the inclusion of ICS pharmacogenomic studies Asthma and/or Lung funcon Corcosteroids Genecs (n=126) Asthma and/or Lung function Corcosteroids Pharmacogenomics (n=77) Asthma and/or Lung funcon Corcosteroids Pharmacogenecs (n=66) Aer reading the full-text, duplicated and unrelated arcles were excluded and ﬁnal list was formed Selected arcles for review (n=46) Fig. 1 Flowchart for the inclusion of ICS pharmacogenomic studies the GWAS data with a series of dose-dependent pharma- cological phenotypic data and detected associations of genome-wide significance between dose-dependent pul- monary function response to ICS, and five loci: rs6924808 on chromosome 6 (p = 5.315 × 10−7), rs10481450 on chromosome 8 (p = 2.614 × 10−8), rs1353649 onchromo- some 11 (p = 3.924 × 10−9), rs12438740 on chromosome 15 (p = 4.499 × 10−8), and rs2230155 onchromosome 15 (p = 1.798 × 10−7). These loci are mapped to candidate genes related to cellular functions [20, 21]. Asthmat- ics who were homozygous for the mutant alleles had 30–300% higher % Forced expiratory volume in one sec- ond (FEV1) values at an intermediate dose of glucocor- ticoids compared to the homozygotes for the wild-type alleles and heterozygotes for all of the associated SNPs (with the exception of rs6924808). Methods Articles published from 1995 January through the end of August 2018 were searched in PubMed using three key- words. Conference abstracts, articles assessing adverse drug effects and articles that had not been published in English were excluded. First, we eliminated articles from the titles, and then by abstracts. Then the remaining arti- cles were read in full. We also screened review articles for possible missed publications. Between 2015 and 2018, three additional GWAS were published [16–18], of which one was conducted by the SHARP group. Changes in lung function were consid- ered as the primary outcome in two of these GWAS and asthma exacerbations were considered the primary out- come in one study. None of the new GWAS could iden- tify the previously found genes/SNPs in candidate gene or GWAS. A search using the keywords “Asthma and/or Lung function, Corticosteroids, Genetics”yielded 1106 studies; “Asthma and/or Lung function, Corticosteroids, Phar- macogenetics” yielded 93 studies; “Asthma and/or Lung function Corticosteroids Pharmacogenomics” yielded 122 studies. Of these, 46 met the inclusion criteria with 38 being candidate gene studies and 7 being GWAS and 1 being exome sequencing study (Fig. 1). Nine articles remained after excluding the studies before 2015 June. In the GWAS by Wang et al. [16], 120 mild-to-moder- ate adult asthmatics were included. In this study, patients received different doses of glucocorticoids (125, 250, 500, 1000 mcg) and the dosage of ICS was increased each week. The hypothesis underlying this approach was that integration of pharmacodynamic properties of corticos- teroids into pharmacogenetic GWASs containing drug responses to different doses may increase the statistical power of significant association detection [19]. For the first time, a mechanistic model was applied to analyse From all the publications selected, the following data were extracted: ICS type and duration of treatment, genes and SNPs, outcome, study design, ethnic backgrounds of patients, sample size, age range and duration of the study. From GWAS, information regarding the in vitro or in sil- ico analysis after the association analysis was extracted. Page 3 of 25 Keskin et al. Clin Transl Allergy (2019) 9:2 Keskin et al. Evidence of functional activity of the previously identified genes and SNPs in GWAS GLCCI1 GLCCI1is a protein-coding gene located within the chromosome 7p21.3. The role of GLCCI1 as one of the target genes of glucocorticoids was described by Chap- man et al. more than two decades ago. They showed an increased expression of this gene in two different cell lines upon the administration of dexamethasone [22]. Glucocorticoids can induce apoptosis in several immune-inflammatory cells such as eosinophils and lymphocytes. Therefore, researchers concluded that reduced expression of GLCCI1 because of rs37973 could result in reduced apoptosis of these cells. Consequently, reduced apoptosis of these cells could decrease ICS effi- cacy. To validate the functional role of the gene and its SNP in asthma control, an in vitro study was performed using lymphoblastoid B cells derived from children in the Childhood Asthma Management Program (CAMP) study Methods Some of the SNPs were sensitive to a small change in low drug doses and some showed variation after larger dose changes. Significant associations were also demonstrated in three additional independent replication populations. Two SNPs, chr6 rs6924808 and chr11 rs1353649, displayed an increased significance level (p = 6.661 × 10−16 and 5.670 × 10−11) in Keskin et al. Clin Transl Allergy (2019) 9:2 Page 4 of 25 the pooled analysis of first GWAS results with these three replication trials. This GWAS underlined the importance of the optimal ICS dose for individualized asthma treat- ment based on a patient’s genetic makeup.hi [12]. This study showed that dexamethasone significantly increased GLCCI1 expression. Specifically, in patients homozygous for the mutant allele (G), the expression was significantly lower compared to homozygotes for the wild-type allele (A) in the presence of dexamethasone. Increased expression of the GLCCI1 gene was associated with a better response to ICS. Furthermore, Hu et al. [23] in their mRNA analysis showed an increased expression in GLCCI1 after ICS. Taken together, GG carriers appear to be less sensitive to corticosteroids at the cellular level. The first GWAS that studied asthma exacerbations as a primary outcome was performed by Dahlin et al. in 2015. In this study, 806 asthmatic children with Cauca- sian ethnicity were included from two population-based biobanks [17]. The results of the GWASs performed in these two populations were meta-analysed. However, none of the SNPs from the meta-analysis met the thresh- old for genome-wide significance. The most significant result was reported for 6 SNPs (rs2395672 and rs279728, rs4271056, rs6467778, rs2691529, and rs9303988) within three different genes CMTR1 (Cap methyltransferase 1), TRIM24 (Tripartite Motif Containing 24) and MAGI2 (Membrane Associated Guanylate Kinase). Among these, the rs2395672 SNP within the CMTR1 gene had the smallest p value from the meta-analysis [joint Odds Ratio (OR) = 1.07, 95% CI 1.03–1.11; p = 2.3 × 10−6].h FBXL7 FBXL7, located in chromosome 5, is a member of Skp1- Cul1-F-box complex which is involved in ubiquitylation and degradation of proteins in the cells [24, 25]. In-vitro studies show that FBXL7 induces cell apoptosis and its cellular abundance is regulated by FBX18, another SCF protein [26]. Therefore, increased FBXL7 expression can induce cell and tissue injury. In fact, studies on lung epi- thelial cells show that overexpression of FBXL7 results in mitochondrial damage [27]. FBXL7 mediates this func- tion through a decrease in the amount another protein (survivin) involved in cell survival and apoptosis. To date, more than 50 F-box proteins have been identified and these proteins appear to play major roles in inflammation and immunity by interacting with each other [28, 29]. A study in murine lung epithelial cells showed that F-box family members interact with proteins that can induce cytokine release in immune cells [26]. In the GWAS of asthma symptoms by Park et al. [14], the variant allele of the rs10044254 SNP was associated with poor symp- tom control. A further in vitro analysis using immor- talized B cells obtained from the CAMP participants demonstrated that the variant allele was associated with a decrease in FBXL7 expression in response to dexameth- asone. Although not statistically significant, increased dexamethasone-induced expression of FBXL7 was asso- ciated with poor asthma symptom scores. Despite these promising findings, no further studies have tried to eluci- date the role of these proteins in asthma control. The latest and so far largest GWAS of ICS response has been published by Mosteller et al. [18]. In this study, 2672 patients (12 years and older) were included from seven randomized, double-blind, placebo-controlled, multi- center clinical studies that were performed in a total of 26 countries. Changes in FEV1 at week 8 and 12 follow- ing fluticasone furoate (FF) or fluticasone propionate (FP) treatment were considered as the study outcome. The analysis was performed with more than 9.8 mil- lion genetic variants (minor allele frequency ≥ 1%), and none of the SNPs reached the genome-wide significance threshold. An overview of published GWAS is provided in Table 1. T gene T gene, located within the chromosome 6, is a member of the genes containing the T locus as a common pro- tein motif [30]. The product of the T locus seems to be involved in the development of all vertebrate organisms. Furthermore, T gene expression has been previously shown in healthy adult lung tissue. In the GWAS study by Tantisira et al., researchers performed a pathway anal- ysis in order to unravel the potential influence of the T gene on response to corticosteroids. To that end, using the program GeneMANIA (http://genemania.org/), they focused on the T gene–NR3C1 (Nuclear Receptor Page 5 of 25 Keskin et al. Clin Transl Allergy (2019) 9:2 Keskin et al. Clin Transl Allergy Table 1 GWAS analysis of response to ICS Referernces Genotyping platform and number of SNPs after pruning Discovery phase population Study design and duration Number of asthmatic patients Medication in the discovery phase Replication population Definition of response SNPs chosen for replication (gene, chromosome position) Study outcome Tantisira et al. [12] Human Hap 550v3 Bead Chip (Illumina) 534 290 SNPs Caucasian chil‑ dren (CAMP) Clinical trial, 16 months 118 child parent trios Budesonide 200 μg twice daily Caucasian patients (n = 935) Children: (CARE trial, n = 101, fluticasone propionate 100 μg twice daily) Adults: (Adult study, n = 385, 1000 μg daily (increased up to 2000 μg if necessary)) (LOCCS, n = 185, fluticasone 100 μg twice daily) (SOCS/SLIC, n = 264, triamcinolone 400 μg twice daily) Changes in FEV1 from baseline rs37972 (GLCCI1,7p21.3) In two replication populations patients homozy‑ gous forthe wild-type allele (C) hadapproximately 12% improve‑ ments in FEV1% compared with the 4% increasein TT carriers after 4–8 weeks of treatment with ICS (combined p = 7×10−4) Tantisira et al. T gene [13] Affymetrix (Santa Clara, CA) using a Human SNP array 6.0 444 088 SNPs Caucasians children: (CAMP and CARE trials) (n = 239) Adults: (ACRN trial) (n = 179) Clinical trial, 6–8 weeks 418 CAMP: bude‑ sonide 200 μg twice daily CARE: fluticasone propionate 100 μg twice daily ACRN: Triamci‑ nolone 400 μg twice daily Adults (n = 407) Flunisolide 1000 μg once daily (increased to 2000 μg if neces‑ sary) Changes in FEV1% pred from baseline rs3099266, rs1134481 and rs2305089(T gene, 6q27) Patients homozy‑ gous for the wild-type allele of all three SNPs had a two to threefold increase in FEV1%pred compared to homozygotes for the mutant allele Combined p values of study populations for the rs1134481, rs2305089 and rs3099266 were 1.57 × 10−5, 2.3 × 10−4 and 1.1 × 10−4 respec‑ tively Page 6 of 25 Keskin et al. Clin Transl Allergy (2019) 9:2 Keskin et al. Clin Transl Allergy continued) s Genotyping platform and number of SNPs after pruning Discovery phase population Study design and duration Number of asthmatic patients Medication in the discovery phase Replication population Definition of response SNPs chosen for replication (gene, chromosome position) Study outcome 4] Human Hap 550v3 Bead Chip or Infin‑ ium HD Human610- Quad Bead Chip (Illumina, San Diego, CA) 440 862 SNPs Caucasian chil‑ dren (CAMP) Clinical trial, 8 weeks 124 Budesonide 200 μg twice daily Caucasian Children: (CARE, n = 77, fluticasone pro‑ pionate 1000 μg daily (increased up to 2000 μg if necessary)) Adults: (LOCCS, n = 110, fluticasone 100 μg twice daily) (ACRN, n = 110, Triamci‑ nolone 400 μg twice daily) Self-reported asthma symp‑ toms based on diary cards. Scores ranged from 0 (absent) to 3 (severe) rs1558726 (RMST,12q21), rs2388639 (LOC728792) and rs10044254 (FBXL7, 5p15.1) The combined p val‑ ues of rs2388639, rs10044254 and rs1558726 SNPs for the pediatric CAMP and CARE subjects were 8.56 × 10−9, 9.12x10−8 and 1.02x10−5 respectively. T gene Homozygotes for the mutant allele for rs10044254 had significantly poorer responses to treat‑ ment compared to the patients homozygous or heterozygous for the wild-type allele (increase of 1.14 (as median score) in homozygotes for the mutant allele versus 0.28 in homozygotes for the reference allele) 5] Illumina Human 660 W BeadChip (Illumina, San Diego, USA) 430 487 SNPs Korean adults with moder‑ ate severe asthma Clinical trial, 4 weeks 189 1000 µg of fluticasone pro‑ pionate daily Same population with the discovery phase Changes in FEV1% 14 SNPs within ALLC (from GWAS) and 11 additional SNPs in ALLC (2q35) rs17017879, rs7558370, rs11123610, rs6754459, rs17445240 and rs13418767 were significantly associated with change in FEV1% (p value < 1.0 × 10−5) Referernces Genotyping platform and number of SNPs after pruning Discovery phase population Study design and duration Number of asthmatic patients Medication in the discovery phase Replication population Definition of response SNPs chosen for replication (gene, chromosome position) Study outcome Park et al. [14] Human Hap 550v3 Bead Chip or Infin‑ ium HD Human610- Quad Bead Chip (Illumina, San Diego, CA) 440 862 SNPs Caucasian chil‑ dren (CAMP) Clinical trial, 8 weeks 124 Budesonide 200 μg twice daily Caucasian Children: (CARE, n = 77, fluticasone pro‑ pionate 1000 μg daily (increased up to 2000 μg if necessary)) Adults: (LOCCS, n = 110, fluticasone 100 μg twice daily) (ACRN, n = 110, Triamci‑ nolone 400 μg twice daily) Self-reported asthma symp‑ toms based on diary cards. Scores ranged from 0 (absent) to 3 (severe) rs1558726 (RMST,12q21), rs2388639 (LOC728792) and rs10044254 (FBXL7, 5p15.1) The combined p val‑ ues of rs2388639, rs10044254 and rs1558726 SNPs for the pediatric CAMP and CARE subjects were 8.56 × 10−9, 9.12x10−8 and 1.02x10−5 respectively. Homozygotes for the mutant allele for rs10044254 had significantly poorer Page 7 of 25 Keskin et al. Clin Transl Allergy (2019) 9:2 Keskin et al. Clin Transl Allergy ble 1 (continued) erernces Genotyping platform and number of SNPs after pruning Discovery phase population Study design and duration Number of asthmatic patients Medication in the discovery phase Replication population Definition of response SNPs chosen for replication (gene, chromosome position) Study outcome ng et al. T gene [16] Affymetrix 6.0 arrays 909 622 SNPs After pruning 266 944 SNPs were involved in the analysis 120 Mild-to- moderate adult asth‑ matics Clinical trial, each dose of ICS was used for 1 week 120 Inhaled multiple different doses of glucocorti‑ coids-budeso‑ nide (125, 250, 500, 1000 mcg), in which, each dose was used for 1 week and the dose was doubled for the subsequent week The IMPACT trial (n: 225, mild, persistent adult asthma, open- label budesonide or prednisone as guided by the symptom-based action plan. The run- in and treatment phases both ended with a 14-day period of intense combined therapy) Salmeterol off corticoster‑ oids (SOCS) and salmeterol ± inhaled corticosteroids (SLIC) trials include 79 and 106 adult asthma,respectively, at the end of the 6-week run-in period on ICS, the milder patients were allocated to SOCS and the more moderate patients allocated to SLIC Changes in FEV1% rs6924808 on chromosome 6 rs10481450 on chromosome 8 rs1353649 on chromosome 11 rs12438740 on chromosome 15 rs2230155 on chromosome 15 The following loci produce associations of genome-wide sig‑ nificancewith phys‑ iological response to glucocorticoid therapy;rs6924808 on chromo‑ some 6 with wild-type allele C andmutant T (p = 5.315 × 10−7), rs10481450 on chromosome 8 withwild- type allele A and mutant T (p = 2.614 × 10− 8), rs1353649 on chromosome 11 with wild- type allele G and mutant A (p = 3.924 × 10−9), rs12438740 on chromosome 15 with wild- type allele C and mutant T (p = 4.499 × 10−8), and rs2230155 onchromo‑ some15 with wild-type allele C and mutant T (p = 1.798 × 10− 7) Page 8 of 25 Keskin et al. Clin Transl Allergy (2019) 9:2 Keskin et al. Clin Transl Allergy continued) es Genotyping platform and number of SNPs after pruning Discovery phase population Study design and duration Number of asthmatic patients Medication in the discovery phase Replication population Definition of response SNPs chosen for replication (gene, chromosome position) Study outcome . [17] Illumina’sOmni2.5 Exome BeadChip (Illumina, Inc., San Diego, CA) BioVU (731,390 SNPs) PMRP (662,256 SNPs) were first‑ mergedandpruned‑ toobtain 740,924 commonautoso‑ malSNPs. T gene In final dataset 237,726 common, inde‑ pendentSNPswere‑ included BioVU at VanderbiltU‑ niversityMedi‑ cal Center in Tennessee Patients had initiated ICS treatment prior to the exacer‑ bation event 806 Caucasian asthmatic BioVU N = 369 PMRP N = 437 ICS (beclometha‑ sone, budeson‑ ide, ciclesonide, flunisolide, mometasone, ortriamci‑ nolone) PersonalizedMedici‑ neResearch Project (PMRP) at the‑ MarshfieldClinic in Wisconsin Asthmaexacer‑ bations CMTR1 rs2395672 rs4271056 rs279728 TRIM24 rs6467778, MAGI2 rs2691529, rs9303988 Six novel SNPs associated with differential risk of asthma exacerba‑ tions (p < 10−5). Rs2395672 in CMTR1, was associated with an increased risk of exacerbations in both populations (OR = 1.07, 95% CI 1.03–1.11; joint p = 2.3X10−6). Two SNPs (rs2395672 and rs279728) were associated with increased risk of exacerba‑ tions, four SNPs (rs4271056 (CMTR1), rs6467778 (TRIM24), rs2691529, and rs9303988 MAGI2) were associated with decreased risk t al. 2184 haplotypes from the 1000 Genomes Project > 9.8 million common genetic variants 2672 asthma patients (≥ 12 years) from 7 randomized, double-blind, placebo- controlled “8–12 week” randomized double-blind placebo controlled parallel group multicenter clinical trial 2672 asthma patients (≥ 12 years) Inhaled fluticasone furoate (FF)- or fluticasone propionate (FP) treatment FEV1change at week 8 and 12 follow‑ ing FF- or FP treatment No genetic variant met the prespeci‑ fied threshold for statistical signifi‑ cance eferernces Genotyping platform and number of SNPs after pruning Discovery phase population Study design and duration Number of asthmatic patients Medication in the discovery phase Replication population Definition of response SNPs chosen for replication (gene, chromosome position) Study outcome Dahlin et al. [17] Illumina’sOmni2.5 Exome BeadChip (Illumina, Inc., San Diego, CA) BioVU (731,390 SNPs) PMRP (662,256 SNPs) were first‑ mergedandpruned‑ toobtain 740,924 commonautoso‑ malSNPs. In final dataset 237,726 common inde BioVU at VanderbiltU‑ niversityMedi‑ cal Center in Tennessee Patients had initiated ICS treatment prior to the exacer‑ bation event 806 Caucasian asthmatic BioVU N = 369 PMRP N = 437 ICS (beclometha‑ sone, budeson‑ ide, ciclesonide, flunisolide, mometasone, ortriamci‑ nolone) PersonalizedMedici‑ neResearch Project (PMRP) at the‑ MarshfieldClinic in Wisconsin Asthmaexacer‑ bations CMTR1 rs2395672 rs4271056 rs279728 TRIM24 rs6467778, MAGI2 rs2691529, rs9303988 Six novel SNPs associated with differential risk of asthma exacerba‑ tions (p < 10−5). Rs2395672 in CMTR1, was associated with an increased risk of exacerbations in both populations (OR = 1.07, 95% CI 1 03 1 11; joint Mosteller et al. T gene [18] 2184 haplotypes from the 1000 Genomes Project > 9.8 million common genetic variants 2672 asthma patients (≥ 12 years) from 7 randomized, double-blind, placebo- controlled, parallel group, multi- center clinical studies in 26 countries “8–12 week” randomized double-blind placebo controlled parallel group multicenter clinical trial 2672 asthma patients (≥ 12 years) Inhaled fluticasone furoate (FF)- or fluticasone propionate (FP) treatment Mosteller et al. [18] 2184 haplotypes from the 1000 Genomes Project > 9.8 million common genetic variants 2672 asthma patients (≥ 12 years) from 7 randomized, double-blind, placebo- controlled, parallel group, multi- center clinical studies in 26 countries “8–12 week” randomized double-blind placebo controlled parallel group multicenter clinical trial 2672 asthma patients (≥ 12 years) Inhaled fluticasone furoate (FF)- or fluticasone propionate (FP) treatment Page 9 of 25 Keskin et al. Clin Transl Allergy (2019) 9:2 Keskin et al. Clin Transl Allergy Table 1 (continued) Referernces Genotyping platform and number of SNPs after pruning Discovery phase population Study design and duration Number of asthmatic patients Medication in the discovery phase Replication population Definition of response SNPs chosen for replication (gene, chromosome position) Study outcome Leusink et al. [67] (Exome array) Infinium Human Exome chip (Illu‑ mina, San Diego, CA, USA), version 1.1, which contains 242 902 variants For common SNP analysis: MAF ≥ 1%, 36,519 SNPs For rare SNP analysis: MAF < 1%. 24,944 SNPs CATO study 110 children with asthma that was not well con‑ trolled despite ICS 2-year rand‑ omized clinical multicenter trial Partici‑ pants were fol‑ lowed up for 2 years, with the symptom- free days in the 2 weeks befo‑ reeach visit, FEV1%, airway hyperrespon‑ siveness (AHR) to methacho‑ line (MchPD20) every three‑ months. Treat‑ ment dosage was adjusted according to the algorithm of the study 110 children with asthma L1-100-μg Fluti‑ casone L2-200-μg Fluti‑ casone L3-200-μg Fluticasone and 100-μg salmeterol L-4 500-μg Fluticasone and 100-μg salmeterol L5-1000-μg Fluticasone and 100-μg salmeterol FEV1%, AHR (Mch PD20) and ICS response outcomes measured by the increase or decrease of FEV1% and AHR Strongest asso‑ ciation for rs72821893 in KRT25 with FEV1% (p = 3.75x10−5), Mch PD20 (p = 0.00095) and MchPD20-based treatment out‑ come (p = 0.006) The 17q12-21 region was found associated with FEV1%pred and AHR, and ICS treat‑ ment response Table adapted from Farzan et al. [10] Keskin et al. ALLCh The study by Park et al. [15], was the first study ever to report an association between the ALLC gene and a disease or treatment response. By performing in silico analysis, Park et al. reported correlations between the ALLC function and three SNPs (rs13418767, rs6754459, and rs13409104) in high LD with the most significant SNP (rs11123610) from the GWAS. Furthermore, using the TFSEARCH program, they showed that rs13418767 is a binding site for Sp1, a member of the Skp1-Cul1-F- box complex. A recent study that investigated the asso- ciation between occupational exposure to pesticides and genome-wide DNA methylation sites found differential DNA methylation in the ALLC gene [34]. In patients with airway obstruction who were exposed to high doses of pesticides, two different CpG sites of the ALLC gene were significantly hypo-methylated. ALLC is an enzyme that has lost its uricolytic activity during vertebrae evolu- tion. However, animal studies suggest rather than a non- functionality, there seems to be low expression level and low substrate affinity of this gene in animals [35]. There is limited knowledge regarding the role of this gene and its variations on asthma and treatment response. While ALLC located within chromosome 2 seems to have a lost function in humans, this chromosome harbors several genes that have been found to be associated with FEV1 [36, 37] and IgE levels [38]. Between 2015 and 2018, six additional candidate gene studies were published. These studies attempted to rep- licate the previously studied markers within the GLCCI1 [23, 43–45]; ADRB2 (Adrenoceptor Beta 2) [46, 47], NR3C1 [43], CRHR1 [43], 17q21 locus [48] and TBX21 [43]. SNPs located in the GLCCI1, NR3C1,and 17q21 were positively replicated in independent populations. An overview of replicated candidate gene studies is pro- vided in Table 2 [23, 43–61]. Candidate gene studies In total 29 candidate gene studies were included in the systematic review by Farzan et al. [10]. In summary, SNPs within the CRHR1 (Corticotropin Releasing Hor- mone Receptor 1), GLCCI1, FCER2 [Fc fragment of IgE, low-affinity II, receptor for (CD23)], NR3C1, STIP1, and TBX21(T-box 21) were studied the most. However, despite the large number of studied SNPs (> 500) within 120 genes, the most consistent results were reported only for one SNP, rs28364072, within the FCER2 gene. rs28364072 was significantly associated with all three outcomes (lung function, symptoms, and exacerbations) in pediatric asthma populations. T gene Clin Transl Allergy (2019) 9:2 Page 10 of 25 Page 10 of 25 gene (hMTr1) is involved in defense mechanisms against viral infections. Increased CMTR1 expression has been found to be associated with T cell mediated immune response mechanisms in human peripheral blood mon- onuclear cells [41]. Using independent microarrays, Dahlin et al. [17], evaluated the expression level of the top genes identified in their GWAS. They collected nasal lavage samples from children during and 1–2 weeks after asthma exacerbations. The expression level of CMTR1, but not TRIM24 or MAGI2,was significantly reduced 1–2 weeks after exacerbations. Since viral infections are the main causes of asthma exacerbations in children and expression of CMTR1 has been shown to be upregulated in children during asthma exacerbations [42], this gene and its product might be promising new therapeutic tar- gets that treat viral infections. Subfamily 3 Group C Member 1) interactions [13]. The results showed that the T gene is co-expressed with the NRIP1, FOXA2, and TTPA genes. The same analysis showed that these genes directly interact or are predicted to interact with NR3C1. Considering the important role of corticosteroids in lung development, it has been suggested that alterations in corticosteroid-responsive genes during development mighthave an influence on bothasthma susceptibilityand treatment response. Previous studies have shown that a decrease in T gene expression inhibits chondrogenesis, the process of cartilage development, mediated by BMP2 and FGFR3 [31]. These two proteins have been shown to be associated with corticosteroids resistance [32, 33]. As suggested by Tantisira et al. [13], these findings might provide a mechanistic basis for the T gene in response to ICS. However, despite these findings no further studies have tried to explore the functional role of this gene in ICS response. Replicated genes and SNPs GLCCI1 (glucocorticoid induced 1) By 2015, three candidate gene studies had studied GLCCI1 in order to replicate the findings of a GWAS published by Tantisira et al. [12]. In short, the GLCCI1 rs37973 was positively replicated only in one of the three candidate gene studies. The positive replication study included 224 adult asthmatic patients of Japanese ethnic- ity. The SNP was significantly associated with an annual decline in FEV1 of 30 ml/year or greater [54]. After 2015, four additional studies, including one study by our own research group, have assessed the association between rs37973 SNP and ICS response [23, 43–45]. CMTR1 CMTR1, located within the chromosome 6p21, is involved in mRNA capping [39]. Capping of mRNA stimulates the stability of mRNA and increases efficient mRNA translation [40]. Furthermore, the product of this Page 11 of 25 Keskin et al. Clin Transl Allergy (2019) 9:2 Keskin et al. Clin Transl Allergy ble 2 Replicated genes of ICS pharmacogenomic studies erences SNPs Study population Design MAF Medication Defination of response Study outcome tisira et al. [49] 131 SNPs genotyped in 14 candidate genes in the steroid pathway ALOX15 (4 SNPs) CRH (4 SNPs) CRHBP (5 SNPs) CRHR1 (17 SNPs) FCER2 (15 SNPs) GATA3 (9 SNPs) HSD11B1 (10 SNPs) IL18BP (7 SNPs) MAPK8 (4 SNPs) NFATC4 (11 SNPs) NR3C1 (16 SNPs) POMC (6 SNPs) POMC (6STAT3 (10 SNPs) POMC (6STAT5A (10 SNPs) Caucasian children and adults: Adult study (Adults, n = 415) CAMP (Children, n = 201) ACRN (Adults, n = 224) Three independent 6–8 week clinical trials rs242941 0.3 (T) rs1876828 (T) Flunisolide 1000– 2000 μg once daily Budesonide 200 μg twice daily Tramcinolone aceto‑ nide 400 μg twice daily Changes in FEV1% from baseline (CRHR1, NM_004382), was associated with treatment response in all three populations Individuals homozy‑ gous for the variants manifested a doubling to quadrupling of the lung function response to ICS compared with lack of the variants (p values ranging from 0.006 to 0.025 for three asth‑ matic populations). rs1876828, rs242939, and rs242941, were each associated with ICS treatment response in both the Adult Study and CAMP. rs1876828, was also strongly associ‑ ated with improved FEV1 over the 6-week triamcinolone treat‑ ment in ACRN adult asthmatics kstra et al. [50] CRHR1(17q21.31) rs1876828 rs242939 rs242941 281 adult patients with symptomatic asthma under 45 years of age Asthma cohort fol‑ lowed for 22 years, clinical trial (Neth‑ erland) rs242941 0.4 (T) rs1876828 0.2 (T) 749 μg/days (426–1152) Mean daily dose of ICS was calculated to an equivalent daily dose of beclometha‑ sone. Immediate effect: Changes in FEV1 from baseline within 3–6 months Long-term effect:Rate of decline in FEV1 annually during 13.0 (7–19) years CRHR1 polymorphisms are not associated with immediate or long-term improve‑ ment in FEV1 by ICSs or with prevention of accelerated FEV1 decrease in adult asthma Page 12 of 25 Keskin et al. Clin Transl Allergy (2019) 9:2 Keskin et al. CMTR1 Clin Transl Allergy ble 2 (continued) ferences SNPs Study population Design MAF Medication Defination of response Study outcome gers et al. [51] CRHR1-rs242941 FCER2-T2206C 311 children (5–12 years) African-American and Caucasian Children (CAMP) CAMP 4-year clinical trial CRHR1 rs242941 0.26 (T) FCER2 T2206C 0.26 (G) in Caucasians, 0.44 (G) in African- Americans Budesonide 200 μg twice daily Exacerbations: Emergency Depart‑ ment visits Hospitalizations Oral prednisone burst Lung function meas‑ urements: poor responders:change in FEV1 %pred: ≤ 7.5% Lower bronchodilator response to albuterol and the minor alleles of RS242941 in CRHR1 (OR 1.6, CI 95% 1–2.7, p = 0.05) and T2206C in FCER2 (OR: 2.1, 95%CI 1.2–3.5, p = 0.006) are associ‑ ated with poor lung function response The minor allele of rs28364072 in FCER2 was associated with recurrent exacerba‑ tions in white subjects (OR: 1.9 for minor allele, p < 0.05) but it did not reach signifi‑ cance in multivariate analysis ugey et al. [52] CRHR1 (17q21.31) rs1876828 rs242941 Caucasian children, adolescence and adults continuing ICS (n = 65) 16 weeks Clinical trial rs242941 0.34 (T) rs1876828 0.15 (T) Fluticasone 100 μg twice daily Change in FEV1% pred Asthma symptoms: Slopes of plots of ACQ scores versus time Minor allele of rs1876828 was associ‑ ated with improve‑ ments in FEV1% pred (p = 1.89 × 10−4) Minor allele of rs242941 was associated with decrement in FEV1% pred (p = 2.07 × 10−3) Table 2 (continued) Page 13 of 25 Keskin et al. Clin Transl Allergy (2019) 9:2 Keskin et al. Clin Transl Allergy able 2 (continued) ferences SNPs Study population Design MAF Medication Defination of response Study outcome skin et al. CMTR1 [43] NR3C1(rs41423247) CRHR1(rs242939, rs242941, rs1876828) TBX21 (rs2240017) GLCCl1 (rs37973, rs3099266, rs2305089) 82 children with asthma exacerbation Single high dose ICS study in children with moderate- severe asthma exacerbation, Clinical trial NR3C1 rs41423247 0.8 (G) CRHR1 rs242939 0.08 (G) rs242941 0.27 (T) rs1876828 0.87 (G) TBX21 rs2240017 0.04 (G) GLCCl1 rs37973 0.47 (G) rs3099266 0.39 (A) rs2305089 0.35 (A) Single-dose Inhaled 4000 mcg Flutica‑ sone propionate +Nebulized albuterol solution Changes in FEV1 at 4th hour Homozygosity for the G allele at rs41423247 of the NR3C1 gene is associated with a higher improvement in FEV1 at 4 h in chil‑ dren with moderate- to-severe asthma exacerbation treated with high-dose ICS (p = 0.006) No genotype-related significant differ‑ ence was observed in SNPs CRHR1 gene rs242939, s242941,and rs1876828, TBX21 rs2240017; GLCCl1 rs37973; and T gene rs3099266 and rs2305089 in FEV1 change osking et al. [53] GLCCI1 (7p21.3) Rs37973 (A/G) Non-Hispanic white adolescents and adults (n = 1916) Pooled data of seven studies, six studies of 8 week trials and one 12-week clinical trial 0.44 (G) Various doses of Fluticasone furoate ranged between 25 and 800 μg daily, Fluticasone propion‑ ate 100–500 μg twice daily Changes in FEV1 from baseline after 8 weeks in 6 studies and at week 12 in one study There was no significant association between changes in FEV1 and rs37973 genotypes uhara et al. [54] GLCCI1 (7p21.3) Rs37973 (A/G) Adult Japanese (n = 224) Asthma cohort receiv‑ ing ICS fot at least 4 years 0.44 (G) ICS maintenance dose varied between patients Annual decline in FEV1 30 ml/year or more rs37973 GG was associ‑ ated with a decline in FEV1 of 30 ml/year or more (estimated effect: 1.10: 0.02 to 2.18, p = 0.047) There was no associa‑ tion between rs37973 genotypes and the outcome, when decline in FEV1 was analyzed as a continu‑ ous variable References SNPs Study population Design MAF Medication Defination of response Study outcome Keskin et al. CMTR1 [43] NR3C1(rs41423247) CRHR1(rs242939, rs242941, rs1876828) TBX21 (rs2240017) GLCCl1 (rs37973, rs3099266, rs2305089) 82 children with asthma exacerbation Single high dose ICS study in children with moderate- severe asthma exacerbation, Clinical trial NR3C1 rs41423247 0.8 (G) CRHR1 rs242939 0.08 (G) rs242941 0.27 (T) rs1876828 0.87 (G) TBX21 rs2240017 0.04 (G) GLCCl1 rs37973 0.47 (G) rs3099266 0.39 (A) rs2305089 0.35 (A) Single-dose Inhaled 4000 mcg Flutica‑ sone propionate +Nebulized albuterol solution Changes in FEV1 at 4th hour Homozygosity for the G allele at rs41423247 of the NR3C1 gene is associated with a higher improvement in FEV1 at 4 h in chil‑ dren with moderate- to-severe asthma exacerbation treated with high-dose ICS (p = 0.006) No genotype-related significant differ‑ ence was observed Page 14 of 25 Keskin et al. Clin Transl Allergy (2019) 9:2 Keskin et al. Clin Transl Allergy Table 2 (continued) References SNPs Study population Design MAF Medication Defination of response Study outcome Vijverberget al. [55] GLCCI1 (7p21.3) Rs37972 (T/C) North European children and young adults BREATHE (n = 1037) PACMAN (n = 431) PAGES (n = 323) Meta-analysis of three pediatric asthma cohorts BREATH:0.45 (T) PACMAN: 0.44 (T) PAGES: 0.41(T) ICS maintenance dose varied between patients Exacerbations: Hospital visits OCS use Poor asthma symp‑ toms: ACT scores ≤ 19 ACQ-scores ≥ 1.5 There was no significant association between increased risk of OCS use, increased risk of asthma exacerba‑ tions and rs37972 genotypes Hu et al. [23] GLCCI1 (7p21.3) Rs37972 (T/C) Rs11976862, (A/G) Rs37973 (A/G) Chinese population of 182 asthmatic patients and 180 healthy controls The association of GLCCI1 variations with ICS response was analyzed in 30 mild-to-moderate asthmatics Case control: study 24 SNPs of GLCCI1 were genotyped in 182 asthmatic patients and 180 healthy controls. –Treatment trial: 2-week run-in period and maintan‑ ance ICS therapy for 12 weeks. Rs37972 (T/C) Asthmatics:0.67 (T) Control: 0.12 (T) Rs11976862 Asthmatics: 0.07 (G) Control: 0.37 (G) Rs37973 Asthmatics: 0.55 (G), Control: 0.37 (G) Inhaled fluticasone propionate (125 mg, twice a day) Changes in FEV1 FEV1 change was significantly correlated with rs37972, rs37973 and rs11976862 at 4 weeks ICS (p = 0.021 for rs37972 and rs37973; p = 0.043 for rs11976862), at 8 weeks (p = 0.021, p = 0.025 and p = 0.035, respec‑ tively) and at 12 weeks (p = 0.040 for rs37972 and rs37973, p = 0.020 for rs11976862) Xu et al. CMTR1 [44] GLCCI1 (7p21.3) Rs37973 (A/G) Chinese population of 418 asthmatics Inhaled fluticasone propionate/salme‑ terol combination (250/50 mg, twice daily) for the next 24 weeks. Follow-up visits occurred at 4, 12, and 24 weeks, and asthma control tests were reviewed and lung function tests were per‑ formed 0.49 (G) Long term ICS treat‑ ment Changes in FEV1 rs37973 was indepen‑ dently associated with poorer clinical therapeutic response to ICS Homozygotes for the wild-type allele who had a percent FEV1 change greater than 5% were more common than were homozygotes of the rare allele (rs37973, AA 67.01% vs. GG 49.49%, p < .05) Xu et al. [44] Page 15 of 25 Keskin et al. Clin Transl Allergy (2019) 9:2 Table 2 (continued) References SNPs Study population Design MAF Medication Defination of response Study outcome Rijavec et al. [45] GLCCI1 (7p21.3) Rs37973 (A/G) 208 Slovenian adults with atopic and nonatopic, mild-to- moderate persistent asthma ICS (alone or in combi‑ nation with a LABA, depending on the degree of asthma control Follow-up visits with spirometry testing after 3 months (short-term) and after at least 3 years (long-term) of ICS treatment 0.29 (G) ICS or ICS + LABA, depending on the degree of asthma control FEV1% change after ICS treatment (3 months) and at least 3 years Treatment was defined as suc‑ cessful when FEV1 decreased by < 30 mL/year After 3 months of ICS treatment, the change in FEV1% was higher in patients with the GG genotype than in patients with the AG + AA genotype (7.5% vs. 4%, p = .049) Szczepankiewicz et al. [56] GR polymorphisms rs6190 rs41423247 rs6195 rs10052957 113 asthmatic children (6 to 18 years of age) (54 of children were with severe, difficult- to-treat asthma) 123 healthy control Analysis of a relation‑ ship between the GR polymorphisms and poor response to glucocorticoids in asthmatic patients (Polish) rs6190 0.03 (G) rs41423247 0.36 (G) rs10052957 0.39 (T) rs6195 0.08 (G) ICS The dose of ICS needed to achieve asthma control Worse response was defined as a neces‑ sity of taking high doses of ICS i.e. > 800 mcg of budesonide and > 500 mcg of fluticasone propionate No association of GR polymor‑ phisms with the dose of ICS needed to achieve asthma con‑ trol was reported ICS Page 16 of 25 Keskin et al. CMTR1 Clin Transl Allergy (2019) 9:2 ble 2 (continued) erences SNPs Study population Design MAF Medication Defination of response Study outcome erberget al. [57] 50 tag SNPs were selected for 17 genes for screening: Ten genes were selected based on their involvement in the glucocorti‑ coid (GC) receptor complex (NR3C1, HSPCA, HSPA4, FKBP4, ST13), GC transport (SERPINA6) or GC- mediated signalling (CREBBP, TBP, NCOA3, SMAD3) Seven genes were selected based on a previously reported association with asthma susceptibility, severity or asthma medication response (ARG1, 17q21 locus, IL2RB IL18R1, PDE4D, HLA-DQ, BCL2) Children and young adults PACMAN (n = 357) BREATHE (n = 820) PAGES (n = 391) Validation cohorts; CAMP (n = 172) GALA II (n = 745) PASS (n = 391) Meta-analysis of three cohorts >0.2 Based on BTS guid‑ linesfortreatment step 2: SABA + ICS step 3: SABA + ICS + LABA step 4: SABA + ICS + LABA + LRA Exacerbations: hospi‑ tal visits Oral corticosteroid (OCS) use in the previous year In a meta–analysis of six studies: ST13 rs138335 remained associated with an increased risk of asthma-related hospital visits and OCS use in the previ‑ ous year,; OR = 1.22 (p = 0.013) and OR = 1.22 (p = 0.0017) but did not passed Bonferronicorrection test None of the other genes including NR3C1, were associ‑ ated at a nominal level with an increased risk of exacerbations in asthmatics using ICS in the three cohorts tisira et al. [49] TBX21 rs2240017 (H33Q) 701 children aged 5–12 years with mild to moderate asthma enrolled in CAMP CAMP, 4 year clinical trial Minor allele frequency of H33Q: 4.5% no minor homozygotes were detected Budesonide 200 μg twice daily Bronchial Hyperreac‑ tivity (Methacholine) (PC20) 3.5-fold greater mean increase in log- transformed PC20 for methacholine after four years of inhaled budesonide in asthmatic children with glutamine vari‑ ants when compared with either H33H homozygotes or in dividuals not taking ICS (p = 0.0002) Table 2 (continued) References Page 17 of 25 Keskin et al. Clin Transl Allergy (2019) 9:2 Keskin et al. Clin Transl Allergy ( Table 2 (continued) References SNPs Study population Design MAF Medication Defination of response Study outcome Ye et al. CMTR1 [46] ADRB2 (G16R A > G) ADCY9 (I772 M T > C) NK2R (G231E G > A) TBX21 (H33Q C > G) 53 mild-to-moderate adult asthmatics Asthmatics adults (Korean) 5–12 weeks ICS treat‑ ment, Clinical trial MAF of four selected polymorphisms were > 5 5–12 weeks of ICS Asthma control status and FEV1 NK2R G231E G > A and TBX21 H33Q C > G polymorphisms were significantly associ‑ ated with asthma control status at 5–12 weeks of ICS treatment (p = 0.041, power = 81.419% and p = 0.006, power = 98.564%, respectively) The NK2R G231E G > A polymorphism also showed significant associations with the mean changes in FEV1% during 12 weeks of ICS treat‑ ment (p < 0.05) Page 18 of 25 Keskin et al. Clin Transl Allergy (2019) 9:2 Table 2 (continued) References SNPs Study population Design MAF Medication Defination of response Study outcome Lopert et al. [59] TBX21 (17q21.32) rs9910408 208 adult patients with atopic and non-atopic, mild to moderate persistent asthma 3 years clinical trial rs9910408 0.38 (G) Treatment with ICS for 3 years (alone or in combina‑ tion with long- acting beta agonists (LABA),according to achieved asthma control) According to the response to ICS therapy patients were divided into ‘‘poor’’ and ‘‘good’’ responders BHR:Poor response was defined as an increase of PD20 for methacholine that was smaller than one doubling dose compared to the initial PD20 Lung function: Poor response decrease in FEV1 by more than 30 ml/year Asthma control: Poor response was defined as less than a three-point increase in the ACT score after at least 3 years of treatment Asthma-related quality of life: Poor response was defined as less than a 16-point increase from the initial AQLQ score The frequency of AA genotype was signifi‑ cantly higher in good responders (p = 0.049) This genotype related response was even more evident in the subgroups of non- smokers (p = 0.008) and in non-atopic patients (p = 0.009) AA genotype was over‑ represented among good responder sac‑ cording to changes in FEV1 in the subgroups of non-smokers (p = 0.013) and in non-atopic patients (p = 0.048) Table 2 (continued) References SNPs Study population Design MAF Medication Defination of response Study outcome Lopert et al. CMTR1 [59] TBX21 (17q21.32) rs9910408 208 adult patients with atopic and non-atopic, mild to moderate persistent asthma 3 years clinical trial rs9910408 0.38 (G) Treatment with ICS for 3 years (alone or in combina‑ tion with long- acting beta agonists (LABA),according to achieved asthma control) According to the response to ICS therapy patients were divided into ‘‘poor’’ and ‘‘good’’ responders BHR:Poor response was defined as an increase of PD20 for methacholine that was smaller than one doubling dose compared to the initial PD20 Lung function: Poor response decrease in FEV1 by more than 30 ml/year Asthma control: Poor response was defined as less than a three-point increase in the ACT score after at least 3 years of treatment Asthma-related quality of life: Poor response was defined as less than a 16-point increase from the initial AQLQ score The frequency of AA genotype was signifi‑ cantly higher in good responders (p = 0.049) This genotype related response was even more evident in the subgroups of non- smokers (p = 0.008) and in non-atopic patients (p = 0.009) AA genotype was over‑ represented among good responder sac‑ cording to changes in FEV1 in the subgroups of non-smokers (p = 0.013) and in non-atopic patients (p = 0.048) Page 19 of 25 Keskin et al. Clin Transl Allergy (2019) 9:2 Keskin et al. Clin Transl Allergy ble 2 (continued) ferences SNPs Study population Design MAF Medication Defination of response Study outcome tisira et al. [60] FCER2(19p13.3) rs889182 rs2287867 rs12980031 rs8110128 rs4804773 rs7249320 rs2277989 rs1042428 T2206C rs4996974 311 children (CAMP) African-American and Caucasian children CAMP 4-year Clinical trial T2206C 0.26 (G) in Caucasians,-0.44 (G) in African-Americans Budesonide 200 μg twice daily Severe asthma exacer‑ bation risk Relative risk, expressed as hazard ratios, for exacerbations in those homozygous for the T2206C mutant allele were (3.95; 95% CI 1.64–9.51); and (3.08;95% CI 1.00–9.47).more likely to have a severe exac‑ erbation compared to all other T2206C genotypes in both white and African-American children ter et al. CMTR1 [61] FCER2 (19p13.3) rs28364072 (A/G) Caucasianchildren PACMAN (n = 386) BREATHE (n = 939) CAMP (n = 311) PACMAN& BREATHE: Asthma cohorts CAMP: 4-year clinical trial PACMAN 0.27 (G) BREATHE 0.26 (G) For BREATHE and PAC‑ MAN: ICS maintenance dose varied between patients CAMP: Budesonide 200 µg twice daily Exacerbations: Emergency depart‑ ment visits Hospitalization Asthma control: ACQ- scores Respiratory symptoms Asthma-related sleep‑ disturbances Asthma-related limitations in Daily activities Additional (airway) medication use dur‑ ing the preceding 12 month The rs28364072 variant was associ‑ ated with increased risk of asthma-related hospital visits in the meta-analysis (OR 2.38, 95% CI 1.47–3.85, p = 0.0004) The variant was associ‑ ated with increased risk of uncontrolled asthma measured by ACQ scores (OR 2.64, 95% CI 1.00–6.98) and was associated with increased daily steroid dose (OR 2.46, 95% CI 1.38–4.39) Page 20 of 25 Keskin et al. Clin Transl Allergy (2019) 9:2 Keskin et al. Clin Transl Allergy continued) s SNPs Study population Design MAF Medication Defination of response Study outcome [68] ORMDL3 (17q21) rs2872507 (G/A) -213 asthmatics who were regularly treated with ICSs In asthmatics who were regularly treated with ICSs, spirometry was repeated after 4–6 weeks of treat‑ ment. Bronchial hyperreactivity was assessed with a methacholine chal‑ lenge test 0.39 (A) 4–6 weeks of ICS: Children < 12 years of age: 200 mcg of fluticasone dry powder daily Children > 12 years of age: 400 mcg fluti‑ casone dry powder daily Changes in FEV1 Asthmatics with genotype AA had an 11.1 ± 16.0% mean increase in FEV1 after 4–6 weeks of ICS, compared with 4.6 ± 9.6% in GG homozygotes (p = 0.0463) Carriers of A allele had a 8.5 ± 13.8% mean increase in FEV1 after ICS treat‑ ment, which was significantly higher than 5.5 ± 10.7% in asthmatics with G allele (p = 0.0150) . CMTR1 [10] 17q21 locus rs7216389 (C/T) 14 PiCA (Pharma‑ cogenomics in Childhood Asthma) populations (4529 steroid treated children and young adults) Ten PiCA studies included patients with non‐Hispanic European origins, two included His‑ panic patients, one African American, one East Asian patients the association between variation in the 17q21 locus, and asthma exacerba‑ tions despite ICS use retrospective reporting of exacerbations in the observational cohort studies Prospective study in CAMP population 0.54–0.81 (T) ICS Asthma‐related hos‑ pitalizations/emer‑ gency department visit (ED) (ii) short courses of oral corticosteroid (OCS) use reported by the parent/child at the study visit or based on completed study questionnaires In the meta‐analysis of 13 studies, rs7216389 was statistically signifi‑ cantly associated with asthma‐related ED visits/hospitalizations, (summary OR per increase in risk allele: 1.32, 95% CI 1.17–1.49, p < .0001, I2 = 3.9%) In the meta‐analysis of the nine studies, the rs7216389‐T was statistically signifi‑ cantly associated with an increased risk of OCS use/high‐dose ICS (summary OR per increase in variant allele: 1.19, 95% CI 1.04‐1.36, p = .01, I2 = 22.8%) ed from Farzan et al [10] References SNPs Study population Design MAF Medication Defination of response Study outcome Berce et al. [68] ORMDL3 (17q21) rs2872507 (G/A) -213 asthmatics who were regularly treated with ICSs In asthmatics who were regularly treated with ICSs, spirometry was repeated after 4–6 weeks of treat‑ ment. Bronchial hyperreactivity was assessed with a methacholine chal‑ lenge test 0.39 (A) 4–6 weeks of ICS: Children < 12 years of age: 200 mcg of fluticasone dry powder daily Children > 12 years of age: 400 mcg fluti‑ casone dry powder daily Changes in FEV1 Asthmatics with genotype AA had an 11.1 ± 16.0% mean increase in FEV1 after 4–6 weeks of ICS, compared with 4.6 ± 9.6% in GG homozygotes (p = 0.0463) Carriers of A allele had a 8.5 ± 13.8% mean increase in FEV1 after ICS treat‑ Table 2 (continued) Keskin et al. Clin Transl Allergy (2019) 9:2 Keskin et al. Clin Transl Allergy (2019) 9:2 Page 21 of 25 Page 21 of 25 studies [63, 64]. By 2015, two studies had investigated the role of SNPs in the NR3C1 in glucocorticoid sensi- tivity and response. However, only one study in adults hadfound an association between anSNP within this gene (rs41423247) and response (defined by changes in FEV1 predicted) to prednisolone but not ICS. CMTR1 In-vitro and in vivo models have shown that the G allele of the rs41423247 locus at the NR3C1 gene was associated with hypersensitivity to glucocorticoids [65, 66].i In a Chinese population of 418 adult asthmatics receiv- ing 24 weeks of ICS treatment, rs37973 was indepen- dently associated with lower ICS response measured with FEV1 changes [44]. Percentages of homozygotes for the wild-type allele who had > 5% FEV1 change were more common than homozygotes of the rare allele (AA 67.01% vs. GG 49.49%, p < .05) [44]. In another Chinese study, Hu et al. evaluated the asso- ciation of 24 SNPs within the GLCCI1 gene with ICS response. They included 180 healthy individuals and 182 adult asthmatic patients from Chinese Han population. In line with the previous findings, patients homozygous for the G allele had significantly poorer improvements in their lung function compared to the heterozygotes and homozygotes for the A allele after 12 weeks of treat- ment with ICS [23]. Contrasting findings were shown in a study in 208 Slovenian adults with atopic and nonatopic asthma. FEV1% predicted was higher in patients with GG genotype than patients with the AG or AA geno- type; after 3 months treatment FEV1% predicted change: 7.5% in patients homozygous for the G allele versus 4% (patients with AG/AA genotype), p value: 0.049. After 3 years of treatment, similar effects were observed; FEV1 %pred 7% (GG) versus 3.5% (AG/AA), p-value: 0.041) [45]. Recently, our group reported a significant association between rs41423247 and improvements in FEV1 after 4 h upon a single high-dose of ICS (4000 µg FP) in 82 Turk- ish children with moderate-to-severe asthma exacerba- tions [43]. Children with the GG genotype at rs41423247 had a higher improvement in FEV1 [24.2% (11.5–36.3)] compared to those with CG + CC, [7.9% (6.1–24.6) (p = 0.006)]. 17q21locus 17q21 is the most consistently identified locus associated with asthma onset in children and severe asthma, and it has also been linked to ICS response. By 2015, two stud- ies examined the association between SNPs within the 17q21 locus and response to ICS [67, 68], though dif- ferent variants were studied. A Slovenian study showed that asthmatic children treated with ICS showed a bet- ter improvement in FEV1% when they were homozygous for the AA genotype at rs2872507 at 17q21 compared to patients with AG or GG genotypes (13.3% vs. 7.0% vs. 4.9% respectively). In addition, a post hoc exome array analysis of Dutch children who participated in the Chil- dren Asthma Therapy Optimal (CATO) trial, showed that several variants in the 17q12-21 locus were found nomi- nally associated with treatment response to ICS (based on FEV1% improvement or BHR during ICS treatment). The strongest association in this region was found for rs72821893 in KRT25. However, in a study we performed in 82 Turkish chil- dren with moderate-severe asthma exacerbation, we were unable to show any association between FEV1 increase after 4 h of single-high-dose ICS and variation at GLCCI1 rs37973, rs3099266, rs2305089 genotypes [43]. Therefore, seven candidate gene studies have tried to replicate the results of the original GWAS. Interest- ingly, three studies that included East Asian popula- tions (Japanese and Chinese) could positively replicate the results of the GWAS. Although the exact definition of the outcomes was different (annual decline in FEV1 and improvements in FEV1), all three showed that the G allele was associated with poor lung function outcomes. Interestingly, the minor allele frequency of rs37973 in East Asian patients was comparable to patients of Cauca- sian ethnicity. However, in contrast to the non-Hispanic white subjects in the GWAS, where LD was almost per- fect between rs37972 and rs37973, in the study by Hu et al. this LD was far from perfect. In 2018, one of the largest pharmacogenomics studies [48] so far was performed by the international Pharma- cogenomics in Childhood Asthma (PiCA) consortium [69]. In this study, more than 4000 children were included from 13 different studies. Genetic variation at position rs7216389 in the17q21 locus was found to be associated with an increased risk of OCS use and asthma-related hospitalizations/ER visits despite ICS use. Discussion This approach will allow the discovery not only of pharmacologic agents that directly target the disease asthma but also other path- ways and biomarkers that are indirectly associated with or increase the risk of the disease such as atopy, eosino- phils, bronchial hyperreactivity, pulmonary functions, and NO [73]. Consistent significant associations were replicated between FCεR2 rs28364072 and poor ICS response meas- ured by exacerbations in two long follow up childhood studies [60, 61], asthma symptoms measured by ACQ in one long follow up childhood study [61], and lung func- tion in one study [51] in asthmatic children. Moreover, supporting clinical findings, functional data related with FCER2 provided a mechanistic basis for the observed associations with severe exacerbations. This variation in FCER2, T2206C, was associated with decreased FCER2 expression and can adversely affect normal negative feedback in the control of IgE synthesis and action [60]. Moreover, it has been shown that the highest IgE levels were found in subjects both homozygous for the T2206C variant and taking ICSs, demonstrating a significant ster- oid-genotype interaction. This may actually be connected with the observation that higher IgE levels are associated with increased frequency of exacerbations [70], and hos- pitalizations [71, 72] in children with asthma. Pharmacogenomics may produce very important infor- mation for the practicing clinician. Predicting the drug response based on genetic testing has implications with respect to not only providing the best treatment but also preventing the adverse events that may be associ- ated especially with higher doses and systemic CS [74, 75]. On a larger scale it may have tremendous effect on pharmacoeconomics by decreasing unnecessary medica- tion use as well as by having a positive impact on the cost of the disease, as has been shown in other disease areas [76]. Even though ongoing international attempts such as “Ubiquitous Pharmacogenomics (U-PGx)” project “An Horizon2020 Program to Drive Pharmacogenom- ics into Clinical Practice” that is going on in seven Euro- pean countries [77] holds promise, we are far from using pharmacogenomics data in clinical asthma practice. In contrast to long-acting beta-2 agonists [78], response to corticosteroids might be too complex to be mainly driven by a few genetic variants. Nevertheless, pharma- cogenomics studies still might provide useful insights in underlying pathways or identify novel drug targets, especially when combined with other-omics layers (e.g. Discussion Glucocorticoids bind to their receptors in the cytosol and after the transfer of glucocorticoid receptor com- plex to the nucleus, they regulate expression of the genes involved in the inflammatory pathways [62, 63]. Therefore, due to its central role in glucocorticoid sign- aling pathway, NR3C1, the gene encoding GR, has been the center of attention in numerous pharmacogenomics In the past 3 years, nine additional pharmacogenom- ics studies on ICS have been published, of which three GWAS. Although there is few overlap between identified variants and applied methodologies vary largely, in vitro and/or in silico analyses provide additional evidence that genes discovered in GWAS (e.g. GLCCI1, FBXL7, T gene, Keskin et al. Clin Transl Allergy (2019) 9:2 Page 22 of 25 Page 22 of 25 ALLC, CMTR1) might play a direct or indirect role in asthma/treatment response pathways. ALLC, CMTR1) might play a direct or indirect role in asthma/treatment response pathways. studies have emphasized the importance of defining the correct phenotype in successful asthma treatment. A good characterization of patients as well has a stand- ardized definition of treatment response is therefore extremely important. Since most pharmacogenetic stud- ies are underpowered, collaboration is inevitable. Novel consortia, such as the Pharmacogenomics in Childhood Asthma (PiCA) consortium, are emerging [69, 72] and are able to conduct large-scale GWAS meta-analyses, while performing sensitivity analyses for specific sub- groups. This might lead to novel insights on the general- izability of findings between different populations, as well as more power to identify novel genetic variants. Candidate gene analysis, on the other hand identi- fied five SNPs within four genes (CRHR1: rs242941, rs1876828; GLCCI1: rs37973; FCεR2: rs28364072 and TBX21: rs2240017) that were positively replicated at least once. In line with the previous systematic review by Farzan et al., most consistent results were obtained for FCεR2 (rs28364072) and TBX21(rs2240017) [46, 51, 58, 60, 61]. The only gene that showed a positive association in both GWAS and candidate gene analyses was GLCCI1 [12, 23, 44, 45, 54]. However, it should be noted that this may be partly due to the fact the candidate gene stud- ies were undertaken after identification of GLCCI1 in a GWAS analysis. In order to increase the utility of the pahramacog- enomic studies in asthma the data that have emerged from GWAS, candidate gene, and mechanistic candi- dates should be evaluated together. Discussion epigenomics, transcriptomics, microbiomics, breathom- ics) or assessing the interaction with the environmental factors using genome-wide interaction studies in well characterized patient populations [79]. Especially with the emergence of novel expensive biologics for patients with a poor response to ICS, it is of great importance to assess at an early stage which patients have an intrinsic poor response to ICS and will be eligible candidates for p Both studies investigating TBX21 H33Q and ICS response showed significant association measured by two different outcomes: improvement in BHR in one child- hood study of 4 years duration [58], and asthma control status in a 5–12 weeks adult study [46]. Since BHR and asthma control level are related to the quality of life in asthma patients and prognosis of asthma, genetic varia- tion in TBX21 may be important for asthma phenotypes. This finding was further supported by a functional study that showed that the TBX21 variant increases T helper 1 and decreases T helper 2 cytokine expression compared to wild type [58]. However, we were not able show any association between TBX21 H33Q and FEV1 4 h after single-high-dose ICS in children with moderate-severe asthma exacerbation [43]. This difference may be due to different outcome parameter or different study design or different doses and duration of ICS. f Asthma is a very heterogeneous disease with various phenotypes and underlying disease pathways. Recent Page 23 of 25 Page 23 of 25 Keskin et al. Clin Transl Allergy (2019 these novel targeted treatments. Genomics might, at least partly, help to answer this question. these novel targeted treatments. Genomics might, at least partly, help to answer this question. 25. Tyers M, Willems AR. One ring to rule a superfamily of E3 ubiquitin ligases. Science. 1999;284:601, 603–4. Authors’ contributions OZK, OMK, EB, CK, HA: made substantial contributions to conception and design, acquisition of data, analysis and interpretation of data. OZK, OMK, EB, CK, HA: been involved in drafting the manuscript. OZK, OMK, NF, AHMvdZ, MEW, SJV been involved in revising the manuscript critically for important intellectual content and given final approval of the version to be published. Each author have participated sufficiently in the work to take public respon‑ sibility for appropriate portions of the content; and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All authors read and approved the final manscript. 9. Palmer LJ, Silverman ES, Weiss ST, Drazen JM. Pharmaco asthma. Am J Respir Crit Care Med. 2002;165:861–6. 10. Farzan N, Vijverberg SJ, Arets HG, Raaijmakers JA, Maitland-van der Zee AH. Pharmacogenomics of inhaled corticosteroids and leukotriene modi‑ fiers: a systematic review. Clin Exp Allergy. 2017;47:271–93. 11. Moher D, Liberati A, Tetzlaff J, Altman DG, PRISMA Group. 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Abbreviations ICS: inhaled corticosteroid; SNP: single nucleotide polymorphism; GWAS: genome wide association studies; SHARP: Single-Nucleotide Polymorphism Health Association-Asthma Resource Project; FEV1: forced expiratory volume in one second; FP: fluticasone propionate; FF: fluticasone furoate; CAMP: Childhood Asthma Management Program; GLCCI1: glucocorticoid-induced transcript 1 protein; FBXL7: F-box and leucine-rich repeat protein 7; ALLC: allantoicase gene; CMTR1: cap methyltransferase 1; TRIM24: tripartite motif containing 24; MAGI2: Membrane Associated Guanylate Kinase; NR3C1: nuclear receptor subfamily 3 group C member 1; CRHR1: corticotropin releas‑ ing hormone receptor 1; FCER2: Fc fragment of IgE, low-affinity II, receptor for (CD23); ADRB2: Adrenoceptor beta 2; TBX21: T-box 21. 5. Chung KF, Godard P, Adelroth E, et al. 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https://openalex.org/W2002841103	https://bmcinfectdis.biomedcentral.com/counter/pdf/10.1186/1471-2334-10-181	English	null	Altered blood glucose concentration is associated with risk of death among patients with community-acquired Gram-negative rod bacteremia	BMC infectious diseases	2,010	cc-by	6,582	RESEARCH ARTICLE Open Access * Correspondence: gpf@mundivia.es 1 Instituto de Investigación y Formación Marques de Valdecilla (IFIMAV), 5 Planta de la Escuela Universitaria de Enfermería, Avda de Valdecilla s/n, 39008, Santander, Spain Full list of author information is available at the end of the article Abstract Abstract Background: Altered blood glucose concentration is commonly observed in patients with sepsis, even among those without hypoglycemic treatments or history of diabetes mellitus. These alterations in blood glucose are potentially detrimental, although the precise relationship with outcome in patients with bacteremia has not been yet determined. Background: Altered blood glucose concentration is commonly observed in patients with sepsis, even among those without hypoglycemic treatments or history of diabetes mellitus. These alterations in blood glucose are potentially detrimental, although the precise relationship with outcome in patients with bacteremia has not been yet determined. Methods: A retrospective cohort study design for analyzing patients with Gram negative rod bacteremia was Background: Altered blood glucose concentration is commonly observed in patients with sepsis, even among those without hypoglycemic treatments or history of diabetes mellitus. These alterations in blood glucose are potentially detrimental, although the precise relationship with outcome in patients with bacteremia has not been yet determined. Methods: A retrospective cohort study design for analyzing patients with Gram negative rod bacteremia was employed, with the main outcome measure being in-hospital mortality. Patients were stratified in quintiles accordingly deviation of the blood glucose concentration from a central value with lowest mortality. Cox proportional-hazards regression model was used for determining the relationship of same day of bacteremia blood glucose and death. detrimental, although the precise relationship with outcome in patients with bacteremia has not been yet determined. Methods: A retrospective cohort study design for analyzing patients with Gram negative rod bacteremia was employed, with the main outcome measure being in-hospital mortality. Patients were stratified in quintiles accordingly deviation of the blood glucose concentration from a central value with lowest mortality. Cox proportional-hazards regression model was used for determining the relationship of same day of bacteremia blood glucose and death. Results: Of 869 patients identified 63 (7.4%) died. Same day of bacteremia blood glucose concentration had a U- shaped relationship with in-hospital mortality. The lowest mortality (2%) was detected in the range of blood glucose concentration from 150 to 160 mg/dL. Greater deviation of blood glucose concentration from the central value of this range (155 mg/dL, reference value) was directly associated with higher risk of death (p = 0.002, chi for trend). The low- risk group (quintile 1) had a mortality of 3.3%, intermediate-risk group (quintiles 2, 3 and 4) a mortality of 7.1%, and the high-risk group (quintile 5) a mortality of 12.05%. © 2010 Peralta 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 In a multivariable Cox regression model, the hazard ratio for death among patients in the intermediate-risk group as compared with that in the low risk group was 2.88 (95% confidence interval, 1.01 to 8.18; P = 0.048), and for the high risk group it was 4.26 (95% confidence interval, 1.41 to 12.94; P = 0.01). Conclusions: Same day of bacteremia blood glucose concentration is related with outcome of patients with Gram- negative rod bacteremia. Lowest mortality is detected in patients with blood glucose concentration in an interval of 150-160 mg/dL. Deviations from these values are associated with an increased risk of death. Conclusions: Same day of bacteremia blood glucose concentration is related with outcome of patients with Gram- negative rod bacteremia. Lowest mortality is detected in patients with blood glucose concentration in an interval of 150-160 mg/dL. Deviations from these values are associated with an increased risk of death. Research article Altered blood glucose concentration is associated with risk of death among patients with community-acquired Gram-negative rod bacteremia Galo Peralta*1, M Blanca Sánchez2, J Carlos Garrido3, Begoña Ceballos4, Fátima Mateos3, Inés De Benito5 and M Pía Roiz6 Peralta et al. BMC Infectious Diseases 2010, 10:181 Peralta et al. BMC Infectious Diseases 2010, 10:181 Peralta et al. BMC Infectious Diseases 2010, 10:181 http://www.biomedcentral.com/1471-2334/10/181 Background viously selected for defining outcome in observational studies with different populations as patients with stroke [10], head injuries [11], severe trauma [12], myocardial infarction [13], community acquired pneumonia [14], or patients admitted in a hospital through an emergency department [15]. Community acquired Gram-negative bacillus bacteremia (GNB) is a leading cause of hospitalization, sepsis and mortality [1]. Altered blood glucose concentration is fre- quently detected in patients with sepsis and has been associated with adverse outcome [2-9]. Variable cut-off values of hospital admission blood glucose concentra- tions with ranges from 100 to 200 mg/dL have been pre- Based on the negative effects of hyperglycemia in criti- cally ill patients, detected in observational studies, several clinical trials that have tried to prove the benefits of its prevention with intensive insulin therapy with disap- pointing results. One of the reasons of these results is the potential effect of tight control of glucose blood concen- Peralta et al. BMC Infectious Diseases 2010, 10:181 http://www.biomedcentral.com/1471-2334/10/181 Page 2 of 9 informed consent due to the retrospective nature of the study. trations causing hypoglycemia [16-18]. In fact hypoglyce- mia is also associated with negative effect over prognosis. In this situation there is a debate about the relevance of control blood glucose range in critically ill patients in general and specifically in patients with sepsis [19,20]. Hospital setting and patients The Sierrallana Hospital is an adult acute-care center in Torrelavega, a city in the North of Spain, which forms part of a health district of 160000 inhabitants. It is a com- munity teaching hospital with 250 beds that has approxi- mately 8000 admissions and 65000 patient visits to the emergency service annually. It includes most major departments and specialties, except transplantation, burns, thoracic surgery, cardiovascular surgery and neu- rosurgery units. Blood cultures are performed at the hos- pital in around 1800 patients per year. A retrospective cohort study design was employed, with the main outcome measure being in-hospital mor- tality. Patients with Gram-negative rod bacteremia (GNB) from January 1997 to December 2006 were identified by using the microbiology laboratory database and their charts were reviewed retrospectively with a standardized data collection form. Only the community acquired epi- sodes considered accordingly CDC criteria [21] were included in this study cohort. Only the first episode of bacteremia on each admission was included. One hun- dred and twenty six nosocomial episodes and 23 episodes with no blood glucose measurement recorded during the same day of the bacteremia were excluded. The following data were collected: age; sex; comorbid conditions; dates of hospital admission and discharge; presence of septic shock at the moment of blood culture extraction; specific antimicrobials administered during hospitalization; dates of start and end of antimicrobial administration; surgical procedures and hospitalization in an intensive care unit. The presence of each of the following comorbid condi- tions was assessed at the time of bacteremia: hepatic dys- function, malignancy, diabetes mellitus, renal insufficiency, human immunodeficiency virus infection, neutropenia, corticosteroid use, previous transplantation, use of an immunosuppressive agent in the preceding 30 days. The study was approved by the Institutional Review Board of the hospital (Comité Ético de Investigación Clínica de Cantabria), which waived the need for Blood cultures The common practice for blood cultures at our hospital is to obtain 20 mL of venous blood and to inoculate it in equal parts into one aerobic (BacT/ALERT FA aerobic, bioMérieux Corporation, Durham, North Carolina) and one anaerobic blood culture bottle (BacT/ALERT FN, bioMérieux). Blood drawn is performed by nurses from a peripheral vein three times at intervals of 30 minutes. As a routine practice at our hospital a positive finding of microbial growth in the blood culture is reported to the attending physicians before the results of antimicrobial susceptibility test are known and organism identification is established. Definitions A localized focus infection was considered to be the source or primary focus of bacteremia. Bacteremia was considered to have been nosocomially acquired if it appeared 48 h after admission and no evidence of infec- tion was present on admission. The bacteremia was cate- gorized as polymicrobial if additional microorganisms were recovered from the blood cultures. The source of the bacteraemia was determined on the basis of the isola- tion of the same microorganism from the presumed por- tal of entry and clinical evaluation. Renal insufficiency was indicated by a creatinine value of 2.0 mg/dL. Neutro- penia was defined as an absolute neutrophil count ≤500 cells/mm3 at the onset of the bacteraemia. Immunosup- pression was defined as the presence of neutropenia or HIV infection (with CD4 count ≤350 cells/mm3), or receipt of immunosuppressive agents. Comorbidities were assessed by using the Charlson comorbidity score [22]. Septic shock was defined as proposed by Bone et al [23]. Empirical antimicrobial therapy was judged to be either adequate or inadequate on the basis of the in vitro susceptibility of an isolated organism, and/or the initia- tion of antibiotic treatment within 24 h of blood culture extraction. Oxyimino-b-lactams (cefuroxime, cefotaxime, ceftriaxone, ceftazidime and aztreonam) were considered to be inappropriate regardless of the MIC for the treat- ment of infections caused by ESBL-producing Gram-neg- ative microorganisms. Therapy with urinary antiseptics such as norfloxacin, fosfomycin, pipemidic acid or nali- dixic acid was considered inadequate [24]. As there is limited information about outcome in rela- tion with blood glucose concentrations in patients with sepsis and/or bacteremia, we conducted a hospital-based retrospective cohort study in order to investigate the association between same day of bacteremia blood glu- cose concentration (SDBGC) and mortality in patients with GNB. Same day of bacteremia blood glucose measurement In patients with more than one blood glucose measure- ment obtained on the same day of the bacteremia only the first one was considered for the analysis. Blood glucose tests were performed at the clinical chemistry laboratory on venous samples by the glucose oxidase method. Peralta et al. BMC Infectious Diseases 2010, 10:181 http://www.biomedcentral.com/1471-2334/10/181 Peralta et al. BMC Infectious Diseases 2010, 10:181 http://www.biomedcentral.com/1471-2334/10/181 Page 3 of 9 Table 1: Demographic and clinical characteristics of patients with bloodstream infections due to Gram- negative bacilli. Characteristics Value Demographics Male sex 458 (52.7) Age >65 y 635 (73.1) Comorbidity Diabetes mellitus 139 (16) Neoplasm 81 (9.3) COPD 81 (9.4) Dementia 71 (8.2) Liver cirrhosis 48 (5.5) Stroke 51 (5.9) Immunosuppression 37 (4.3) Chronic renal failure 29 (3.4) Charlson index ≥3 145 (16.7) Microorganism E. coli 710 (81.7) Klebsiella spp. 53 (6.1) Proteus spp. 44 (5.1) Salmonella spp. 39 (4.5) Enterobacter spp. 22 (2.5) Polymicrobial bacteremia 67 (7.7) Adequate empirical treatment 648 (74.6) Presentation Patient population During the study period, blood cultures were performed in 18094 patients and were positive in 2678. Among these, 1018 patients were identified as having GNB. After considering inclusion/exclusion criteria, 869 cases were analyzed. The demographic and clinical characteristics of these patients are listed in Table 1. The median age of the patients was 75 years (interquartile range, 64-82 years). In 721 (83%) blood cultures were obtained at the emergency department, and in 821 (94.5%) blood glucose determina- tion was performed in samples obtained in the emer- gency department. Of the selected patients, 672 (77.3%) were hospitalized and the remainder were diagnosed of bacteremia after the patients had been discharged from the emergency department and re-evaluated in an outpa- tient clinic. The main sites of infection were as follows: urinary in 484 patients (55.7%), biliary in 157 (18.1%); unknown in 116 (13.3%); abdominal in 37 (4.3%); enteric in 28 (3.2%); pneumonia in 16 (1.8%), skin in 13 (1.5%) and spontane- ous bacterial peritonitis in 8 (0.9%). Statistical analysis Table 1: Demographic and clinical characteristics of patients with bloodstream infections due to Gram- negative bacilli. Bivariable analyses were conducted to determine the association between potential risk factors and mortality. Of primary interest was the association between blood glucose concentration and in hospital mortality. Categor- ical data were compared by the chi-square or Fisher's exact tests. Quantitative data were compared by Student's t test or the Mann-Whitney U test, as appropriate. To evaluate the effect of different levels of blood glucose concentration on mortality, relative risks and 95 percent confidence intervals were calculated as hazard ratios derived from the Cox proportional-hazards regression model. Multivariable models were fitted with use vari- ables with a P value < 0.1 in the univariate. The level of significance was set at P < 0.05. The SPSS (version 14) software package was used for all analyses. Discussion cose concentration respect the reference value and mor- tality was detected (Figure 2). In this cohort study of patients with GNB, SDBGC was independently associated with prognosis. SDBGC had a U-shaped relationship with in-hospital mortality with the lowest mortality in patients with SDBGC of 150-160 mg/ dL. Based on the deviation of blood glucose concentra- tion from the value with the lowest mortality, we classi- fied patients in groups with low, intermediate, and high risk categories of death. As compared with the low risk blood glucose concentration category, intermediate risk concentration category and high risk category were asso- ciated with hazard ratios for death of 2.88 and 4.6, respec- tively. The increased risk was similar for patients without diabetes mellitus. Although several other studies have made risk assessments for patients with GNB, this is, to our knowledge, the first observational study to evaluate the effect of the deviation from a reference level of blood glucose concentration on outcome in this population. On the basis of the findings presented in figure 2, we combined subgroups into three categories designated as low-risk (quintile 1, corresponding to blood glucose con- centration from 140 to 170 mg/dL), intermediate-risk (quintiles 2, 3 and 4, corresponding to blood glucose con- centration from 93 mg/dL to 139 mg/dL and from 171 mg/dL to 217 mg/dL), and high-risk (quintile 5, corre- sponding to blood glucose concentration <93 mg/dL or greater than 217 mg/dL). Mortality in the intermediate- risk category was 7.1% (95% Confidence interval: 5% to 9.3%), which was similar to the average mortality of 7.4% for the entire cohort. Blood glucose concentrations The range of SDBGC was 52 to 582 mg/dL. The relation- ship of mortality and blood glucose concentration showed an asymmetric U shape distribution with the lowest mortality among patients with glucose blood con- centration of 151-160 mg/dL (Figure 1). Patients were divided into subgroups according the dif- ference of the blood glucose concentration with the cen- tral value of the interval with lowest mortality (155 mg/ dL, reference value). Characteristics of these subgroups (quintiles) are reflected in table 2. The proportion of patients with diabetes mellitus or liver cirrhosis was much higher among those classified in the 5th quintile. A relationship between the absolute difference of blood glu- Page 4 of 9 Peralta et al. BMC Infectious Diseases 2010, 10:181 http://www.biomedcentral.com/1471-2334/10/181 Septic shock 9 (1.0) Outcome Required ICU 29 (3.3) In hospital mortality 63 (7.2) Data are no. (%) of patients. ICU: Intensive care unit. Table 1: Demographic and clinical characteristics of patients with bloodstream infections due to Gram- negative bacilli. (Continued) Septic shock 9 (1.0) Outcome Required ICU 29 (3.3) In hospital mortality 63 (7.2) Data are no. (%) of patients. ICU: Intensive care unit. Table 1: Demographic and clinical characteristics of patients with bloodstream infections due to Gram- negative bacilli. (Continued) Table 1: Demographic and clinical characteristics of patients with bloodstream infections due to Gram- negative bacilli. (Continued) blood glucose concentration as compared with that in the low risk group was 2.88 (95 percent confidence interval, 1.01 to 8.18; P = 0.048), and for the high risk group it was 4.26 (95 percent confidence interval, 1.41 to 12.94; P = 0.01). Other factors associated with death are reflected in table 4. When we selected non-diabetic patients for the analysis the hazard ratio for death among patients in the intermediate-risk group according to blood glucose con- centration as compared with that in the low risk group was 2.76 (95 percent confidence interval, 0.96 to 7.98; P = 0.06), and for the high risk group 5.11 (95 percent confi- dence interval, 1.55 to 16.92; P = 0.008). Data are no. (%) of patients. ICU: Intensive care unit. Predictors of mortality Risk factors for death in a univariate analysis are reflected in table 3. Kaplan-Meier estimates of survival for all sub- jects according to blood glucose concentration risk cate- gories are shown in figure 3. In a multivariable Cox regression model, the hazard ratio for death among patients in the intermediate-risk group according to Hyperglycemia associated with insulin resistance is common in critically ill patients and, although it can be considered an adaptive response, it has been associated with outcome [2,7,16-18]. Several observational studies have demonstrated that elevated initial blood glucose is independently associated with prognostic of severe acute illness [25]. However, the specific consequences of hyper- glycemia in patients with sepsis have not quite been addressed [7,8,19,26]. Hypoglycemia is also a manifesta- tion of sepsis [2-5] and is associated with a worse out- come in hospitalized patients with acute illnesses [4,6] and specifically in patients with E. coli bacteremia [5]. Figure 1 In hospital mortality according to blood glucose con- centration. The numbers inside the bars reflect the number of pa- tients in each group. >190 181- 190 171- 180 161- 170 151- 160 141- 150 131- 140 121- 130 111- 120 101- 110 ≤100 Blood glucose concentration (mg/dL) 16 14 12 10 8 6 4 2 0 Mortality (%) 155 27 34 48 50 73 82 100 98 73 66 20 3 3 2 1 3 4 6 7 7 7 Surviving Exitus >190 181- 190 171- 180 161- 170 151- 160 141- 150 131- 140 121- 130 111- 120 101- 110 ≤100 Blood glucose concentration (mg/dL) 16 14 12 10 8 6 4 2 0 Mortality (%) 155 27 34 48 50 73 82 100 98 73 66 20 3 3 2 1 3 4 6 7 7 7 Surviving Exitus As the initial altered blood glucose has been considered detrimental in the last years several studies have evalu- ated the effect of controlling blood glucose concentra- tions in critically ill patients with insulin therapy over mortality. A prospective randomized non-blind trial showed that critical medical intensive care unit patients had a slight and not significant reduction in ICU mortal- ity of 2.8% when treated with intensive insulin therapy to achieve target levels of blood glucose concentration from 80 to 100 mg/dL when compared with patients receiving conventional glucose control (target blood glucose level Figure 1 In hospital mortality according to blood glucose con- centration. Predictors of mortality 2.2 3.7 1.2 1.2 4.2 0.25 Adequate empirical treatment 82.6 80.3 85.8 81.8 83.7 0.7 Polymicrobial bacteremia 8.2 7.4 9.2 5.4 8.4 0.74 Presentation Septic shock 0 0.5 1.8 1.2 1.8 0.35 Outcome Required ICU 4.4 1.6 3.1 4.2 3.6 0.58 Death 3.3 6.3 7.4 7.8 12.0 0.03 Data are % of patients. ICU: Intensive care unit. Table 2: Baseline characteristics of patients with GNB according to quintiles of the absolute difference of the SDBGC respect the reference value of lowest mortality (155 mg/dL). (Continued) e characteristics of patients with GNB according to quintiles of the absolute difference of the SDBGC rence value of lowest mortality (155 mg/dL). (Continued) Data are % of patients. ICU: Intensive care unit. 180-220 mg/dL) [17]. The study only demonstrated a beneficial effect after the first three days of the intensive insulin therapy [17]. Another multicenter randomized prospective trial of intensive versus conventional glyce- mic control in severe sepsis was closed prematurely because of a nearly six fold increase in severe hypoglyce- mia among the patients in the intensive glycemic control treated group [26]. Moreover, the selection of a strict tar- get blood glucose concentration is not supported by some observational studies, which reveal that optimal blood glucose concentration for these critically ill patients may be higher [25]. On the other hand intensive insulin ther- apy is potentially detrimental per se [16,17,27]. Although hypoglycemia in hospitalized patients is harmful [4,6,26,27], no information is reflected in the literature about the optimal management of hypoglycemia in patients with sepsis, except for causal treatment when feasible. 5 4 3 2 1 Quintile of the difference of blood glucose concentration 14 12 10 8 6 4 2 0 Mortality (%) 146 154 151 178 177 20 13 12 12 6 Surviving Exitus Figure 2 Mortality according to deviation of blood glucose con- centration with the reference blood glucose concentration of 155 mg/dL which is the central value of the interval with the lowest mortality. Patients are distributed in quintiles of the difference in blood glucose concentration. The numbers inside the bars reflect the number of patients in each group (p = 0.002 chi square for linear trend). Predictors of mortality The numbers inside the bars reflect the number of pa- tients in each group. Figure 1 In hospital mortality according to blood glucose con- centration. The numbers inside the bars reflect the number of pa- tients in each group. Figure 1 In hospital mortality according to blood glucose con- centration. The numbers inside the bars reflect the number of pa- tients in each group. Peralta et al. BMC Infectious Diseases 2010, 10:181 Peralta et al. BMC Infectious Diseases 2010, 10:181 http://www.biomedcentral.com/1471-2334/10/181 Page 5 of 9 Table 2: Baseline characteristics of patients with GNB according to quintiles of the absolute difference of the SDBGC respect the reference value of lowest mortality (155 mg/dL). 1st Quintile n = 183 2nd Quintile n = 190 3rd Quintile n = 163 4th Quintile n = 167 5th Quintile n = 166 p Range of difference in BGC with the reference value (mg/dL) ≤15 16-29 30-42 43-62 ≥63 - Demographics Male sex 59.0 53.7 54.0 54.5 41.6 0.02 Age >65 y 74.9 76.3 75.5 61.4 77.1 0.006 Comorbidity Diabetes mellitus 10.4 11.1 6.8 4.2 49.1 <0.001 Neoplasm 10.4 10.5 9.3 10.2 6.1 0.59 COPD 9.3 13.2 9.9 7.8 6.1 0.2 Dementia 6.0 6.8 8.0 7.9 12.7 0.19 Liver cirrhosis 4.4 3.2 6.2 4.2 10.3 0.04 Stroke 5.5 6.3 3.1 6.6 7.9 0.44 Immunosuppression 4.4 2.1 3.7 6.0 5.5 0.39 Chronic renal failure 2.7 2.1 4.3 2.4 5.5 0.37 Charlson ≥3 11.5 16.1 13.8 18.5 14.8 0.57 Origin of infection Urinary 49.5 55.0 55.3 63.0 59.1 0.12 Biliary 20.3 21.2 22.4 13.9 12.8 0.07 Unknown 17.6 12.2 12.4 9.1 15.9 0.16 Intra-abdominal 4.4 3.7 4.3 4.8 4.3 1 Pneumonia 1.1 1.6 1.2 2.4 3.0 0.64 Microorganism E. coli 82.0 83.2 82.2 82.6 78.3 0.8 cteristics of patients with GNB according to quintiles of the absolute difference of the SDBGC alue of lowest mortality (155 mg/dL). Peralta et al. BMC Infectious Diseases 2010, 10:181 Peralta et al. BMC Infectious Diseases 2010, 10:181 http://www.biomedcentral.com/1471-2334/10/181 Page 6 of 9 Klebsiella spp. 6.0 3.7 6.7 6.6 7.8 0.56 Proteus spp. 4.4 5.8 6.1 4.8 4.2 0.9 Salmonella spp. 5.5 3.7 3.7 4.2 5.4 0.86 Enterobacter spp. Predictors of mortality 5 4 3 2 1 Quintile of the difference of blood glucose concentration 14 12 10 8 6 4 2 0 Mortality (%) 146 154 151 178 177 20 13 12 12 6 Surviving Exitus Our results are consistent with some previous studies. Finney et al [25] found in an observational study that a target blood glucose level of around 145 mg/dL may be associated with a better outcome in critically ill patients. We have detected similar differences in mortality of 2.7% than a previous randomized clinical trial, when we com- pare patients with blood glucose concentration of 80-100 mg/dL and 180-200 mg/dL that were the end points defined in that trial [17]. However, the optimal blood glu- cose levels may be different after the first days of hospital admission [17] and our data are referred to the day of the bacteremia. One recent observational study from Austra- lia in critically ill patients has detected also a U shape relationship of blood glucose concentration with mortal- Quintile of the difference of blood glucose concentration Figure 2 Mortality according to deviation of blood glucose con- centration with the reference blood glucose concentration of 155 mg/dL which is the central value of the interval with the lowest mortality. Patients are distributed in quintiles of the difference in blood glucose concentration. The numbers inside the bars reflect the number of patients in each group (p = 0.002 chi square for linear trend). Figure 2 Mortality according to deviation of blood glucose con- centration with the reference blood glucose concentration of 155 mg/dL which is the central value of the interval with the lowest mortality. Patients are distributed in quintiles of the difference in blood glucose concentration. The numbers inside the bars reflect the number of patients in each group (p = 0.002 chi square for linear trend). Peralta et al. BMC Infectious Diseases 2010, 10:181 Peralta et al. BMC Infectious Diseases 2010, 10:181 http://www.biomedcentral.com/1471-2334/10/181 Page 7 of 9 Table 3: Univariate analysis of factors associated with mortality in patients with GNB. Predictors of mortality Surviving N = 806 Death N = 63 RR (95%CI) P Demographics Male 426 (52.9) 32 (50.8) 0.93 (0.58-1.49) 0.43 Age >65 y 581 (72.2) 54 (85.7) 2.20 (1.11-4.39) 0.01 Comorbidity Diabetes mellitus 130 (16.2) 9 (14.3) 0.87 (0.56-2.41) 0.43 Neoplasm 74 (9.2) 7 (11.1) 1.2 (0.57-2.57) 0.38 Immunosuppression 31 (3.9) 6 (9.5) 2.36 (1.08-5.11) 0.046 Liver cirrhosis 41 (5.1) 7 (11.1) 2.13 (1.02-4.41) 0.052 Chronic renal failure 45 (5.6) 6 (9.5) 1.68 (0.76-3.71) 0.16 Charlson index ≥3 129 (16) 21 (33.3) 2.4 (1.46-3.92) 0.001 Microorganism Escherichia coli 664 (82.4) 46 (73) 0.61 (0.36-1.03) 0.05 Klebsiella spp. 48 (6.0) 5 (7.9) 1.33(0.56-3.17) 0.34 Proteus spp. 40 (5.0) 4 (6.3) 1.27 (0.48-3.34) 0.4 Salmonella spp. 34 (4.2) 5 (7.9) 1.84 (0.78-4.32) 0.14 Enterobacter spp. 19 (2.4) 3 (4.8) 1.93 (0.65-5.67) 0.21 Polymicrobial bacteremia 56 (6.9) 11 (17.5) 2.56 (1.4-4.64) 0.006 Adequate empirical treatment 610 (83.7) 38 (70.4) 0.5 (0.28-0.86) 0.014 Origin of infection Urinary 466 (58.3) 18 (29.0) 0.32 (0.19-0.54) <0.0001 Biliary 147 (18.4) 10 (16.1) 0.86 (0.45-1.66) 0.4 Unknown 101 (12.6) 15 (24.2) 2.05 (1.19-3.54) 0.013 Intra-abdominal 32 (4.09 5 (8.1) 1.95 (0.83-4.58) 0.12 Table 3: Univariate analysis of factors associated with mortality in patients with GNB. Peralta et al. BMC Infectious Diseases 2010, 10:181 http://www.biomedcentral.com/1471-2334/10/181 Page 8 of 9 Pneumonia 10 (1.3) 6 (9.7) 5.66 (2.86-11.18) <0.001 Presentation Septic Shock 4 (0.5) 5 (7.9) 8.24 (4.37-15.54) <0.0001 Data are no. (%) of patients. RR: Relative risk Table 3: Univariate analysis of factors associated with mortality in patients with GNB. (Continued) ble 3: Univariate analysis of factors associated with mortality in patients with GNB. (Continued) Data are no. (%) of patients. RR: Relative risk tality would be the same after the initial phases of GNB. However as we select initial blood glucose concentration for the analysis, those are not influenced by therapies as insulin, glucose-containing solutions or parenteral nutri- tion used during hospitalizations. ity, with lowest levels of mortality in patients with blood glucose concentrations among 100-150 mg/dL [28]. Simi- larly with this study the U shape relationship of blood glucose concentration with mortality that we have detected is asymmetric and there was more increase in mortality in patients with high than with low blood glu- cose with respect the range with lowest mortality. Predictors of mortality If sep- sis related mortality has a direct relationship with high or low blood glucose concentration, it can be speculated that increasing blood glucose concentration to achieve an optimal target level could be of potential benefit. The threshold to indicate correction of low blood glucose concentration should first be determined [28]. In summary, our study indicates that low and high blood glucose concentrations are associated with higher mortality of patients with GNR bacteremia regardless of the presence of diabetes mellitus. Studies to determine the precise correlation between blood glucose concentra- tion and mortality with suspected bacteremia are needed. Conclusions Our study has several limitations. Data are retrospec- tive and the results came from a population with specific characteristics: patients with GNB, a low proportion of immunosuppression, stay in ICU, septic shock or death, so the extrapolation of these results must be made with caution. As has been commented, the measurements of blood glucose concentration were made at the beginning of the illness, so it remains unknown whether the blood glucose concentrations associated with the lowest mor- Same day of bacteremia blood glucose concentration is related with outcome of patients with Gram-negative rod bacteremia. Lowest mortality is detected in patients with blood glucose concentration in an interval of 150-160 Figure 3 Adjusted estimates of overall survival in patients with GNB according to risk groups. The three risk groups are based in the quintiles of the absolute difference in glucose blood concentration with the central value of the interval with lowest mortality: low risk group (quintile 1), intermediate risk group (quintiles 2, 3 and 4), high risk group (quintile 5). Survival estimates have been adjusted for the presence of age >65 years, Charlson index ≥3, presence of immuno- suppression, liver cirrhosis, urinary origin, lung origin, unknown origin, E. coli bacteremia, polymicrobial bacteremia, presence of shock and adequacy of empirical antibiotic treatment. P = 0.036 by the log-rank test for the overall comparison between groups. Table 4: Results of Cox analyses examining risk factors for mortality associated with bacteraemia due to Gram- negative bacilli. Mortality risk factor HR (95% CI) p value Charlson index ≥3 2.04 (1.12-3.69) 0.02 Urinary origin 0.44 (0.23-0.82) 0.01 Lung origin 4.14 (1.54-11.08) 0.005 Polymicrobial bacteremia 2.6 (1.28-5.28) 0.008 Adequate empirical treatment 0.43 (0.23-0.79) 0.006 Shock 9.31 (3.42-25.38) <0.001 Blood glucose category (high vs low risk) 4.26 (1.4-12.9) 0.01 HR: hazard ratio Table 4: Results of Cox analyses examining risk factors for mortality associated with bacteraemia due to Gram- negative bacilli. Figure 3 Adjusted estimates of overall survival in patients with GNB di t i k Th th i k b d i th Figure 3 Adjusted estimates of overall survival in patients with GNB according to risk groups. The three risk groups are based in the Figure 3 Adjusted estimates of overall survival in patients with GNB according to risk groups. Author Details 20. Inzucchi SE: Clinical practice. Management of hyperglycemia in the hospital setting. N Engl J Med 2006, 355:1903-11. 1Instituto de Investigación y Formación Marques de Valdecilla (IFIMAV), 5 Planta de la Escuela Universitaria de Enfermería, Avda de Valdecilla s/n, 39008, Santander, Spain, 2Clinical Pharmacology Service, Hospital Universitario "Marqués de Valdecilla", Santander, Spain, 3Biochemistry Service, Hospital Sierrallana, Torrelavega, Spain, 4Emergency Service, Hospital Sierrallana, Torrelavega, Spain, 5Microbiology Service, Hospital Sierrallana, Torrelavega, Spain and 6Microbiology Service, Hospital Universitario Marques de Valdecilla, Santander, Spain 1Instituto de Investigación y Formación Marques de Valdecilla (IFIMAV), 5 Planta de la Escuela Universitaria de Enfermería, Avda de Valdecilla s/n, 39008, Santander, Spain, 2Clinical Pharmacology Service, Hospital Universitario "Marqués de Valdecilla", Santander, Spain, 3Biochemistry Service, Hospital Sierrallana, Torrelavega, Spain, 4Emergency Service, Hospital Sierrallana, Torrelavega Spain 5Microbiology Service Hospital Sierrallana Torrelavega 21. Garner JS, Jarvis WR, Emori TG, Horan TC, Hughes JM: CDC definitions for nosocomial infections. Am J Infect Control 1988, 16:128-40. 22. Charlson ME, Pompei P, Ales KL, MacKenzie CR: A new method of classifying prognostic comorbidity in longitudinal populations: development and validation. J Chronic Dis 1987, 40:373-8. g p g y p Torrelavega, Spain, 5Microbiology Service, Hospital Sierrallana, Torrelavega, S d 6 b l S l d ld ll 23. Bone RC, Sprung CL, Sibbald WJ: Definitions for sepsis and organ failure. Crit Care Med 1992, 20:724-6. Spain and 6Microbiology Service, Hospital Universitario Marques de Valdecilla, Santander, Spain 24. Peralta G, Sanchez MB, Garrido JC, De Benito I, Cano ME, Martinez- Martinez L, Roiz MP: Impact of antibiotic resistance and of adequate empirical antibiotic treatment in the prognosis of patients with Escherichia coli bacteraemia. J Antimicrob Chemother 2007, 60:855-63. mg/dL. Deviations from these values are associated with an increased risk of death. 12. Laird AM, Miller PR, Kilgo PD, Meredith JW, Chang MC: Relationship of early hyperglycemia to mortality in trauma patients. J Trauma 2004, 56:1058-62. 13. Hsu CW, Chen HH, Sheu WH, Chu SJ, Shen YS, Wu CP, Chien KL: Initial serum glucose level as a prognostic factor in the first acute myocardial infarction. Ann Emerg Med 2007, 49:618-26. Competing interests Galo Peralta has been a consultant in the past for Janssen-Cilag, Wyeth, Bristol- Myers Squibb, and Boehringer Ingelheim, and has also served as a speaker for Wyeth and GlaxoSmithKline. 14. McAlister FA, Majumdar SR, Blitz S, Rowe BH, Romney J, Marrie TJ: The relation between hyperglycemia and outcomes in 2,471 patients admitted to the hospital with community-acquired pneumonia. Diabetes Care 2005, 28:810-5. References 1. Weinstein MP, Towns ML, Quartey SM, Mirrett S, Reimer LG, Parmigiani G, Reller LB: The clinical significance of positive blood cultures in the 1990s: a prospective comprehensive evaluation of the microbiology, epidemiology, and outcome of bacteremia and fungemia in adults. Clin Infect Dis 1997, 24:584-602. 26. Brunkhorst FM, Engel C, Bloos F, Meier-Hellmann A, Ragaller M, Weiler N, Moerer O, Gruendling M, Oppert M, Grond S, Olthoff D, Jaschinski U, John S, Rossaint R, Welte T, Schaefer M, Kern P, Kuhnt E, Kiehntopf M, Hartog C, Natanson C, Loeffler M, Reinhart K, German Competence Network Sepsis (SepNet): Intensive insulin therapy and pentastarch resuscitation in severe sepsis. N Engl J Med 2008, 358:125-39. 2. Mizock BA: Alterations in carbohydrate metabolism during stress: A review of the literature. Am J Med 1995, 98:75-84. 27. Krinsley JS: Glycemic control, diabetic status and mortality in a heterogeneous population of critically ill patients before and during the era of tight glycemic control. Semin Thorac Cardiovasc Surg 2006, 18:317-325. 3. Miller SI, Wallace RJ Jr, Musher DM, Septimus EJ, Kohl S, Baughn RE: Hypoglycemia as a manifestation of sepsis. Am J Med 1980, 68:649-54. 4. Shilo S, Berezovsky S, Friedlander Y, Sonnenblick M: Hypoglycemia in hospitalized nondiabetic older patients. J Am Geriatr Soc 1998, 46:978-82. 4. Shilo S, Berezovsky S, Friedlander Y, Sonnenblick M: Hypoglycemia in hospitalized nondiabetic older patients. J Am Geriatr Soc 1998, 46:978-82. 28. Bagshaw SM, Egi M, George C, Bellomo R, Australia New Zealand Intensive Care Society Database Management Committee: Early blood glucose control and mortality in critically ill patients in Australia. Crit Care Med 2009, 37:463-70. 5. Alamgir S, Volkova NB, Peterson MW: Prognostic value of low blood glucose at the presentation of E. coli bacteremia. Am J Med 2006, 119:952-7. 5. Alamgir S, Volkova NB, Peterson MW: Prognostic value of low blood glucose at the presentation of E. coli bacteremia. Am J Med 2006, 119:952-7. 6. Kagansky N, Levy S, Rimon E, Cojocaru L, Fridman A, Ozer Z, Knobler H: Hypoglycemia as a predictor of mortality in hospitalized elderly patients. Arch Intern Med 2003, 163:1825-9. 6. Kagansky N, Levy S, Rimon E, Cojocaru L, Fridman A, Ozer Z, Knobler H: Hypoglycemia as a predictor of mortality in hospitalized elderly patients. Arch Intern Med 2003, 163:1825-9. Conclusions The three risk groups are based in the quintiles of the absolute difference in glucose blood concentration with the central value of the interval with lowest mortality: low risk group (quintile 1), intermediate risk group (quintiles 2, 3 and 4), high risk group (quintile 5). Survival estimates have been adjusted for the presence of age >65 years, Charlson index ≥3, presence of immuno- suppression, liver cirrhosis, urinary origin, lung origin, unknown origin, E. coli bacteremia, polymicrobial bacteremia, presence of shock and adequacy of empirical antibiotic treatment. P = 0.036 by the log-rank test for the overall comparison between groups. g g p g p quintiles of the absolute difference in glucose blood concentration with the central value of the interval with lowest mortality: low risk group (quintile 1), intermediate risk group (quintiles 2, 3 and 4), high risk group (quintile 5). Survival estimates have been adjusted for the presence of age >65 years, Charlson index ≥3, presence of immuno- suppression, liver cirrhosis, urinary origin, lung origin, unknown origin, E. coli bacteremia, polymicrobial bacteremia, presence of shock and adequacy of empirical antibiotic treatment. P = 0.036 by the log-rank test for the overall comparison between groups. Page 9 of 9 Peralta et al. BMC Infectious Diseases 2010, 10:181 http://www.biomedcentral.com/1471-2334/10/181 Received: 3 November 2009 Accepted: 22 June 2010 Published: 22 June 2010 This article is available from: http://www biomedcentral com/1471-2334/10/181 © 2010 Peralta 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 re BMC Infectious Diseases 2010 10:181 25. Finney SJ, Zekveld C, Elia A, Evans TW: Glucose critically ill patients. JAMA 2003, 290:2041-7. 25. Finney SJ, Zekveld C, Elia A, Evans TW: Glucose control and mortality in critically ill patients. JAMA 2003, 290:2041-7. Authors' contributions GP, MPR, MBS, and JCG were involved in the study conception, BC, FT and IDB were involved in the coordination, and data acquisition, GP performed the data analyses, all authors were involved in the interpretation and validation of the results, GP, MPR and MBS were involved in the drafting of the manuscript and all authors read and approved the final manuscript. 15. Cheung NW, Li S, Ma G, Crampton R: The relationship between admission blood glucose levels and hospital mortality. Diabetologia 2008, 51:952-5. 16. Van den Berghe G, Wouters P, Weekers F, Verwaest C, Bruyninckx F, Schetz M, Vlasselaers D, Ferdinande P, Lauwers P, Bouillon R: Intensive insulin therapy in critically ill patients. N Engl J Med 2001, 345:1359-1367. Acknowledgements 17. Van den Berghe G, Wilmer A, Hermans G, Meersseman W, Wouters PJ, Milants I, Van Wijngaerden E, Bobbaers H, Bouillon R: Intensive insulin therapy in the medical ICU. N Engl J Med 2006, 354:449-61. The authors are indebted to all who participated in this study. Thanks to all the health care professionals of the Sierrallana Hospital, and specifically those from the Internal Medicine Service, the Emergency Service, the Microbiology Ser- vice and the Biochemistry Service with a special mention to Dra. Rodriguez- Lera. 18. Vanhorebeek I, Langouche L, Van den Berghe G: Tight blood glucose control with insulin in the ICU: facts and controversies. Chest 2007, 132:268-78. 19. Russell JA: Management of sepsis. N Engl J Med 2006, 355:1699-713. Pre-publication history h bl h The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-2334/10/181/prepub The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-2334/10/181/prepub p 7. Bader MS: Hyperglycemia and mortality in elderly patients with Staphylococcus aureus bacteremia. South Med J 2007, 100:252-6. p 7. Bader MS: Hyperglycemia and mortality in elderly patients with Staphylococcus aureus bacteremia. South Med J 2007, 100:252-6. doi: 10.1186/1471-2334-10-181 Cite this article as: Peralta et al., Altered blood glucose concentration is associated with risk of death among patients with community-acquired Gram-negative rod bacteremia BMC Infectious Diseases 2010, 10:181 8. Frankenfield DC, Omert LA, Badellino MM, Wiles CE, Bagley SM, Goodarzi S, Siegel JH: Correlation between measured energy expenditure and clinically obtained variables in trauma and sepsis patients. JPEN J Parenter Enteral Nutr 1994, 18:398-403. 9. Krinsley JS: Association between hyperglycemia and increased hospital mortality in a heterogeneous population of critically ill patients. Mayo Clin Proc 2003, 78:1471-8. 10. Capes SE, Hunt D, Malmberg K, Pathak P, Gerstein HC: Stress hyperglycemia and prognosis of stroke in nondiabetic and diabetic patients. Stroke 2001, 32:2426-2436. p 11. Jeremitsky E, Omert LA, Dunham CM, Wilberger J, Rodriguez A: The impact of hyperglycemia on patients with severe brain injury. J Trauma 2005, 58:47-50. 11. Jeremitsky E, Omert LA, Dunham CM, Wilberger J, Rodriguez A: The impact of hyperglycemia on patients with severe brain injury. J Trauma 2005, 58:47-50.
https://openalex.org/W2789124133	https://escholarship.org/content/qt75b140sg/qt75b140sg.pdf?t=pnl3pn	English	null	Efficient pedigree recording for fast population genetics simulation	bioRxiv (Cold Spring Harbor Laboratory)	2,018	cc-by	13,091	UC Irvine UC Irvine Previously Published Works Title Efficient pedigree recording for fast population genetics simulation Permalink https://escholarship.org/uc/item/75b140sg Journal PLOS Computational Biology, 14(11) ISSN 1553-734X Authors Kelleher, Jerome Thornton, Kevin R Ashander, Jaime et al. Publication Date 2018 DOI 10.1371/journal.pcbi.1006581 Copyright Information This work is made available under the terms of a Creative Commons Attribution License availalbe at https://creativecommons.org/licenses/by/4.0/ Peer reviewed UC Irvine UC Irvine Previously Published Works Title Efficient pedigree recording for fast population genetics simulation Permalink https://escholarship.org/uc/item/75b140sg Journal PLOS Computational Biology, 14(11) ISSN 1553-734X Authors Kelleher, Jerome Thornton, Kevin R Ashander, Jaime et al. Publication Date 2018 DOI 10.1371/journal.pcbi.1006581 Copyright Information This work is made available under the terms of a Creative Commons Attribution License availalbe at https://creativecommons.org/licenses/by/4.0/ Peer reviewed UC Irvine UC Irvine Previously Published Works Title Efficient pedigree recording for fast population genetics simulation Permalink https://escholarship.org/uc/item/75b140sg Journal PLOS Computational Biology, 14(11) ISSN 1553-734X Authors Kelleher, Jerome Thornton, Kevin R Ashander, Jaime et al. Publication Date 2018 DOI 10.1371/journal.pcbi.1006581 Copyright Information This work is made available under the terms of a Creative Commons availalbe at https://creativecommons.org/licenses/by/4.0/ Peer reviewed RESEARCH ARTICLE Editor: Sergei L. Kosakovsky Pond, Temple University, UNITED STATES Editor: Sergei L. Kosakovsky Pond, Temple University, UNITED STATES Received: January 26, 2018 Accepted: October 8, 2018 Published: November 1, 2018 Copyright: © 2018 Kelleher et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: The only relevant data is our computer code; the main tools are available at https://github.com/jeromekelleher/ msprime or https://pypi.python.org/pypi/msprime – and other scripts used in the paper are available at https://github.com/petrelharp/ftprime_ms. Funding: Work on this project was supported by funding from the Sloan Foundation and the National Science Foundation (under DBI-1262645) to PLR; the Wellcome Trust (grant 100956/Z/13/Z) to Gil McVean; the NIH (R01GM115564) to KRT; and the USF&WS to H. Bradley Shaffer. The Jerome KelleherID1, Kevin R. ThorntonID2, Jaime AshanderID3, Peter L. RalphID4* Jerome KelleherID1, Kevin R. ThorntonID2, Jaime AshanderID3, Peter L. RalphID4* 1 Big Data Institute, University of Oxford, Oxford, United Kingdom, 2 Ecology and Evolutionary Biology, University of California, Irvine, Irvine, California, United States of America, 3 Ecology and Evolutionary Biology, University of California, Los Angeles, Los Angeles, United States of America, 4 Institute for Ecology and Evolution, University of Oregon, Eugene, Oregon, United States of America * plr@uoregon.edu a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Abstract In this paper we describe how to efficiently record the entire genetic history of a population in forwards-time, individual-based population genetics simulations with arbitrary breeding models, population structure and demography. This approach dramatically reduces the computational burden of tracking individual genomes by allowing us to simulate only those loci that may affect reproduction (those having non-neutral variants). The genetic history of the population is recorded as a succinct tree sequence as introduced in the software pack- age msprime, on which neutral mutations can be quickly placed afterwards. Recording the results of each breeding event requires storage that grows linearly with time, but there is a great deal of redundancy in this information. We solve this storage problem by providing an algorithm to quickly ‘simplify’ a tree sequence by removing this irrelevant history for a given set of genomes. By periodically simplifying the history with respect to the extant population, we show that the total storage space required is modest and overall large efficiency gains can be made over classical forward-time simulations. We implement a general-purpose framework for recording and simplifying genealogical data, which can be used to make sim- ulations of any population model more efficient. We modify two popular forwards-time simu- lation frameworks to use this new approach and observe efficiency gains in large, whole- genome simulations of one to two orders of magnitude. In addition to speed, our method for recording pedigrees has several advantages: (1) All marginal genealogies of the simulated individuals are recorded, rather than just genotypes. (2) A population of N individuals with M polymorphic sites can be stored in O(N log N + M) space, making it feasible to store a simu- lation’s entire final generation as well as its history. (3) A simulation can easily be initialized with a more efficient coalescent simulation of deep history. The software for recording and processing tree sequences is named tskit. Powered by the California Digital Library University of California eScholarship.org eScholarship.org OPEN ACCESS Citation: Kelleher J, Thornton KR, Ashander J, Ralph PL (2018) Efficient pedigree recording for fast population genetics simulation. PLoS Comput Biol 14(11): e1006581. https://doi.org/10.1371/ journal.pcbi.1006581 Introduction Since the 1980’s, coalescent theory has enabled computer simulation of the results of popula- tion genetics models identical to that which would be produced by large, randomly mating populations over long periods of time without actually requiring simulation of so many gener- ations or meioses. Coalescent theory thus had three transformative effects on population genetics: first, giving researchers better conceptual tools to describe gene trees and thus bring- ing within-population trees into better focus; second, producing analytical methods to esti- mate parameters of interest from genetic data; and finally, providing a computationally feasible method to produce computer simulations of population genetics processes. However, these powerful advances came with substantial caveats: the backwards-in-time processes that are described by coalescent theory are only Markovian, and thus feasible to work with, under the following important assumptions: (a) random mating, (b) neutrality, (c) large population size, and (d) small sample size relative to the population size. The first two assumptions can be side-stepped to a limited extent [1, 2], but it remains a challenge to map results of coalescent models onto species that are distributed across continuous geographical space [3, 4] and/or have large numbers of loci under various sorts of selection. Usually, the relationship between the life history of a species—fecundity and mortality schedules, density-dependent effects on fitness, and demographic fluctuations—are all absorbed into a single compound parameter, the coalescence rate. More mechanistic models are possible using “forwards–backwards” sim- ulations, that first simulate population size changes forwards in time and then thread a coales- cent backwards [5], but these still require the assumptions above to be met for each subpopulation. The last assumption is no longer safe, either—for example, a recent study [6] simulated 600,000 samples of human chromosome 20 to examine biases in GWAS. Several studies have now shown that in samples approaching the size of the population, genealogical properties may be distorted relative to the coalescent expectation [7–9]. These considerations, and increasing computational power, have led to a resurgence of interest in large forwards- time, individual-based simulations. For instance, Harris and Nielsen [10] used SLiM [11] to simulate ten thousand human exomes to assess the impact of genetic load and Neanderthal introgression on human genetic diversity. Sanjak et al. Author summary Sexually reproducing organisms are related to the others in their species by the complex web of parent-offspring relationships that constitute the pedigree. In this paper, we 1 / 21 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1006581 November 1, 2018 Pedigree recording for simulation funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. describe a way to record all of these relationships, as well as how genetic material is passed down through the pedigree, during a forwards-time population genetic simulation. To make effective use of this information, we describe both efficient storage methods for this embellished pedigree as well as a way to remove all information that is irrelevant to the genetic history of a given set of individuals, which dramatically reduces the required amount of storage space. Storing this information allows us to produce whole-genome sequence from simulations of large populations in which we have not explicitly recorded new genomic mutations; we find that this results in computational run times of up to 50 times faster than simulations forced to explicitly carry along that information. Competing interests: The authors have declared that no competing interests exist. Competing interests: The authors have declared that no competing interests exist. This is a PLoS Computational Biology Methods paper. Pedigree recording for simulation individuals, using the output to explore the relationship between the genotype/phenotype model and GWAS outcomes. Modern computing power easily allows simulations of birth, death and reproduction in a population having even hundreds of millions of individuals. However, if our interest lies in the resulting genetic patterns of variation—and often, the point of such simulations is to compare to real data—then such simulations must record each individual’s genome. As samples of most species’ genomes harbor tens or hundreds of millions of variant sites, carrying full genotypes for even modest numbers of individuals through a simulation can quickly become prohibitive. To make matters worse, a population of size N must be simulated across many multiples of N generations to produce stable genetic patterns [14, 15]. Because of this computational burden, even the fastest simulation frameworks such as SLiM 2 [16] and fwdpp [13] can “only” simu- late tens of megabases of sequence in tens of thousands of individuals for tens of thousands of generations. In practice, current state-of-the-art simulation software may take on the order of weeks to simulate models of large genomic regions without selection [13, 17], and existing sim- ulation engines differ in how efficiently they calculate fitnesses in models with selection [13]. These population and region sizes are still substantially short of whole genomes (hundreds to thousands of megabases) for many biological population sizes of interest. However, it is thought that most genetic variation is selectively neutral (or nearly so). By definition, neutral alleles carried by individuals in a population do not affect the population process. For this reason, if one records the entire genealogical history of a population over the course of a simulation, simply laying down neutral mutations on top of that history afterwards is equivalent to having generated them during the simulation: it does not matter if we generate each generation’s mutations during the simulation, or afterwards. To add mutations after the fact, we need to know the genealogical trees relating all sampled individuals at each position along the genome. Combined with ancestral genotypes and the origins of new mutations, these trees completely specify the genomic sequence of any individual in the population at any time. Introduction [12] used fwdpp [13] to simulate a series of models of quantitative traits under mutation-selection balance with population sizes of 2 × 104 diploids in stable populations and populations growing up to around 5 × 105 Since the 1980’s, coalescent theory has enabled computer simulation of the results of popula- tion genetics models identical to that which would be produced by large, randomly mating populations over long periods of time without actually requiring simulation of so many gener- ations or meioses. Coalescent theory thus had three transformative effects on population genetics: first, giving researchers better conceptual tools to describe gene trees and thus bring- ing within-population trees into better focus; second, producing analytical methods to esti- mate parameters of interest from genetic data; and finally, providing a computationally feasible method to produce computer simulations of population genetics processes. However, these powerful advances came with substantial caveats: the backwards-in-time processes that are described by coalescent theory are only Markovian, and thus feasible to work with, under the following important assumptions: (a) random mating, (b) neutrality, (c) large population size, and (d) small sample size relative to the population size. The first two assumptions can be side-stepped to a limited extent [1, 2], but it remains a challenge to map results of coalescent models onto species that are distributed across continuous geographical space [3, 4] and/or have large numbers of loci under various sorts of selection. Usually, the relationship between the life history of a species—fecundity and mortality schedules, density-dependent effects on fitness, and demographic fluctuations—are all absorbed into a single compound parameter, PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1006581 November 1, 2018 2 / 21 Results The strategy described above is only of interest if it is computationally feasible. Therefore, we begin by benchmarking the performance improvement achieved by this method, implemented using the forwards-time simulation library fwdpp [13] and tree sequence tools implemented in tskit. Then, we describe the conceptual and algorithmic foundations for the method: (a) a format, implemented in the tskit Python API, for recording tree sequences efficiently in several tables; (b) an algorithm to record these tables during a forwards-time simulation; and (c) an algorithm to simplify a tree sequence, i.e., remove redundant information. Finally, we analyze the run time and space complexity of our general-purpose method. Pedigree recording for simulation marginal genealogies of individuals living at the end of the simulation. These marginal geneal- ogies enable fast data storage and processing, but also provide additional information that can be used to better understand the notoriously complex dynamics of population genetics. Although we were motivated by a need for more efficient genomic simulations, these tools may prove more widely useful. This work originated as improvements to the algorithmic tools and data structures in the coalescent simulator msprime. The software tools described here for working with tree sequences are referred to as tskit; they are currently bundled with the Python package msprime, but will soon be separately available as a Python API and an embeddable C library. To obtain this information, we record from forward simulation the population pedi- gree—the complete history of parent-offspring relationships of an entire population going back to a remote time—and the genetic outcomes of each ancestral meiosis, periodically dis- carding all information irrelevant to the genetic history of the extant population. The informa- tion in this embellished pedigree is stored as a succinct tree sequence (or, for brevity, “tree sequence”), which contains all the information necessary to construct the genealogical tree that relates each individual to every other at each position on the genome. The idea of storing genealogical information to speed up simulations is not new. It was implemented in AnA-FiTS [18], but without the critical step of discarding irrelevant genealogi- cal information. Padhukasahasram et al. [19] obtained impressive speedups for a Wright– Fisher simulation by keeping track of genealogies over the preceding 8 generations and only tracking neutral genotypes for those segments having descendants across this window. Our approach is similar, but uses genealogies across the entire duration of the simulation. The embellished pedigree is equivalent to the ancestral recombination graph, or ARG [20, 21], which has been the subject of substantial study [22–25]. However, it is unclear if an ARG-based approach would share the computational advantages of the data structures we use here [26]. In this paper, we describe a storage method for succinct tree sequences (and hence, genome sequence) as well as an algorithm for simplifying these. The data structure is succinct in the sense that its space usage is close to optimal, while still allowing efficient retrieval of informa- tion (see, e.g., [27]). We also describe how these tools can efficiently record, and later process, the embellished population pedigree from a forwards-time simulation. While providing sub- stantial savings in computational time and space, our methods provide in principle much more information than simply simulating the genomes—the tree sequence encodes all PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1006581 November 1, 2018 3 / 21 Simulation benchmarks To measure the performance gains from recording the pedigree we ran simulations both with and without recording. (Although we record more than just the parent–offspring relationships of the pedigree, for brevity we refer to the method as “pedigree recording”). All simulations used fwdpp to implement a discrete-time Wright-Fisher population of N diploid individuals, simulated for 10N generations (details below). Simulations without pedigree recording intro- duced neutral mutations at a rate equal to the recombination rate, so μ = r, where μ and r are the expected per-generation number of mutations per gamete and recombination breakpoints per diploid, respectively. Simulations with pedigree recording introduced neutral mutations at the same rate retrospectively, as described below, resulting in statistically identical simulation results. We ran simulations with different values of N and varied the size of the genomic region according to the scaled recombination parameter ρ = 4Nr. Deleterious mutations were introduced at rate ρ/100 per generation, drawing scaled selec- tion coefficients (2Ns) from a Gamma distribution with a mean of -5 and a shape parameter of 1. This distribution of fitness effects results in thousands of weakly-deleterious mutations seg- regating in the population, many of which drift to intermediate frequencies. The case of many mutations with selection is a non-trivial violation of exchangeability assumptions of the coales- cent [2]. Therefore, these selected mutations must be explicitly tracked in our forward simula- tion and the time savings due to pedigree recording come from not having to record neutral mutations. Pedigree tracking dramatically reduced runtimes, as shown in Fig 1, producing a relative speedup of up to around 50 fold relative to standard simulations that track neutral mutations (Fig 2). Pedigree tracking results in greater relative speedups for larger N and we observe increasing relative speedups as 4Nr increases for a given N (Fig 2). Importantly, runtimes are approximately linear in region size ρ when pedigree tracking (partially obscured by the log scale of the horizontal axis in Fig 1). In a more limited set of neutral simulations we found the same qualitative behavior, and a numerically larger speedup by using pedigree tracking (see S1 Text). 4 / 21 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1006581 November 1, 2018 Pedigree recording for simulation Fig 1. Total run time per single simulation replicate as a function of region length. Line color represents different diploid population sizes (N). Simulation benchmarks The left figure shows run times for standard simulations including neutral mutations. The right column shows run times of simulations that recorded the pedigree and added neutral mutations afterwards. The dashed line in the right panel shows results for an implementation using fwdpy11 where the pedigree simplification steps were handled in a separate thread of execution and fitness calculations were parallelized across four cores. Simulations with N = 5 × 104 timed out for region sizes larger than 103. https://doi.org/10.1371/journal.pcbi.1006581.g001 Fig 1. Total run time per single simulation replicate as a function of region length. Line color represents different diploid population sizes (N). The left figure shows run times for standard simulations including neutral mutations. The right column shows run times of simulations that recorded the pedigree and added neutral mutations afterwards. The dashed line in the right panel shows results for an implementation using fwdpy11 where the pedigree simplification steps were handled in a separate thread of execution and fitness calculations were parallelized across four cores. Simulations with N = 5 × 104 timed out for region sizes larger than 103. https://doi.org/10.1371/journal.pcbi.1006581.g001 https://doi.org/10.1371/journal.pcbi.1006581.g001 In our implementation, simulations with pedigree recording used substantially more RAM than simple forward simulations (see S1 Text). This is unsurprising: unsimplified tree sequences grow quickly, and so storing history can use arbitrarily much memory. However, this is not a requirement of the method, only a straightforwards consequence of a speed– memory tradeoff: the amount of required memory is mostly determined by the interval between simplification steps, but less frequent simplification reduces overall computation time (see S1 Text). In fact, our method could in some situations reduce the amount of memory required, if memory usage in the forwards simulation was dominated by the cost of maintain- ing neutral genetic variants. Tables for succinct tree sequences We now explain what we actually did to achieve this 50× speedup. The “pedigree recording” simulations above recorded information about each new individual in a collection of tables that together define a succinct tree sequence (or, simply “tree sequence”). A tree sequence is an encoding for a sequence of correlated trees, such as those describing the history of a sexual population. Tree sequences are efficient because branches that are shared by adjacent trees are stored once, rather than repeatedly for each tree. The topology of a tree sequence is defined via Computational Biology \| https://doi.org/10.1371/journal.pcbi.1006581 November 1, 2018 5 / 21 Pedigree recording for simulation Fig 2. Relative speedup of simulations due to pedigree recording. Each line shows the ratio of total run times of standard simulations to those of simulations with pedigree recording. Data points are taken from Fig 1 for simulations that ran to completion in both cases. https://doi.org/10.1371/journal.pcbi.1006581.g002 Fig 2. Relative speedup of simulations due to pedigree recording. Each line shows the ratio of total run times of standard simulations to those of simulations with pedigree recording. Data points are taken from Fig 1 for simulations that ran to completion in both cases. https://doi.org/10.1371/journal.pcbi.1006581.g002 Fig 2. Relative speedup of simulations due to pedigree recording. Each line shows the ratio of total run times of standard simulations to those of simulations with pedigree recording. Data points are taken from Fig 1 for simulations that ran to completion in both cases. https://doi.org/10.1371/journal.pcbi.1006581.g002 Fig 2. Relative speedup of simulations due to pedigree recording. Each line shows the ratio of total run times of standard simulations to those of simulations with pedigree recording. Data points are taken from Fig 1 for simulations that ran to completion in both cases. https://doi.org/10.1371/journal.pcbi.1006581.g002 its nodes and edges, while information about variants is recorded as sites and mutations; we give an example in Fig 3. This formulation is derived from the “coalescence records” encoding of tree sequences [26], normalised to remove redundancy and generalised to include a more general class of tree topologies. The nodes of a tree sequence correspond to the vertices in the individual genealogies along the sequence. Each node refers to a specific, distinct ancestor, and so has a unique “time”, thought of as the node’s birth time, which determines the height of any vertices the node is associated with. Tables for succinct tree sequences The leftmost panels show the tree sequence pictorially in two different ways: (top) a sequence of tree topologies; the first tree extends from genomic position 0 to 5, and the second from 5 to 10; and (bottom) the edges that define these topologies, displayed over their corresponding genomic segment (for instance, the edge from node 2 to node 4 is present only on the interval from 0 to 5). The remaining panels show the specific encoding of this tree sequence in the four tables (nodes, edges, sites and mutations). https://doi.org/10.1371/journal.pcbi.1006581.g003 https://doi.org/10.1371/journal.pcbi.1006581.g003 Recovering the sequence of trees from this information is straightforward: each point along the genome at which the tree topology changes is accompanied by the end of some edges and the beginning of others. Since each edge records the genomic interval over which a given node inherits from a particular ancestor, to construct the tree at a certain point in the genome we need only retrieve all edges overlapping that point and construct the corresponding tree. To modify the tree to reflect the genealogy at a nearby location, we simply remove those edges whose intervals do not overlap that location, and add those new edges whose intervals do. Inci- dentally, this property that edges naturally encode differences between nearby trees (e.g., as “subtree prune and regraft” moves) allows for efficient algorithms to compute statistics of the genome sequence that take advantage of the highly correlated nature of nearby trees [26]. Given the topology defined by the nodes and edges, sites and mutations encode the sequence information for each sample in an efficient way. Each site records two things: its position on the genome and an ancestral state. For example, in Fig 3 we have two sites, one at position 2.5 with ancestral state ‘A’ and the other at position 7.5 with ancestral state ‘G’. If no mutations occur at a given site, all nodes inherit the ancestral state. Each mutation records three things: the site at which it occurs, the first node to inherit the mutation, and the derived state. Thus, all nodes below the mutation’s node in the tree will inherit this state, unless further mutations are encountered. Three mutations are shown in Fig 3, illustrated by red stars. The first site, in the left-hand tree, has a single mutation, which results in node 2 inheriting the state ‘T’. Tables for succinct tree sequences (Note that since each node time is equal to the amount of time since the birth of the corresponding parent, time is measured in clock time, not in meioses). The example of Fig 3 has five nodes: nodes 0, 1 and 2 occur at time 0 and are the samples, while nodes 3 and 4 represent those ancestors necessary to record their genealogy, who were born one and two units of time in the past, respectively. The edges define how nodes relate to each other over specific genomic intervals. Each edge records the endpoints [ℓ, r) of the half-open genomic interval defining the spatial extent of the edge; and the identities p and c of the parent and child nodes of a single branch that occurs in all trees in this interval. The spatial extent of the edges defining the topology of Fig 3 are shown in the bottom left panel. For example, the branch joining nodes 1 to 3 appears in both trees, and so is recorded as a single edge extending over the whole chromosome. It is this method of capturing the shared structure between adjacent trees that makes the tree sequence encoding compact and algorithmically efficient. 6 / 21 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1006581 November 1, 2018 Pedigree recording for simulation Fig 3. An example tree sequence with three samples over a chromosome of length 10. The leftmost panels show the tree sequence pictorially in two different ways: (top) a sequence of tree topologies; the first tree extends from genomic position 0 to 5, and the second from 5 to 10; and (bottom) the edges that define these topologies, displayed over their corresponding genomic segment (for instance, the edge from node 2 to node 4 is present only on the interval from 0 to 5). The remaining panels show the specific encoding of this tree sequence in the four tables (nodes, edges, sites and mutations). https://doi.org/10.1371/journal.pcbi.1006581.g003 Fig 3. An example tree sequence with three samples over a chromosome of length 10. Pedigree recording for simulation tables (as described above) for this simulation consumed 157MiB of storage space. Using the tskit Python API, the time required to load this file into memory was around 1.5 seconds, and the time required to iterate over all 1 million trees was 2.7 seconds. In contrast, recording the topological information in Newick format would require around 20 TiB and storing the genotype information in VCF would require about 1 TiB (giving a compression factor of 144,000 in this instance). Working with either the Newick or VCF encoding of this dataset would likely require several days of CPU time just to read the information into memory. Validity of a set of tables. Given a set of node and edge tables as described above, there are only two requirements that ensure the tables describe a valid tree sequence. These are: 1. Offspring must be born after their parents (and hence, no loops). 2. The set of intervals on which each individual is a child must be disjoint. A pair of node and edge tables that satisfy these two requirements is guaranteed to uniquely describe at each point on the genome a collection of directed, acyclic graphs—in other words, a forest of trees. For some applications it is necessary to check that at every point there is only a single tree. Checking this is more difficult, but is implemented in tskit’s API. For effi- ciency, tskit makes several other sortedness requirements on the tables, that are not neces- sarily satisfied by tables emitted by a forwards-time simulation. tskit’s API includes tools to rectify this by first sorting and then using the simplify algorithm described below, which works on sorted tables and is guaranteed to produce a valid, tskit-ready tree sequence. Tables for succinct tree sequences The second site, in the right hand tree, has two mutations: one occurring over node 3 changing the state to ‘C’, and a back mutation over node 1 changing the state to ‘G’. This encoding of a sequence of trees and accompanying mutational information is very concise. To illustrate this, we used msprime to simulate 500,000 samples of a 200 megabase chromosome with human-like parameters: Ne = 104 and per-base mutation and recombination rates of 10−8 per generation. This resulted in about 1 million distinct marginal trees and 1.1 million infinite-sites mutations. The HDF5 file encoding the node, edge, site and mutation PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1006581 November 1, 2018 7 / 21 Pedigree recording for simulation and edge data. This information is then periodically copied to the tskit Tables API, where it is sorted and simplified. Flexibility. To demonstrate the flexibility provided by the Tables API and provide an implementation that decouples forward simulation internals from transfer of data to tskit, we also implemented a version of the simulations described in “Simulation benchmarks” sepa- rately in Python, described in S1 Text. In this proof-of-concept implementation, the simula- tion engine (we use simuPOP, [32]) invokes callbacks at critical points of the simulation, and we infer nodes and edges from the information that is provided. Rows are appended to the tables one-by-one, and the tables are periodically sorted and simplified to control memory usage. Benchmarking results from this implementation are shown (alongside results from fwdpp) for simulations without selection in S1 Text: a relatively modest speedup of around 5× is achieved, likely due to increased overhead. Recording the pedigree in forwards time To record the genealogical history of a forwards time simulation, we need to record two things for each new chromosome: the birth time; and the endpoints and parental IDs of each dis- tinctly inherited segment. These are naturally stored as the nodes and edges of a tree sequence. To demonstrate the idea, we write out in pseudocode how to run a neutral Wright–Fisher sim- ulation that records genealogical history in this way. The simulation will run for T generations, and has N haploid individuals, each carrying a single chromosome of length L. For simplicity, we sample exactly one crossover per generation. Note that the table recording portion of the algorithm does not depend on the Wright–Fisher nature of the population simulation; next we will describe how to record tables from any simulation. We use RUðAÞ to denote an element of the set A chosen uniformly at random (and all such instances are independent). Given a node table N , the function N :addrowðtÞ adds a new node to the table N with time t and returns the ID of this new node. Similarly, the function E:addrowð‘; r; p; cÞ adds a new edge (ℓeft, right, parent, child) to the edge table E. The function simplifyðP; N ; EÞ (described below) simplifies the history stored in the tables N and E to the minimal information required to represent the genealogies of the list of node IDs P; after sim- plification the nodes appearing in P are relabeled (0, 1, . . ., \|P\| −1). A step-by-step explanation follows the pseudocode. Algorithm W. (Forwards-time tree sequence). Simulates a randomly mating population of N haploid individuals with chromosome of length L for T generations, and returns the node and edge tables (N and E) recording the simulated history. In each generation, the current population is stored in P, while produced offspring are placed in P0. The tables are simplified every s generations, removing genealogical information from N and E irrelevant to the current population. W1. [Initialisation.] Set N NodeTableðÞ, E EdgeTableðÞ, t T, and j 0. For 0 k < N, set Pk N :addrowðTÞ. W2. [Generation loop head: new node.] Set u N :addrowðtÞ and P0 j u. W2. [Generation loop head: new node.] Set u N :addrowðtÞ and P0 j u. W3. [Choose parents.] Set a RUðf0; . . . ; N 1gÞ, b RUðf0; . . . The tskit Tables API The facilities for working with succinct tree sequences are implemented as part of the tskit Python API, which provides a powerful platform for processing tree topology and mutation data. The portions of tskit that we discuss here are dedicated to tree sequence input and output using simple tables of data, as described above, so we refer to this as the “Tables API”. output using simple tables of data, as described above, so we refer to this as the Tables API . The Tables API is primarily designed to facilitate efficient interchange of data between pro- grams or between different modules of the same program. We adopted a ‘columnar’ design, where all the values for a particular column are stored in adjacent memory locations. There are many advantages to columnar storage—for example, since adjacent values in memory are from the same column, they tend to compress well, and suitable encodings can be chosen on a per-column basis [28]. A particular advantage of this approach is that it enables very efficient copying of data, and in principle zero-copy data access (where a data consumer reads directly from the memory of a producer). Our implementation efficiently copies data from Python as a NumPy array [29] into the low-level C library used to manipulate tree sequences. This archi- tecture allows for data transfer rates of gigabytes per second (impossible under any text-based approach), while retaining excellent portability. NumPy’s array interface provides a great deal of flexibility and efficiency, and makes it straightforward to transfer data from sources such as HDF5 [30] or Dask [31]. For small scale data and debugging purposes, a simple text based for- mat is also supported. The tskit Python Tables API provides a general purpose toolkit for importing and pro- cessing succinct tree sequences, and a collection of tutorials are being developed at https:// github.com/tskit-dev/tutorials. Interoperation with Python simulators is then straightforward. The implementation we benchmark here uses pybind11 (https://github.com/pybind/ pybind11/) to interface with the fwdpp C++ API [13]. No modifications were required to the fwdpp code base; rather, we simply need to bookkeep parent/offspring labels, and perform simple processing of the recombination breakpoints from each mating event to generate node PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1006581 November 1, 2018 8 / 21 W6. [Simplify.] Call simplifyðP0; N ; EÞ, and set P0 k k for 0 k < N. W7. [Generation loop.] Set t t −1. If t = 0 terminate. Set P P0, j 0, and go to W2. W7. [Generation loop.] Set t t −1. If t = 0 terminate. Set P P0, j 0, and go to W2. We begin in W1 by creating new node and edge tables, and setting our population P (a vec- tor of N node IDs) to the initial population. This initial population is a set of N nodes with birth time T generations ago. We also initialise our generation clock t and individual index j. Step W2 replaces the jth individual (with node ID Pj) by creating a new node with birth time t (and ID u). In step W3 we determine the new node’s ancestry by choosing two indexes a and b uniformly, giving us parental IDs Pa and Pb, and choose a chromosomal breakpoint x. We record the effects of this event by storing two new edges: one recording that the parent of node u from 0 to x is Pa, and another recording that the parent of u from x to L is Pb. Step W5 then iterates these steps for each of the N individuals for each generation. At the end of a generation, we then check if we need to simplify (done every s generations). If simplification is required, we do this in step W6 by calling the simplify function on the node and edge tables with the cur- rent set of population IDs P0 as the samples. This updates the tables in-place to remove all redundant information, and remaps the specified sample IDs to 0, . . ., N −1 in the updated tables. Hence, we set our current population IDs to 0, . . .N −1 after simplify has completed. Step W7 loops these steps until the required number of generations have been simulated. This algorithm records only topological information about the simulated genealogies, but it is straightforward to add mutational information. Mutations that occur during the simulation can be recorded by simply storing the node in which they first occur, the derived state, and (if not already present) the genomic position of the site at which it occurs. This allows selected mutations, that the forwards time simulation must generate, to be recorded in the tree sequence. Neutral mutations can be generated after the simulation has completed, thus avoid- ing the cost of generating the many mutations that are lost in the population. Recording the pedigree in forwards time ; N 1gÞ and x RUðð0; LÞÞ. W4. [Record edges.] Call E:addrowð0; x; Pa; uÞ and E:addrowðx; L; Pb; uÞ. W5. [Individual loop.] Set j j + 1. If j < N go to W2. Otherwise, if t mod s 6¼ 0 go to W7. W6. [Simplify.] Call simplifyðP0; N ; EÞ, and set P0 k k for 0 k < N. PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1006581 November 1, 2018 9 / 21 https://doi.org/10.1371/journal.pcbi.1006581.g004 Pedigree recording for simulation Pedigree recording for simulation from parent to child and only some small subset will correspond to coalescences within the marginal trees. The second source of redundancy in the (unsimplified) output of Algorithm W is due to the fact that lineages die out: a large number of individuals in the simulation leave no descendants in the present day population. Node 26 in Fig 4a, for example, leaves no ancestors in the current population, and so the entire path tracing back to its common ancestor with 27 is redundant. Essential steps in tree sequence recording. Since a tree sequence can record the history of genetic inheritance in any situation (requiring only unambiguous inheritance and no time travel), any individual-based population genetics simulator can maintain a tree sequence with only a little bookkeeping. We have furthermore provided several tools to minimize this book- keeping, only requiring one-way output at the birth of each new individual. Concretely, to record a tree sequence, including mutations, a simulator must record for each new genome (so, twice for each new diploid individual): • the birth time of the genome in the Node Table, • the birth time of the genome in the Node Table, • the segments the genome inherits from its parental genomes in the Edge Table, • the locations of any new mutations in the Site Table, • the locations of any new mutations in the Site Table, • and the derived state of these new mutations in the Mutation Table (as well as the identity of this genome the mutations appeared in). • and the derived state of these new mutations in the Mutation Table (as well as the identity of this genome the mutations appeared in). Each of these can be simply appended to the ends of the respective tables. Besides this, simplifi- cation should be run every once in a while (e.g., every 100 generations). Before simplification, time in the Node Table must be translated to “time ago” if it is not already. (This was avoided in Algorithm W since there t denoted “time until the end of the simulation”). There are also several “cleaning” steps, for which we provide functions in the Tables API: sorting according to several criteria for algorithmic efficiency; and removing any duplicate sites from the Site Table. After simplification, since simplifyðP0; N ; EÞ results in the N individuals in P0 being relabeled in tskit’s tables as 0, 1, . . ., N −1, there must be bookkeeping that keeps in sync the individ- ual IDs as recorded by the simulator with the node IDs recorded in the tables. In other words, to record a tree sequence, a simulation needs only to know (a) which genomes recombined to produce each new genome, and how; (b) the locations and results of any new mutations on each genome; and (c) the identities of every currently alive individual at each time simplification occurs. Storing metadata. Applications may also want to store more information not fitting into an existing column of the tables, such as the selection coefficient of a mutation, or the sex of an individual. This (and, indeed, arbitrary information) can be stored in the metadata columns present in Node, Site, and Mutation tables. W7. [Generation loop.] Set t t −1. If t = 0 terminate. Set P P0, j 0, and go to W2. This is straight- forward to do because we have access to the marginal genealogies. Fig 4 shows an example of a marginal genealogy produced by a forwards-time Wright– Fisher process like Algorithm W. On the left is the tree showing all the edges output by the simulation, while on the right is the minimal tree representing the ancestry of the current set of samples. Clearly there is a great deal of redundancy in the topological information output by the simulation. This redundancy comes from two sources. First, there are a large number of nodes in the tree that have only one child. In Algorithm W we do not attempt to identify coa- lescence events, but simply record all parent-child relationships in the history of the popula- tion. As such, many of these edges will record the simple passing of genealogical information Fig 4. An example of a marginal genealogy from a Wright-Fisher simulation with N = 5. (A) the original tree including all intermediate nodes and dead-ends, and (B) the minimal tree relating all of the currently-alive individuals (27–31). htt //d i /10 1371/j l bi 1006581 004 Fig 4. An example of a marginal genealogy from a Wright-Fisher simulation with N = 5. (A) the original tree including all intermediate nodes and dead-ends, and (B) the minimal tree relating all of the currently-alive individuals (27–31). https://doi.org/10.1371/journal.pcbi.1006581.g004 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1006581 November 1, 2018 10 / 21 2. Within the marginal trees, all non-sample vertices must have at least two children (i.e., unary tree vertices are removed). 2. Within the marginal trees, all non-sample vertices must have at least two children (i.e., unary tree vertices are removed). 3. Any nodes and edges not ancestral to any of the sampled nodes are removed. 4. There are no adjacent redundant edges, i.e., pairs of edges (ℓ, x, p, c) and (x, r, p, c) which can be represented with a single edge (ℓ, r, p, c). 4. There are no adjacent redundant edges, i.e., pairs of edges (ℓ, x, p, c) and (x, r, p, c) which can be represented with a single edge (ℓ, r, p, c). Simplification is essential not only for keeping the information recorded by forwards simu- lation manageable, but also is useful for extracting subsets of a tree sequence representing a very large dataset. We implement simplification by starting at the end of the simulation, and moving back up through history, recording in the new tree sequence only that information necessary to con- struct the tree sequence of the specified individuals. This process of tracing ancestry back through time in a pedigree was the motivation for Hudson’s coalescent simulation algorithm [33], so it is unsurprising that simplification uses many of the same tools as the implementa- tion of Hudson’s algorithm in msprime [26]. The main difference is that events in a coales- cent simulation are random, while in our simplification algorithm they are predetermined by history. An implementation in pseudocode is provided in S1 Text, and a python implementa- tion as supplementary information. Conceptually, this works by (a) beginning by painting the chromosome in each sample a distinct color; (b) moving back through history, copying the colors of each chromosome to the portions of its parental chromosomes from which it was inherited; (c) each time we would paint two colors in the same spot (a coalescence), record that information as an edge and instead paint a brand-new color; and (d) once all colors have coalesced on a given segment, stop propagating it. This “paint pot” description misses some details—for instance, we must ensure that all coalescing segments in a given individual are assigned the same new color—but is reasonably close. Fig 5 shows an example tree sequence, before and after simplification, and Fig 6 depicts the “paint pot” state of the algorithm during the process of simplifying this tree sequence. Tree sequence simplification It is desirable for many reasons to remove redundant information from a tree sequence. To formalize this: suppose that we are only interested in a subset of the nodes of a tree sequence (which we refer to as our ‘samples’), and wish to reduce this input tree sequence to the smallest one that still completely describes the history of the specified samples, having the following properties: 1. All marginal trees must match the subtree of the corresponding tree in the input tree sequence that is induced by the samples. Computational Biology \| https://doi.org/10.1371/journal.pcbi.1006581 November 1, 2018 11 / 21 Pedigree recording for simulation Pedigree recording for simulation Fig 5. An example of tree sequence simplification. (A) The augmented pedigree diagram on the left relates the ten homologous chromosomes of five diploid individuals (BC, DE, FG, HI, and JK) to each other and to a common ancestral chromosome (A); dotted lines connect the two chromosomes of each individual, and solid lines lead to the products of their meioses. The corresponding tables (right) have 11 node records (one for each chromosome) and 15 edge records (one for each distinctly inherited segment). Blue numbers denote crossing over locations—for instance, D and E were parents to G, who inherited the left 70% of the chromosome from E and the remainder from D. B, C, D, and E inherit clonally from A. (B) The five distinct trees found across the chromosome (blue numbers denote locations on the chromosome). Labels after simplification are shown in red (see text). (C) Tables recording the tree sequence after simplification with nodes J and K as samples. The mapping from labels in the forwards time simulation to nodes in the tree sequence is shown in red. https://doi.org/10.1371/journal.pcbi.1006581.g005 Fig 5. An example of tree sequence simplification. (A) The augmented pedigree diagram on the left relates the ten homologous chromosomes of five diploid individuals (BC, DE, FG, HI, and JK) to each other and to a common ancestral chromosome (A); dotted lines connect the two chromosomes of each individual, and solid lines lead to the products of their meioses. The corresponding tables (right) have 11 node records (one for each chromosome) and 15 edge records (one for each distinctly inherited segment). Blue numbers denote crossing over locations—for instance, D and E were parents to G, who inherited the left 70% of the chromosome from E and the remainder from D. B, C, D, and E inherit clonally from A. (B) The five distinct trees found across the chromosome (blue numbers denote locations on the chromosome). Labels after simplification are shown in red (see text). (C) Tables recording the tree sequence after simplification with nodes J and K as samples. The mapping from labels in the forwards time simulation to nodes in the tree sequence is shown in red. https://doi.org/10.1371/journal.pcbi.1006581.g005 Fig 5. An example of tree sequence simplification. 2. Within the marginal trees, all non-sample vertices must have at least two children (i.e., unary tree vertices are removed). Since the method begins with the samples and moves back through time, in the out- put tree sequence, the n samples will be numbered 0, 1, . . ., n −1 and subsequent nodes will be ordered by time since birth. This is seen in the red labels of Fig 5. More concretely, the algorithm works by moving back through time, processing each par- ent in the input tree sequence in chronological order. The main state of the algorithm at each point in time is a set of ancestral lineages, and each lineage is a linked list of ancestral segments. An ancestral segment (ℓ, r, u) is found in a lineage if the output node u inherits the genomic interval [ℓ, r) from that lineage (and so u corresponds to a “color” in the description above). We also maintain a map from input nodes to lineages. Crucially, the time required to run the algorithm is linear in the number of edges of the input tree sequence. Sequential simplification and prior history. Any simulation scheme that records data into tables, as Algorithm W does, has its genealogical history available at any time as a tree sequence. This has two additional advantages: First, simplification can be run periodically through the simulation, if we take the set of samples to be the entire currently alive population. This is important in practice as it keeps memory usage from growing linearly (and quickly) with time. Second, the simulation can be begun with a tree sequence produced by some other method—for instance, by a coalescent simulation with msprime, providing an easy, efficient way to specify prior history. A natural question is now: how often should simplification occur? Limited testing (described in S1 Text) found that different simplification intervals affect run times by approximately 25%, with the lowest run time occurring when simplifying every 103 generations. Thus, there is a memory-versus-speed tradeoff—simplifying more often would keep fewer extinct nodes and edges in memory. PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1006581 November 1, 2018 12 / 21 Following the “paint pot” description in the text, we begin by coloring J and K’s genomes in red and blue respectively, then trace how these colors were inherited back up through the pedigree until they coalesce. To aid in this, the smaller colored chromosomes on either side of each solid arrow show the bits inherited from each of the two parental chromosomes, with genomic position 0.0 on the bottom and 1.0 at the top. Each time a red and a blue segment overlap, a coalescence occurs, two edges are output, and we stop propagating that segment. For instance, both J and K inherit from H between 0.5 and 0.9, which resulted in the first two edges of the simplified table of Fig 5C. Later, both inherit from E between 0.2 and 0.5, along the paths J-H-G-E and K-I-E respectively, resulting in the next two edges. https://doi org/10 1371/journal pcbi 1006581 g006 Fig 6. A depiction of each state of the simplification algorithm as it moves up through the embellished pedigree in the example of Fig 5A. Following the “paint pot” description in the text, we begin by coloring J and K’s genomes in red and blue respectively, then trace how these colors were inherited back up through the pedigree until they coalesce. To aid in this, the smaller colored chromosomes on either side of each solid arrow show the bits inherited from each of the two parental chromosomes, with genomic position 0.0 on the bottom and 1.0 at the top. Each time a red and a blue segment overlap, a coalescence occurs, two edges are output, and we stop propagating that segment. For instance, both J and K inherit from H between 0.5 and 0.9, which resulted in the first two edges of the simplified table of Fig 5C. Later, both inherit from E between 0.2 and 0.5, along the paths J-H-G-E and K-I-E respectively, resulting in the next two edges. Fig 6. A depiction of each state of the simplification algorithm as it moves up through the embellished pedigree in https://doi.org/10.1371/journal.pcbi.1006581.g006 Computational complexity. Figs 1 and 2 show that this method can dramatically improve simulation performance in practice—but, how does it perform in theory? Both computational time and storage space are depend mostly on the number of mutations and edges in the tree sequence. (A) The augmented pedigree diagram on the left relates the ten homologous chromosomes of five diploid individuals (BC, DE, FG, HI, and JK) to each other and to a common ancestral chromosome (A); dotted lines connect the two chromosomes of each individual, and solid lines lead to the products of their meioses. The corresponding tables (right) have 11 node records (one for each chromosome) and 15 edge records (one for each distinctly inherited segment). Blue numbers denote crossing over locations—for instance, D and E were parents to G, who inherited the left 70% of the chromosome from E and the remainder from D. B, C, D, and E inherit clonally from A. (B) The five distinct trees found across the chromosome (blue numbers denote locations on the chromosome). Labels after simplification are shown in red (see text). (C) Tables recording the tree sequence after simplification with nodes J and K as samples. The mapping from labels in the forwards time simulation to nodes in the tree sequence is shown in red. https://doi.org/10.1371/journal.pcbi.1006581.g005 Fig 5. An example of tree sequence simplification. (A) The augmented pedigree diagram on the left relates the ten homologous chromosomes of five diploid individuals (BC, DE, FG, HI, and JK) to each other and to a common ancestral chromosome (A); dotted lines connect the two chromosomes of each individual, and solid lines lead to the products of their meioses. The corresponding tables (right) have 11 node records (one for each chromosome) and 15 edge records (one for each distinctly inherited segment). Blue numbers denote crossing over locations—for instance, D and E were parents to G, who inherited the left 70% of the chromosome from E and the remainder from D. B, C, D, and E inherit clonally from A. (B) The five distinct trees found across the chromosome (blue numbers denote locations on the chromosome). Labels after simplification are shown in red (see text). (C) Tables recording the tree sequence after simplification with nodes J and K as samples. The mapping from labels in the forwards time simulation to nodes in the tree sequence is shown in red. https://doi.org/10.1371/journal.pcbi.1006581.g005 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1006581 November 1, 2018 13 / 21 Pedigree recording for simulation Fig 6. A depiction of each state of the simplification algorithm as it moves up through the embellished pedigree in the example of Fig 5A. Pedigree recording for simulation Then, each time the marginal tree changes along the sequence, four edges end and four new edges begin (except for changes affecting the root, which require fewer; see [26]). Suppose that T x is the marginal tree at genomic position x, and write jT xj for the total length of the tree. For the tree at nearby position x + dx to be different, there must have been a crossing-over between x and x + dx in one of the jT xj meioses that gave birth to those individuals. (Recall that these “individuals” are haploid). Since we measure distance along the genome so that length is equal to the expected number of crossing-overs per generation, the expected distance until the next crossing-over is 1=jT xj. If every crossing-over changed the marginal tree, then this would imply, for consistency, that the expected total number of times that the marginal tree changes along the genome is equal to the total area of the tree sequence (as defined above). Not every such crossing-over changes the marginal tree, but most do, so the total area of the tree sequence, multiplied by four, gives an upper bound on the expected number of edges in the tree sequence beyond those describing the leftmost tree. Since we are considering a chromo- some of length 1, the expected total area is equal to the mean marginal tree length, as above. If T is large relative to N, so that all marginal trees have a single root with high probability, then coalescent theory tells us that the expected total length of the branches of a marginal tree back to the most recent common ancestor is approximately 2N log(N). Therefore, the tree sequence describing the entire population is expected to need no more than 2N + 8N log(N) edges. Not every new edge derives from a never-before-seen node, but the number of nodes is at most equal to the number of edges plus the sample size. Therefore, we would need O(N2) space to store the complete history of the simulation, but only O(N log N) to store the history that is relevant to the extant population. What if T is smaller: how many of the resulting 2NT edges are required after simplification? In other words, how fast does the information in the pedigree become irrelevant? The key quantity to understand for this will be the total “area” of the tree sequence, which is the sum of the lengths of all ancestral genomic segments that some, but not all, of the present population has inherited. It can be found by summing the product of segment length (left minus right coordinates) and edge length (difference in birth times between parent and child), across all edges. This area is also equal to the sum of the total lengths of all marginal trees (i.e., the trees describing inheritance at each position on the genome), so can be computed as the sequence length multiplied by the mean marginal tree length. Since we analyze tree sequences arising from a Wright–Fisher model, statistical properties of a marginal tree are fairly well-described by coalescent theory. Similar arguments to those below go back at least to Wat- terson [34], who also explicitly computed smaller order corrections relevant to whole-popula- tion genealogies of the Wright–Fisher model. The arguments below are mostly self-contained, but for an introduction to coalescent theory, including the basic facts used below, see [15]. First: how much memory do simplified tree sequences require? Consider a simulation of a Wright–Fisher population of N haploid individuals using Algorithm W for T generations. Since every chromosome inherits material from both parents, without simplification this would produce tables of NT nodes and 2NT edges. After simplification, we are left with the tree sequence describing the history of only the current generation of N individuals, back to either T generations ago, or their common ancestor, whichever comes first. The tree sequence must store edges describing the leftmost marginal tree, which requires at most 2N −2 edges. PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1006581 November 1, 2018 14 / 21 Pedigree recording for simulation of edges, this will result in around μ2N log(N) mutations. With N = 2 × 104 and T = 10N, this implies that adding neutral mutations to the simplified tree sequence reduces the number of mutations that must be generated by a factor of 10,000. This could result in substantial time savings, even without considering the computational burden of propagating mutations for- wards across generations. Since each mutation is stored only as a single row in the mutation table, and at most one row in the site table, the space required for M mutations is O(M); combined with the O(N log N) storage for edges and nodes of a simplified tree sequence, this implies that the full results of a simulation of N individuals having M mutations can be stored in O(N log N + M) space. In simulations of whole chromosomes, there are typically a small, bounded number of mutations per chromosome per generation, so M will be O(N log N) as well. How does the computation time required for simplification scale? Simply because it must process each edge, the simplification algorithm is at least linear in the number of edges of the input tree sequence. Empirically the algorithm is exactly linear, as seen in Fig 7B, which shows the time required to simplify increasingly large subsets of a large tree sequence. When simpli- fying the result of a forwards-time sequence, the number of edges is the main contributing fac- tor. Suppose on the other hand we want to simplify an already-minimal but large tree Fig 7. Time and space complexity of simplify. (A) Number of edges in the simplified tree sequence for 10 replicate Wright–Fisher simulations with N = 100 as a function of number of generations. Each line is one simulation, the heavy blue line gives the average, and the dashed line is the upper bound of Eq (1). (B) Time required to simplify the first k edges of a large (4.2GiB) unsimplified tree sequence produced by a forwards-time simulation plotted against k. The time scales linearly with the number of input edges. (C) Time required to simplify the tree sequence resulting from a coalescent simulation of 500,000 samples of a 200 megabase human chromosome to a random subsample of n samples, plotted against n (note the log scale; the time scales logarithmically with n). https://doi.org/10.1371/journal.pcbi.1006581.g007 Fig 7. Now, we need to compute the expected total length of all branches in a coalescent tree up until time T (or the common ancestor, whichever comes first). Again, coalescent theory tells us that the expected length of time for which a coalescent tree has k lineages is 2N/(k(k −1)) = 2N (1/(k −1) −1/k) generations. Since the tree has k branches over this period, it is expected to contribute 2N/(k −1) to the total tree length. By summing over n < k N, the N tips of a tree are expected to descend from only n lineages around 2(N/n −1) generations ago. Inverting this relationship between time and number of roots implies that a marginal tree cut T units of time ago is expected to have around r(T) roots, where r(T) = 2N/(T + 2). The total tree length over this time is 2N PN k¼rðTÞþ1 1=ðk 1Þ 2N logðN=rðTÞÞ, since Px k¼1 1=k logðxÞ þ g. This is a crude estimate for several reasons: first, we should not count the branch leading to the root of the tree (i.e., when r(T) = 1), and second, this does not account for the discrete nature of the Wright–Fisher model. Nonetheless, this leads as above to an upper bound on the num- ber of edges of 2N 1 þ 4 log min N; T þ 2 2 : ð1Þ ð1Þ This implies that the number of edges required to store the last T generations of history for the entire chromosome of a population of size N grows as O(N log T)—proportionally to N at first but rapidly tapering off. The bound holds up reasonably well in practice, as shown in Fig 7. What about mutations? Forwards-time generation of infinite-sites mutations with total mutation rate per generation μ would produce around μNT mutations (and the same number of sites), simply because there was a total of NT meioses. Since mutations that are retained after simplification are precisely those that fall on the marginal trees, the number of mutations is proportional to the total area of the tree sequence. By the same argument as for the number PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1006581 November 1, 2018 15 / 21 Discussion In this paper, we have shown that storing pedigrees and associated recombination events in a forwards-time simulation not only results in having available a great deal more information about the simulated population, but also can speed up the simulation by orders of magnitude. To make this feasible, we have described how to efficiently store this information in numerical tables, and have described a fundamental algorithm for simplification of tree sequences. Con- ceptually, recording of genealogical and recombination events can happen independently of the details of simulation; for this reason, we provide a well-defined and well-tested API in Python for use in other code bases (a C API is also planned). The tree sequences produced by default by this method are very compact, storing genotype and genealogical information in a small fraction of the space taken by a compressed VCF file. The format also allows highly efficient processing for downstream analysis. Efficient process- ing is possible because many statistics of interest for population genetics are naturally expressed in terms of tree topologies, and so can be quickly computed from the trees underly- ing the tree sequence format. For example, pairwise nucleotide diversity π, is the average den- sity of differences between sequences in the sample. To compute this directly from sequence data at m sites in n samples requires computing allele frequencies, taking O(nm) operations. By using the locations of the mutations on the marginal trees, and the fact that these are corre- lated, sequential tree algorithms similar to those in [26] can do this in roughly O(n + m + t log n) operations, where t is the number of distinct trees. The tskit API provides a method to compute π among arbitrary subsets of the samples in a tree sequence, which took about 0.7 seconds when applied to an example simulation of 100 megabases of human-like sequence for 200,000 samples (about 500K sites). The corresponding numeric genotype matrix required about 95GiB of RAM, and calculating π took about 66 seconds with NumPy. Another attractive feature of this set of tools is that it makes it easy to incorporate prior his- tory, simply by seeding the simulation with a (relatively inexpensive) coalescent simulation. This allows for incorporation of deep-time history beyond the reach of individual-based simu- lations. Pedigree recording for simulation sequence with N nodes to a subsample of size n. How does the required time scale with n? In this case, the computation is dominated by the size of the output tree sequence, which grows with log(n), as shown in Fig 7C. sequence with N nodes to a subsample of size n. How does the required time scale with n? In this case, the computation is dominated by the size of the output tree sequence, which grows with log(n), as shown in Fig 7C. Discussion This may not even negatively affect realism, since geographic structure from times lon- ger ago than the mixing time of migration across the range has limited effect on modern genealogies [35], other than possibly changing effective population size [36, 37]. Time and space complexity of simplify. (A) Number of edges in the simplified tree sequence for 10 replicate Wright–Fisher simulations with N = 100 as a function of number of generations. Each line is one simulation, the heavy blue line gives the average, and the dashed line is the upper bound of Eq (1). (B) Time required to simplify the first k edges of a large (4.2GiB) unsimplified tree sequence produced by a forwards-time simulation plotted against k. The time scales linearly with the number of input edges. (C) Time required to simplify the tree sequence resulting from a coalescent simulation of 500,000 samples of a 200 megabase human chromosome to a random subsample of n samples, plotted against n (note the log scale; the time scales logarithmically with n). https://doi org/10 1371/journal pcbi 1006581 g007 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1006581 November 1, 2018 16 / 21 Methods We implemented simulations and the connection to tskit in C++, using fwdpp library functions and interface code using a continuum-sites model for both mutation and recombi- nation. Simulations were run using fwdpy11 (version 0.13.a0), a Python package based on fwdpp (version 0.5.7). The majority of results are presented based on a single-threaded imple- mentation. However, we also implemented a parallelized version using Python’s queue. Queue to run the simplification step in a separate Python thread. Our implementation allows a maximum of four simplification intervals to be in the queue at once. This parallelized version also performed fitness calculation in parallel using two threads of execution in C++. Code for all simulations and figures is available at https://github.com/petrelharp/ftprime_ ms. These made use of the GNU Scientific Library (version 1.16, [46]), pybind11 (version 2.2.1, [47]), and GCC (version 4.8.5). We ran all benchmarks on an Ubuntu Linux (version 16.04) system with two 2.6 GHz Intel E5-2650 CPU with hyperthreading enabled. We ran one simulation at a time and the machine was under minimal load otherwise. We used GNU paral- lel [48] to kill any simulation that did not finish within 72 hours, and the Linux time com- mand to record run time and peak memory usage of each replicate. A final note In preparing this manuscript, we debated a number of possible terms for the embellished pedi- gree, i.e., the “pedigree with ancestral recombination information”, the object through which each tree of a tree sequence is threaded. Etymological consensus [45] has “pedigree” derived from the french “pied de grue” for the foot of a crane (whose branching pattern resembles the bifurcation of a single parent-offspring relationship). An analogous term for the embellished pedigree might then be nedigree, from “nid de grue”, as the nest of a crane is a large jumble of (forking) branches. We thought it would be confusing to use this term throughout the manu- script, but perhaps it will prove useful elsewhere. Pedigree recording for simulation such as the coalescent with gene conversion [39], using the structured coalescent to model var- ious forms of natural selection [40–42], or the coalescent within a known pedigree. For such models, one could in principle generate tables of nodes and edges to be simplified in tskit. The resulting succinct tree sequence object would be in the same format as those generated by msprime’s simulate function, and therefore compatible with existing methods for down- stream analyses. Another application of our methods would be the case of simulating coalescent histories conditional on known pedigrees. The standard description of the Wright-Fisher coalescent averages over pedigrees. However, conditional on a realized pedigree, the distribution of coa- lescent times in the recent past differs from that of the unconditional coalescent [43]. For pop- ulations with known pedigrees (e.g., [44]), it may be of use to simulate transmission along such pedigrees for the purpose of inference. Other applications The methods described here for efficiently storing tree sequences may prove useful in other fields. We have focused on the interpretation of tree sequences as the outcome of the process of recombination, but in principle, we can efficiently encode any sequence of trees which differ by subtree-prune-and-regraft operations. Since each such operation requires a constant amount of space to encode, the total space required is O(n + t) for t trees with n leaves [26]. For instance, the large numbers of large, correlated trees produced by MCMC samplers used in Bayesian phylogenetics (e.g., [38]) might be compactly stored as a tree sequence, which would then allow highly efficient computation of properties of the posterior distribution. In this article, we applied our methods for storing trees to the problem of pedigree record- ing in a forward-time simulation. However, the method applies to any simulation scheme gen- erating nodes and edges. For example, one could use the methods described here to generate succinct tree sequences under coalescent processes not currently implemented in msprime, PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1006581 November 1, 2018 17 / 21 Author Contributions Conceptualization: Jerome Kelleher, Jaime Ashander, Peter L. Ralph. Methodology: Jerome Kelleher, Kevin R. Thornton, Jaime Ashander, Peter L. Ralph. Project administration: Jerome Kelleher, Kevin R. Thornton, Peter L. Ralph. Resources: Jerome Kelleher, Kevin R. Thornton, Peter L. Ralph. Software: Jerome Kelleher, Kevin R. Thornton, Jaime Ashander, Peter L. Ralph. Supervision: Jerome Kelleher, Peter L. Ralph. Validation: Jerome Kelleher, Kevin R. Thornton, Jaime Ashander, Peter L. Ralph. Visualization: Jerome Kelleher, Kevin R. Thornton, Jaime Ashander, Peter L. Ralph. Writing – original draft: Jerome Kelleher, Kevin R. Thornton, Jaime Ashander, Peter L. Ralph. Writing – review & editing: Jerome Kelleher, Kevin R. Thornton, Jaime Ashander, Peter L. Ralph. Pedigree recording for simulation Acknowledgments Thanks to Gil McVean, Jared Galloway, Brad Shaffer, and Evan McCartney–Melstad for useful discussions. Supporting information S1 Text. Additional benchmarks and algorithm listings. The supplementary text contains (A) benchmarking of run time and memory usage on simulations without selection; (B) benchmarking of memory usage with selection; (C) an analysis of the effect of simplification interval on run times; (D) details for the simuPOP implementation; and (E) more details, and a listing, of the simplification algorithm. (PDF) 18 / 21 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1006581 November 1, 2018 Pedigree recording for simulation 9. Bhaskar A, Clark AG, Song YS. Distortion of genealogical properties when the sample is very large. Proc Natl Acad Sci USA. 2014; 111(6):2385–2390. https://doi.org/10.1073/pnas.1322709111 PMID: 24469801 10. Harris K, Nielsen R. 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https://openalex.org/W2599851317	https://europepmc.org/articles/pmc5482596?pdf=render	English	null	Expression of wild-type p53-induced phosphatase 1 in diabetic epiretinal membranes	Oncotarget	2,017	cc-by	4,956	www.impactjournals.com/oncotarget/ Oncotarget, 2017, Vol. 8, (No. 22), pp: 35532-35541 Expression of wild-type p53-induced phosphatase 1 in diabetic epiretinal membranes Jiping Xu1,, Haibin Zhong1,, Ling Cui1,, Qianqian Lan1, Lifei Chen1, Wenjing He1, Yu Wu1, Li Jiang1, Hui Huang1, Xin Zhao1, Li Li1, Siming Zeng1, Min Li1 and Fan Xu1 1 Department of Ophthalmology, People’s Hospital of Guangxi Zhuang Autonomous Region, Nanning, Guangxi, People’s Republic of China These authors have contributed equally to this work Correspondence to: Fan Xu, email: eyefanxu@163.com Correspondence to: Min Li, email: eyeminli@163.com Keywords: Wip1, epiretinal membranes, proliferative diabetic retinopathy, NF-κB, glial cell, Pathology Section Received: January 06, 2017 Accepted: March 11, 2017 Published: March 29, 2017 Copyright: Xu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC-BY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. A S AC Research Paper: Pathology www.impactjournals.com/oncotarget/ Oncotarget, 2017, Vol. 8, (No. 22), pp: 35532-35541 Expression of wild-type p53-induced phosphatase 1 in diabetic epiretinal membranes Jiping Xu1,, Haibin Zhong1,, Ling Cui1,, Qianqian Lan1, Lifei Chen1, Wenjing He1, Yu Wu1, Li Jiang1, Hui Huang1, Xin Zhao1, Li Li1, Siming Zeng1, Min Li1 and Fan Xu1 1 Department of Ophthalmology, People’s Hospital of Guangxi Zhuang Autonomous Region, Nanning, Guangxi, People’s Republic of China These authors have contributed equally to this work Correspondence to: Fan Xu, email: eyefanxu@163.com Correspondence to: Min Li, email: eyeminli@163.com Keywords: Wip1, epiretinal membranes, proliferative diabetic retinopathy, NF-κB, glial cell, Pathology Section Received: January 06, 2017 Accepted: March 11, 2017 Published: March 29, 2017 Copyright: Xu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC-BY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. A S AC Research Paper: Pathology Jiping Xu1,, Haibin Zhong1,, Ling Cui1,, Qianqian Lan1, Lifei Chen1, Wenjing He1, Yu Wu1, Li Jiang1, Hui Huang1, Xin Zhao1, Li Li1, Siming Zeng1, Min Li1 and Fan Xu1 1 Department of Ophthalmology, People’s Hospital of Guangxi Zhuang Autonomous Region, Nanning, Guangxi, People’s Republic of China These authors have contributed equally to this work Correspondence to: Fan Xu, email: eyefanxu@163.com Correspondence to: Min Li, email: eyeminli@163.com Keywords: Wip1, epiretinal membranes, proliferative diabetic retinopathy, NF-κB, glial cell, Pathology Section Received: January 06, 2017 Accepted: March 11, 2017 Published: March 29, 2017 Copyright: Xu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC-BY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Jiping Xu1,, Haibin Zhong1,, Ling Cui1,, Qianqian Lan1, Lifei Chen1, Wenjing He1, Yu Wu1, Li Jiang1, Hui Huang1, Xin Zhao1, Li Li1, Siming Zeng1, Min Li1 and Fan Xu1 1 Department of Ophthalmology, People’s Hospital of Guangxi Zhuang Autonomous Region, Nanning, Guangxi, People’s Republic of China These authors have contributed equally to this work Correspondence to: Fan Xu email: eyefanxu@163 com * These authors have contributed equally to this work ABSTRACT Objective: The aims of the present study were to investigate the expression and distribution of Wild-type p53-induced phosphatase 1 (Wip1) in diabetic patients with proliferative diabetic retinopathy (PDR) with epiretinal membranes (ERMs) meanwhile analyze the colocalization of Wip1 and nuclear factor kappa-B (NF-κB) p65 in ERMs. Methods: ERMs samples were collected from patients with PDR (PDR group) or non-diabetic patients with idiopathic epiretinal membranes (iERMs) (control group) during pars plana vitrectomy. Real-Time PCR analysis was carried out to examine the mRNA expression of Wip1 in ERMs. Immunohistochemical analysis and Immunofluorescent analysis were performed to detect the protein expression of Wip1 in ERMs. Double immunofluorescent staining was performed to detect the colocalization of Wip1 and glial fibrillary acidic protein (GFAP) (retinal glial cells marker), also Wip1 and NF-κB. Results: ERMs were obtained from 17 eyes of 17 patients with PDR (the PDR group) and 9 eyes of 9 nondiabetic patients (the control group) with iERMs. Our results showed high expression levels of Wip1 mRNAs in ERMs after PDR, but low in iERMs. In addition, both immunohistochemistry and immunofluorescence assay showed strong immunoreactivity for Wip1 in PDR ERMs. Furthermore, Wip1 and GFAP were coexpressed in PDR membranes. Finally, the expression of Wip1 was paralleled with NF-κB. Conclusion: These data support the notion that Wip1 contributes to the formation of the ERMs in PDR membranes via NF-κB signaling. RESULTS Wild-type p53-induced phosphatase 1 (Wip1), which is encoded by the PPM1D gene, plays a key role in stress signaling [13]. Wip1 dephosphorylates multiple target proteins in response to various stresses, tumorigenesis and aging [14]. Recent work has demonstrated that Wip1 promotes cell cycle progression, cell survival and proliferation [15]. Importantly, Wip1 might be involved in glia proliferation and inflammation [16, 17]. Additionally, Lowe et al. [18] found that NF-κB p65 was a positive regulator of Wip1 expression. However, little is known about the expression of Wip1 in ERMs of PDR. INTRODUCTION Glial cell proliferation following PDR is suggested to play a crucial role in ERMs formation [4-6]. Glial cell proliferation involves a complex cross-talk among retinal glial cells, including Müller cells and astrocytes [5, 6]. Commonly, the signs of glial cell proliferation are increased immunoreactivity for glial fibrillary acidic protein (GFAP) and glial cell hypertrophy, as well as proliferation and migration of the retinal glial cells [7]. Meanwhile, there is increasing evidence that chronic, low- grade subclinical inflammation play a considerable role in the pathogenesis of DR [8-10]. Furthermore, numerous studies have demonstrated various trophic factor receptors Diabetic retinopathy (DR) is one of the leading causes of decreased vision and blindness in the working- age population of developed countries [1, 2]. Proliferative diabetic retinopathy (PDR) is the more advanced form of DR, characterized by outgrowth of epiretinal membranes at the vitreoretinal interface [3]. These epiretinal membranes (ERMs) may cause distortion of the retinal architecture, vascular leakage, secondary retinal edema and rhegmatogenous retinal detachment [4]. www.impactjournals.com/oncotarget Oncotarget 35532 with PDR with ERMs. Moreover, the colocalization of Wip1 and NF-κB p65 was analyzed by double-staining immunohistochemistry of ERMs. and transcription factors, such as nuclear factor kappa B (NF-kB), are involved in ERMs formation [6, 11, 12]. Unfortunately, the exact mechanisms that regulate the formation of epiretinal membranes in PDR are still incompletely understood. mRNA level of Wip1 was higher in the PDR group Wip1 mRNAs were detected in all ERMs obtained from the PDR group and the control group by qRT-PCR analysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified as an loading control to compare the relative abundance of PCR products. As shown in Figure 1, in the ERMs, the mRNA level of Wip1 was significantly higher in the patients with diabetes compared to the nondiabetic group. The mean relative mRNA level of Wip1 in PDR groups is 0.62 (range from 0.38 to 0.86), while it is 0.25 in iERM groups (range from 0.14 to 0.36). There is a statistically significant difference in Wip1 mRNA expression between PDR and iERM patients, P < 0.01. Patient information Indicates statistically significant result compared to the corresponding data in the control group as shown in Table 1 (P<0.05). * Independent sample t-test. # χ2 test. N.A. = Not applicable. ranged from 2 to 35 years, with a mean of 12.11 ± 5.39 years. The patients in the control group were 4 females and 5 males (P = 1.0) whose ages ranged from 33 to 71 years, with a mean of 58.01 ± 3.61 years (P = 0.47). Nine patients in the PDR group and three patients in the control group had hypertension (P > 0.42). The fasting blood glucose in the control group ranged from 4.6 to 8.9 mM, with a mean of 5.98 ± 0.25 mM (P < 0.01). The diagnoses of the samples in the PDR group included vitreous hemorrhage (n = 11), traction retinal detachment (n = 13), and panretinal photocoagulation history (n = 9). In the control group, no patient had vitreous hemorrhage, traction retinal detachment, and panretinal photocoagulation history. The patients in both groups were enrolled consecutively from March 2014 to December 2015. (Figure 2 E1-F2), with a mean number of 3.8 ± 2.5 (Figure 2G). In the PDR group, Wip1 expressions were detected in all frozen sections (Figure 2 A1-D2), with a mean number of 57.2 ± 6.9 (Figure 2G). Consistent with the results of immunohistochemistry, strong immunoreactivity for Wip1 was detected in the PRD group in all membranes by immunofluorescence assay (Figure 3 A1-D2), with a mean number of 55.7 ± 8.2 (Figure 3G). Unlike the PDR group, there was no or weak immunoreactivity for Wip1 in control group (Figure 3 E1-F2), with a mean number of 4.2 ± 1.3 (Figure 3G). Co-location of Wip1 and GFAP was detected in PDR group Since retinal glial cells are one of the major cellular components of ERMs in PDR, we further detected the co- location of Wip1 and GFAP (retinal glial cells marker). In serial sections, the distribution of glial cells expressing GFAP was similar to the distribution of cells expressing Wip1 in the PRD group in all membranes. Double immunofluorescent staining confirmed that the great majority of these Wip1 cells were GFAP-positive glial cells (Figure 4). The mean numbers of ERMs expressing Wip1 and GFAP (55.8 ± 9.6 and 57.8 ± 7.4, respectively) in PDR group were significantly higher in iERMs (6.7 ± 1.5 and 17.1 ± 3.2, respectively) (P < 0.05). Significant correlations were detected between the numbers of Wip1- and GFAP-positive cells in the ERMs after PDR (r = 0.81, P < 0.01) (Figure 5). Patient information Table 1 includes the clinical and demographic Characteristics of patients enrolled in the study. ERMs were obtained from 17 eyes of 17 patients with PDR (the PDR group) and 9 eyes of 9 nondiabetic patients (the control group) with idiopathic epiretinal membranes (iERMs). The patients in the PDR group were 8 females and 9 males whose ages ranged from 37 to 71 years, with a mean of 57.32 ± 1.21 years. The duration of diabetes The aims of the present study were to investigate the expression and distribution of Wip1 in diabetic patients i j l / Figure 1: qRT-PCR analysis of Wip1 expression in ERMs derived from patients. A. cDNA derived from ERMs with PDR and ERMs were analyzed by PCR using specific primers for Wip1. As controls for cDNA integrity, the cDNA was also amplified with specific rimers for GAPDH. B. Relative mRNA level represented a ratio between the amount of target gene and the amount of GAPDH control. The data are mean ± SEM (*p < 0.01). Figure 1: qRT-PCR analysis of Wip1 expression in ERMs derived from patients. A. cDNA derived from ERMs with PDR and iERMs were analyzed by PCR using specific primers for Wip1. As controls for cDNA integrity, the cDNA was also amplified with specific primers for GAPDH. B. Relative mRNA level represented a ratio between the amount of target gene and the amount of GAPDH control. The data are mean ± SEM (p < 0.01). www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 35533 Table 1: Baseline patient characteristics from epiretinal membranes samples PDR group (n=17) Control group (n=9) p value Age (years) 57.32 ± 1.21 58.01 ± 3.61 0.47 Female Gerder, n (%) 8 4 1.00# Duration of diabetes (years) 12.11 ± 5.39 N.A. N.A. hypertension 9 3 0.42# Fasting blood glucose, mmol/L 10.09 ± 0.64 5.98 ± 0.25 <0.01* Vitreoretinal condition: Vitreous hemorrhage 11/6 0/9 Traction retinal detachment 13/4 0/9 Panretinal photocoagulation history 9/8 0/9 Data are expressed as the mean ± standard deviation or the median and range. Indicates statistically significant result compared to the corresponding data in the control group as shown in Table 1 (P<0.05). * Independent sample t-test. # χ2 test. N.A. = Not applicable. : Baseline patient characteristics from epiretinal membranes samples Data are expressed as the mean ± standard deviation or the median and range. Protein expression of Wip1 was higher in the PDR group Previous studies had demonstrated that NF-κB was involved in the formation of glial cell components of ERMs in PDR. Meanwhile, it has found that NF-κB is a positive regulator of Wip1. Therefore, we further Immunohistochemical analysis was performed to identify the Wip1 protein expressions in both groups. We confirmed weak expression of Wip1 in control group www.impactjournals.com/oncotarget Oncotarget 35534 Oncotarge 35535 www.impactjournals.com/oncotarget Figure 2: Immunohistochemical epressions of Wip1 in ERM with PDR and iERM samples. Low (40×) and higher (100× power views of Wip1 in iERM samples in PDR group A.-D. and control group E.-F. The number of positive cells was scored, and th percentages of Wip1 positive cells were used for statistical comparison G. The data were means ± SEM (n = 3; *p < 0.01, significantl different from the control group). Figure 2: Immunohistochemical epressions of Wip1 in ERM with PDR and iERM samples. Low (40×) and higher (100×) power views of Wip1 in iERM samples in PDR group A.-D. and control group E.-F. The number of positive cells was scored, and the percentages of Wip1 positive cells were used for statistical comparison G. The data were means ± SEM (n = 3; p < 0.01, significantly different from the control group). www.impactjournals.com/oncotarget Oncotarget 35535 Oncotarget 35536 www.impactjournals.com/oncotarget Figure 3: Immunofluorescence expressions of Wip1 in ERM samples. Low (40×) and higher (100×) power views of Wip1 in iERM samples in PDR group A.-D. and control group E.-F. The number of positive cells was scored, and the percentages of Wip1 positive cells were used for statistical comparison G. The data were means ± SEM (n = 3; p < 0.01, significantly different from the control group). Figure 3: Immunofluorescence expressions of Wip1 in ERM samples. Low (40×) and higher (100×) power views of Wip1 in iERM samples in PDR group A.-D. and control group E.-F. The number of positive cells was scored, and the percentages of Wip1 positive cells were used for statistical comparison G. The data were means ± SEM (n = 3; p < 0.01, significantly different from the control group). www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 35536 the formation of the ERMs in PDR membranes via NF-κB signaling. detected the co-location of Wip1 and NF-κB. According to the results of immunofluorescence assay, most of Wip1 positive cells were double labeled with NF-κB (Figure 6). The mean numbers of ERMs expressing Wip1 and NF-κB (57.9 ± 6.8 and 56.7 ± 7.8, respectively) in PDR group were significantly higher in iERMs (4.9 ± 1.2 and 5.3 ± 2.3, respectively) (P < 0.05). Significant correlations were detected between the numbers of Wip1- and NF-κB- positive cells in the ERMs after PDR (r = 0.78, P < 0.01) (Figure 5). g g Although the pathogenesis of DR is still not fully understood, DR may have components of chronic inflammation [8, 10]. Epidemiologic evidence indicates that the occurrence of DR and its complications is closely related to the appearance of inflammatory biomarkers [19]. DR has increased serum levels of inflammatory markers (e.g. C-reactive protein, interleukin-6 (IL-6), and tumor necrosis factor-alpha) [20]. Numbers of studies have confirmed that chronic inflammation can stimulate the proliferation of glial cells, which is the one of the major components of ERMs in PDR [21, 22]. Therefore, the proliferation of glial cells and inflammation might be the important points of penetration to explore the pathogenesis of ERM after PDR. DISCUSSION In the present study, we have revealed the expression profiles and location of Wip1 in ERMs. Our results show high expression levels of Wip1 mRNAs in ERMs after PDR, but low in iERMs. In addition, both immunohistochemistry and immunofluorescence assay showed strong immunoreactivity for Wip1 in PDR membranes. Furthermore, Wip1 and GFAP (retinal glial cells marker) were coexpressed in PDR membranes, suggesting Wip1 mainly located in retinal glial cells. Finally, the expression of Wip1 was paralleled with NF- κB. These data support the notion that Wip1 contributes to NF-κB has long been considered a prototypical proinflammatory factor present in many cell types that mainly regulates proinflammatory cytokine production, leukocyte recruitment, or cell survival, which are essential for the inflammatory response [23]. The activation of retinal glial cells in the onset of various inflammatory retinal diseases has been linked to the activation of the NF-κB signaling pathway [24]. Activated glial cells Figure 4: Double labeling immunofluorescent for Wip1 and GFAP in ERM. Coexpression of Wip1 A. and GFAP B. in the ERM derived from a PDR patient. C. Nuclei were stained using DAPI. D. Merge, magnification 40×. E. Merge, magnification 100×. GFAP, glial fibrillary acidic protein(glial cell marker); DAPI, 4’,6-diamidino-2-phenylindole. Figure 4: Double labeling immunofluorescent for Wip1 and GFAP in ERM. Coexpression of Wip1 A. and GFAP B. in the ERM derived from a PDR patient. C. Nuclei were stained using DAPI. D. Merge, magnification 40×. E. Merge, magnification 100×. GFAP, glial fibrillary acidic protein(glial cell marker); DAPI, 4’,6-diamidino-2-phenylindole. www.impactjournals.com/oncotarget Oncotarget 35537 ERMs [6]. There is compelling clinical, histological and experimental evidence that any type of intraocular inflammation can cause ERMs [25]. Meanwhile, it has been confirmed that NF-κB can contribute to the can induce the up-regulation of numerous NF-κB target genes including pro-inflammatory cytokines (e.g. TNF-α, IL-1βand IL-6), which are important contributors to the pathological process of DR and the formation of Figure 5: The number of positive cells for GFAP and Wip1 was scored in both groups, and the percentages of positive cells were used for statistical comparison. GFAP, glial fibrillary acidic protein. p < 0.01, p < 0.05; NS, non-significant. Figure 5: The number of positive cells for GFAP and Wip1 was scored in both groups, and the percentages of positive cells were used for statistical comparison. GFAP, glial fibrillary acidic protein. *p < 0.01, p < 0.05; NS, non-significant. DISCUSSION Figure 6: Double labeling immunofluorescent for NF-kB and Wip1 in ERM. Coexpression of NF-kB A. and Wip1 B. in the ERM derived from a PDR patient. C. Nuclei were stained using DAPI. D. Merge, magnification 40×. E. Merge, magnification 100×. NF- kB, nuclear factor kappa B; DAPI, 4’,6-diamidino-2-phenylindole. Figure 6: Double labeling immunofluorescent for NF-kB and Wip1 in ERM. Coexpression of NF-kB A. and Wip1 B. in the ERM derived from a PDR patient. C. Nuclei were stained using DAPI. D. Merge, magnification 40×. E. Merge, magnification 100×. NF- kB, nuclear factor kappa B; DAPI, 4’,6-diamidino-2-phenylindole. www.impactjournals.com/oncotarget Oncotarget 35538 proliferation of glial cells [26]. Importantly, previous study has confirmed that NF-κB mRNA expression levels in ERMs from patients with PDR was significantly higher than those in iERMs [12]. Consistent with previous study, we found strong immunoreactivity for NF-κB in ERMs in the PRD group. These results suggest that NF-κB may be involved in the formation of ERMs after PDR. glioma cells. In this study, we observed that Wip1 was mainly located in GFAP-positive (retinal glial cells marker) cells, suggesting Wip1 may also be involved in the glial cells proliferation in ERMs in the PRD group. In conclusion, these data could support the hypothesis that the interactions of NF-κB and Wip1 are involved in the formation of ERMs in PDR. Additional studies are needed to clarify the in-depth mechanisms by which NF-κB/Wip1 signaling pathways regulate inflammatory response and glial cell proliferation in ERMs after PDR, particularly with respect to cross-talk among cellular signaling pathways. Wip1 is one of the members of the Ser/Thr PP2C family, which is encoded by PPM1D gene on chromosome 17q23-24. Lowe et al. [18] have found that PPM1D is a transcriptional target of NF-κB in breast cancer cells. Activation of NF-κB can significantly up-regulate Wip1 protein expression at both the mRNA and protein levels by directly binding to the PPM1D promoter region in LPS and TNF-a-treated splenic B cells [27]. Meanwhile, Wip1 was shown to negatively regulate the expression of NF- κB [28]. These studies described a negative feedback loop involving Wip1 and NF-κB. In the present study, we found that transcriptional and protein expression levels of Wip1 was significantly increased and co-location with NF-κB in ERMs from patients with PDR. Furthermore, the results of analysis of the phenotype of Wip1−/− mice show that Wip1 may involve in the regulation of inflammation [28]. DISCUSSION Wip1−/− mice are more susceptible to infection due to the presence of abnormal lymphoid structure and defective T- and B- cell responses [28]. Meanwhile, Wip1 played an important role in regulation of cell proliferation [29, 30]. Overexpression of Wip1 is observed in human gliomas, and PPM1D silencing suppresses proliferation of human ACKNOWLEDGMENTS This study was supported by the National Natural Science Foundation of China (No. 81460087, 81660168, 81660161 and 81560166), the Natural Science Foundation of Guangxi Zhuang Autonomous Region (No. 2012GXNSFAA276039 and No. 2011GXNSFA018228) and Science Fund Project of People’s Hospital of Guangxi Zhuang Autonomous Region (No. qn2014-1 and qn2014- 2). Immunohistochemical analysis None. Immunoistochemical assay was performed in accordance with previously studys [24, 31]. Briefly, the cryosections (7-µm thick) were cut by a cryostat, mounted on 3-aminopropyltriethoxysilane-coated glass slides, and air-dried at room temperature. Then the cross-sections were fixed in ice-cold acetone and washed with phosphate buffered saline (PBS). The sections were incubated with normal donkey serum for 30 min to block non-specific staining, and then incubated overnight at 4oC with mouse monoclonal anti-Wip1 antibody (1:150; Santa Cruz Biotechnology, Santa Cruz, CA, US). Subjects and sample collection All ERMs were obtained following approval by the Ethics Committee at People’s Hospital of Guangxi Zhuang Autonomous Region, and in accordance with the guidelines of the Declaration of Helsinki for research involving human tissue. Informed consent was obtained from all patients. ERMs samples were collected from patients with PDR (PDR group) or non-diabetic patients with iERMs (control group) during pars plana vitrectomy for the repair of traction retinal detachment or combined traction/rhegmatogenous retinal detachment. The membranes peeled and removed from the retina were fixed Figure 7: The number of positive cells for NF-kB and Wip1 was scored in both groups, and the percentages of positive cells were used for statistical comparison. NF-kB, nuclear factor kappa B. *p < 0.01; NS, non-significant. Figure 7: The number of positive cells for NF-kB and Wip1 was scored in both groups, and the percentages of positive cells were used for statistical comparison. NF-kB, nuclear factor kappa B. *p < 0.01; NS, non-significant. www.impactjournals.com/oncotarget Oncotarget 35539 Statistical analysis in a test tube containing 4% paraformaldehyde (PFA), and were subsequently embedded in optimum cutting temperature (OCT) compound for immunohistochemistry and immunofluorescence. Immunoreactive cells were counted in five random fields, using an eyepiece calibrated grid with 40 magniﬁcation. The cells present in an area of 0.33×0.22 mm were counted. Data were expressed as mean values ± standard deviation (SD) and analyzed by the Mann- Whitney test. Pearson correlation coefficients were computed to investigate the linear relationship between the variables investigated. All collected data were analyzed by SPSS software (SPSS, version 13.0, SPSS, Chicago). All ERM samples were tested in triplicate, and statistical significance was accepted at P < 0.05. Real-time PCR analysis Total RNA was extracted and purified from frozen specimens using the Trizol reagent (Invitrogen Corporation, Carlsbad, CA, USA) and then reverse transcribed to synthesise complementary DNA (cDNA) according to the manufacturer’s protocol. The housekeeping gene GAPDH was used as an internal loading control. The sequences of gene specific primers for Wip1 (forward, 5’-GAAGGATGACTTTGTCAG-3’; reverse, 5’-CCCAGACTTGTTCATT AC-3’) and GAPDH (forward, 5’-ACCACAGTCCAT GCCATCAC-3’; reverse, 5’-TCCACCACCCTGTTGCTGTA-3’) were designed using NCBI Primer-BLAST. 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https://openalex.org/W4362577075	https://etasr.com/index.php/ETASR/article/download/5689/3057	English	null	Torque Control of an In-Wheel Axial Flux Permanent Magnet Synchronous Motor using a Fuzzy Logic Controller for Electric Vehicles	Engineering, Technology and Applied science research/Engineering, Technology and Applied Science Research	2,023	cc-by	4,583	Vol. 13, No. 2, 2023, 10357-10362 Vol. 13, No. 2, 2023, 10357-10362 Engineering, Technology & Applied Science Research 10357 Torque Control of an In-Wheel Axial Flux Permanent Magnet Synchronous Motor using a Fuzzy Logic Controller for Electric Vehicles Vo Thanh Ha Faculty of Electrical and Electronics Engineering, University of Transport and Communications, Vietnam vothanhha.ktd@utc.edu.vn (corresponding author) Received: 17 January 2023 \| Revised: 31 January 2023 \| Accepted: 3 February 2023 Keywords-AFPMSM; fuzzy logic; PI; FOC; Electrical Vehicles (EVs) AFPMSMs and their response to wheels. Torque and speed controllers are controlled based on Direct Torque Control (DTC) and Field-Oriented Control (FOC). These controllers are designed using linear and nonlinear control methods such as PI, LQR, dead beat, sliding mode, flatness, fuzzy [10-14], or hybrid controllers such as fuzzy-neural, fuzzy-sliding, and PI- fuzzy [14-17]. However, the torque response has a slight pulsation, and the actual speed response quickly and accurately tracks the required speed [17-20]. Therefore, the study of intelligent control solutions to improve an integrated in-wheel AFPMSM torque in an EV should be combined with the required components and physical properties, such as brake and accelerator pedals, road inclination, and wind resistance. Consequently, these parameters are necessary to improve the performance and torque of EVs. ABSTRACT This paper presents the control design of an in-wheel axial-flux permanent magnet synchronous motor with one stator and one rotor, using a fuzzy logic controller for electric vehicles. In this controller, the surgeon ambiguous inference file is built by two input vectors, the stator current error and the derivative of the stator error. These input variables include five membership functions: Negative Big (NB), Negative Small (NS), Equal Zero (ZE), Positive Small (PS), and Positive Big (PB). The fuzzy logic controller was implemented using a 5×5 matrix to meet the required output stator voltage of the controller. The fuzzy logic torque controller was compared with the PI controller in stator current response, torque, and speed. The proposed controller was evaluated using simulation results from MATLAB/SIMULINK. a: Torque Control of an In-Wheel Axial Flux Permanent Magnet Synchronous Motor using a Fuzzy … I. INTRODUCTION Electric cars are a revolutionary trend in today transportation. Electric cars have advantages compared to cars with internal combustion engines as they eliminate complicated gearboxes and emissions and are environmentally friendly [1- 2]. The powertrain structure of Electric Vehicles (EVs) tends to use an in-wheel distributed electric drive system consisting of multiple motors, which ensures traction at the front or rear of a car on two or four wheels, using a front, rear, or four-wheel drive system [3-4]. This electric powertrain improves driving performance by differentiating between wheels, makes full use of vehicle energy, improves transmission efficiency, increases range, eases braking, has good heat dissipation, and is more convenient for installation and maintenance [5-6]. The Axial Flux Permanent Magnet Synchronous Motor (AFPMSM) is widely used in in-wheel motor drive systems because it has short shaft length characteristics, is lightweight, has good vibration resistance, and has a long service life, thus improving reliability and safety [7]. Although AFPMSM motors enhance the performance of EVs, each vehicle should have installed multiple motors, resulting in a complex control system [8]. In- wheel motors increase the cost of a vehicle and have high requirements for control procedures, such as power balancing, electronic differential, and energy recovery. In addition, electric car in-wheel motors require small size, light weight, small torque, high efficiency, large overload capacity, and wide speed range [9]. Therefore, scientists have been interested in studying the control of traction and torque of in-wheel This study presents the control design of an in-wheel AFPMSM, one stator, and one rotor, using a Fuzzy Logic Controller (FLC) for EV systems. In this controller, the surgeon ambiguous inference file is built by two input vectors, the stator current error and the derivative of the stator error. These input variables include five membership functions, Negative Big (NB), Negative Small (NS), Equal Zero (ZE), Positive Small (PS), and Positive Big (PB). The FLC was implemented with a 5×5 matrix so that the output stator voltage of the controller is met. The fuzzy logic torque controller was compared with the PI controller in stator current response, torque, and speed. Ha: Torque Control of an In-Wheel Axial Flux Permanent Magnet Synchronous Motor using a Fuzzy … Vol. 13, No. 2, 2023, 10357-10362 Engineering, Technology & Applied Science Research B. Mathematical Model of the Electric Vehicle The following equation results from applying Newton's second law to the parts of the outside force operating on the vehicle's body: 89 9<= = 7 −7, -> −7->?? −89. :. @A( C) (15) (15) ̱ = (6) ̱ = (6) Air resistance is given by: Air resistance is given by: The equation of flux components is given by: The equation of flux components is given by: 7, -> = EFGH $ (5 9 + 5IJ)$ (16) = + = (7) (16) (7) In some cases, the wind speed can be set to IJ = 0. The rolling resistance exists in the case of an underinflated tire and can be given by: In some cases, the wind speed can be set to IJ = 0. The rolling resistance exists in the case of an underinflated tire and can be given by: where isd, isq are the d and q coordinate stator currents, and Lsd, Lsq are the d and q coordinate inductance. Substituting (5) and (6) into (4) and passing through the d-q coordinate system gives the system of equations of the PMSM motor: 7->?? = L-7MN (17) 7MN = 89: OP@( C) (18) (17) (18) (18) = + − = + + + (8) where 7MN is the vertical surface reaction and L- is the rolling resistance coefficient. where 7MN is the vertical surface reaction and L- is the rolling resistance coefficient. (8) B. Mathematical Model of the Electric Vehicle B. Mathematical Model of the Electric Vehicle The gearbox model shows the angular speed and torque relationships according to the gear ratio + ,- ≺1, as shown in: !"+ ,- = !0ℎ 0ℎ= "+ ,- (11) (11) = + + (1) = + − (2) where !" is the motor torque, !0ℎ is the torque acting on the wheel, ! = !0ℎ is the load torque, and J is the inertia torque of the motor. Applying Newton's second law in the rotation of the motor gives: (2) The stator voltage equation is as follows: The stator voltage equation is as follows: ! " −!01 = 2 34 (12) ̱ = . ̱ + ̱ (3) (12) (3) The drive wheel model can be expressed as: where Rs is the stator resistance and ̱ is the stator flux. Then, converting (3) from the phase winding system of the stator to the coordinate system, quasi-rotor flux gives: 501 = 0101 !01 = !6 = 701 (13) (13) ̱ = . ̱ + ̱ + ̱ (4) The vehicle will act on the road surface with a force F while the wheel is resting on it with a force N and is being propelled by a torque Twh. In contrast, the road surface will act against the vehicle with a point of the same value in the opposite direction of Ft. In this scenario, the reasonable force that propels the car at speed is the frictional force Ft given by: (4) The relationship between stator and rotor flux is described by: by: ̱ = ̱ + ̱ (5) (5) 7 = 89. :. ; (14) 7 = 89. :. ; (14) where ̱ is the polar flux vector. Since the d-axis of the coordinate system coincides with the axis of the polar flux, the perpendicular component (q-axis) of ̱ will be zero. Thus, the flux vector has only real components. where μ is the grip coefficient. The following equation results from applying Newton's second law to the parts of the outside force operating on the vehicle's body: where μ is the grip coefficient. www.etasr.com II. MODEL AND CONTROL OF THE ELECTRIC CAR POWER SYSTEM component, not a magnetizing current component, and motor torque is given by: component, not a magnetizing current component, and motor torque is given by: !" = # $ % (10) !" = # $ % A. Mathematical Model of an AFPMSM Ha: Torque Control of an In-Wheel Axial Flux Permanent Magnet Synchronous Motor using a Fuzzy … A. Mathematical Model of an AFPMSM (10) It is possible to utilize the standard PMSM model for an AFPMSM. Stator parameters, such as the inductor calculation, change between the two models. Furthermore, the Back-EMF produced by an excitation coil and a permanent magnet is the same. Therefore, the radial PMSM of a AFPMSM and the model are mathematically related. The stator voltage equation in the d-q frame of reference is given by: III. FLC TORQUE CONTROLLER DESIGN The torque of the motor is described by: The torque of the motor is described by: The FLC controls the system by calculating the necessary voltages usd and usq so that the difference between the currents isd and isq is as tiny as possible. The exactly planned isd and isq stator currents are used to regulate the motor's torque control current. This paper outlines the modern controller design for the isd and the control strategy for an in-wheel AFPMSM, one stator, and one rotor, utilizing an FLC. This controller uses the stator current error and the derivative of the stator error as the two input vectors to build the ambiguous inference file. !" = # $ %& + ' −() (9) (9) The motor torque is comprised of two components: the primary component and the reactive component. To create a control system, the stator current vector must be adjusted so that the vertical current vector is parallel to the polar flux. Therefore, there is a torque-generating current www.etasr.com Vol. 13, No. 2, 2023, 10357-10362 Engineering, Technology & Applied Science Research 10359 (a) (b) (c) Fig. 1. (a), (b) Inputs, (c) output, and fuzzy rules of the FLC controller. TABLE I. MATRIX OF FLC CONTROLLER Input 1 (e) / Input 2 (Δe) NB NS ZE PS PB NB NB NB NB NS ZE NS NB NB NS ZE PS ZE NS NS ZE PS PB PS ZE ZE PS PB PB PB ZE PS PB PB PB (a) A. Building Trajectories of Accelerator, Brake, and Operating Modes of Electric Vehicles The trajectories of accelerators and brakes of electric cars are built according to a function F(x1, x2, …xn). Figure 2 shows the trajectories of the accelerator and brakes. The F function can be obtained experimentally and the output is calculated by looking up or interpolating the defined table using the block parameters according to a method, such as linear (linear gradient), Lagrange (Linear Lagrange), closest point, block spline, and Akima spline interpolation. The Fcos function can range in size from 1 to 30. The first and second inputs define the row and column dimension breakpoints, respectively. Figure 3 shows the determination of the accelerator and brake trajectories. (b) Fig. 2. The trajectories of the accelerator and brakes. (c) Fig. 1. (a), (b) Inputs, (c) output, and fuzzy rules of the FLC controller. (c) ig. 2. The trajectories of the accelerator and brakes. Fig. 3. The parameters trajectories of accelerator and brakes. Fig. 3. The parameters trajectories of accelerator and brakes. (a), (b) Inputs, (c) output, and fuzzy rules of the FLC controller. The input variables include five membership functions: Positive Big (PB), Positive Small (PS), Equal Zero (ZE), Negative Big (NB), and Negative Small (NS). Table I and Figure 1 show the FLC constructed using a 5×5 matrix. The FLC consists of 25 rules, which are implemented as follows: Fig. 3. The parameters trajectories of accelerator and brakes.  If (input 1 is NB) and (input 2 is NB), then (output is NB)  If (input 1 is NB) and (input 2 is NB), then (output is NB)  If (input 1 is NB) and (input 2 is NS), then (output is NB)  If (input 1 is NB) and (input 2 is ZE), then (output is NB)  If (input 1 is PB) and (input 2 is PB), then (output is PB) B. Simulation Results and Evaluation Engineering, Technology & Applied Science Research 10360 10360 The proposed controller was simulated in MATLAB to evaluate its effectiveness for the traction transmission system in electric cars using an in-wheel AFPMSM, with the following simulation scenario:  At time t = 0s, the car starts to accelerate and the accelerator value increases from 0 to 1 after 0.45s. Torque reaches a maximum of 205Nm and remains there for 2s.  At time t = 0s, the car starts to accelerate and the accelerator value increases from 0 to 1 after 0.45s. Torque reaches a maximum of 205Nm and remains there for 2s.  At time t = 0s, the car starts to accelerate and the accelerator value increases from 0 to 1 after 0.45s. Torque reaches a maximum of 205Nm and remains there for 2s.  At time t = 0s, the car starts to accelerate and the accelerator value increases from 0 to 1 after 0.45s. Torque reaches a maximum of 205Nm and remains there for 2s.  At t = 2s, the vehicle starts to decelerate, and the brake reaches a value from 0 to 1 at t = 3.5s. The torque gradually decreases to -205Nm and returns to 0 at t = 4.66s.  At t = 2s, the vehicle starts to decelerate, and the brake reaches a value from 0 to 1 at t = 3.5s. The torque gradually decreases to -205Nm and returns to 0 at t = 4.66s.  Assume the speed of the wind is 0.  The car moves on a flat road, but at t = 3.5s to 4.3s, the car goes downhill.  The car moves on a flat road, but at t = 3.5s to 4.3s, the car goes downhill. C dq CQ dq uvw CQ AFPMSM CQ CQ sd u sq u su  su  si  si  sd i sq i  tv tu tw s  Torrque Controller s  s di Voltage source inveter * sqi sd i sqi Inputs Incline Brake Accelerato * T Wind Vehicle Controller  dc U sd i sqi Fig. 4. The control structure for electric cars using an in-wheel AFPMSM motor, based on a FOC method. Torque Controller Torrque Torque Controller Voltage source inveter Vehicle Controller Inputs Fig. 4. The control structure for electric cars using an in-wheel AFPMSM motor, based on a FOC method. B. Simulation Results and Evaluation  If (input 1 is NB) and (input 2 is NS), then (output is NB) Figure 4 shows the control structure of the traction drive system for electric cars using the in-wheel AFPMSM, and Table II displays the simulation parameters.  If (input 1 is NB) and (input 2 is ZE), then (output is NB)  If (input 1 is PB) and (input 2 is PB), then (output is PB) Ha: Torque Control of an In-Wheel Axial Flux Permanent Magnet Synchronous Motor using a Fuzzy … www.etasr.com Vol. 13, No. 2, 2023, 10357-10362 Engineering, Technology & Applied Science Research Engineering, Technology & Applied Science Research These figures show that the torque of the two controllers has the same form as the isq current. The FLC has less pulse rate (3%) for the torque response than the PI controller (8%). Table V shows the torque and speed response criteria of the PI and the FLC controllers. TABLE V. RESULTS OF EVALUATION RESPONSIBILITY Controller PI FLC Torque responses Shape Same as isq current response Same as isq current response Torque ripple 8% 3% Speed responses Accelerated setting time 2.2 (s) 2.2 (s) Over-adjustment 0% 0% Fig. 8. Torque responses of the PI controller. Fig. 9. Torque responses of the FCL controller. Figure 10 shows the speed responses of an electric car transmission system using the PI controller and the FLC. The actual speed response of an electric car in both cases is in line Engineering, Technology & Applied Science Research Vol. 13, No. 2, 2023, 10357-10362 Engineering, Technology & Applied Science Research www.etasr.com Ha: Torque Control of an In-Wh (a) (b) Fig. 6. Stator current responses (a) isd and (b) isq for the PI controller. (a) (b) Fig. 7. Stator current responses (a) isd and (b) isq for the FLC controller. (a) (b) Fig. 6. Stator current responses (a) isd and (b) isq for the PI controller. Figures 8 and 9 show the comparison of the torque and speed responses of the FLC and the PI controller. These figures show that the torque of the two controllers has the same form as the isq current. The FLC has less pulse rate (3%) for the torque response than the PI controller (8%). Table V shows the torque and speed response criteria of the PI and the FLC controllers. Axial Flux Permanent Magnet Synchronous Motor using a Fuzzy torque response than the PI controller (8%). Table V shows t torque and speed response criteria of the PI and the FL controllers. TABLE V. RESULTS OF EVALUATION RESPONSIBILITY Controller PI FLC Torque responses Shape Same as isq current response Same as isq curren response Torque ripple 8% 3% Speed responses Accelerated setting time 2.2 (s) 2.2 (s) Over-adjustment 0% 0% Fig. 8. Torque responses of the PI controller. Fig. 9. Torque responses of the FCL controller. Figure 10 shows the speed responses of an electric c transmission system using the PI controller and the FLC. Engineering, Technology & Applied Science Research Table III shows the parameters of the simulated PI controller. Figures 6 and 7 show the stator current responses of the FLC and the PI controller. These figures show that the stator current response in the steady-state method is fast (0.4s) and accurate in the steady-state mode (the actual signal follows the set call). However, the proposed FLC gives better results than the PI controller in over-regulating (no over-throttling), while the PI controller with an over-regulating current at an over-regulating time is 10%. Table IV presents the evaluation criteria for the stator current responses of the PI and the FLC controllers. TABLE II. PARAMETERS FOR AFPMSM Motor parameters Value Symbol Value Power Pđm 35Kw Rated speed Nđm 1800rpm Rated voltage Uđm 275V Number of pole pairs Zp 8 Magnetic flux density R 0.0437 Maximum torque Pmax 205Nm Armature resistance Rs 0.0101Ω Shaft inductance d Ld 2.4368e-4H Shaft inductance q Lq 2.9758e-4H Fig. 5. The MATLAB/SIMULINK control structure for electric cars using an in-wheel AFPMSM. TABLE II. PARAMETERS FOR AFPMSM TABLE II. PARAMETERS FOR AFPMSM Motor parameters Value Symbol Value Power Pđm 35Kw Rated speed Nđm 1800rpm Rated voltage Uđm 275V Number of pole pairs Zp 8 Magnetic flux density R 0.0437 Maximum torque Pmax 205Nm Armature resistance Rs 0.0101Ω Shaft inductance d Ld 2.4368e-4H Shaft inductance q Lq 2.9758e-4H Fig. 5. The MATLAB/SIMULINK control structure for electric cars using an in-wheel AFPMSM. TABLE III. PARAMETERS FOR THE PI CONTROLLER Controller Ki Kp Current controller Id 7.103004e+2 0.8779 Current controller Iq 1.0615e+3 1.0744 TABLE IV. RESULTS OF EVALUATION RESPONSIBILITY Controller PI FLC Stator current isd Accelerated setting time (s) 0.4 0.4 Over-adjustment 10% 0% Stator current isq Set-up time (s) 0.4 0.4 Over-adjustment 10% 0% Fig. 5. The MATLAB/SIMULINK control structure for electric cars using an in-wheel AFPMSM. Ha: Torque Control of an In-Wheel Axial Flux Permanent Magnet Synchronous Motor using a Fuzzy … www.etasr.com Engineering, Technology & Applied Science Research Vol. 13, No. 2, 2023, 10357-10362 10361 www.etasr.com Ha: Torque Control of an In-Wheel Axial Flux Permanent Magnet Synchronous Motor using a Fuzzy … (a) (b) Fig. 6. Stator current responses (a) isd and (b) isq for the PI controller. (a) (b) Fig. 7. Stator current responses (a) isd and (b) isq for the FLC controller. Figures 8 and 9 show the comparison of the torque and speed responses of the FLC and the PI controller. Engineering, Technology & Applied Science Research T actual speed response of an electric car in both cases is in li (a) TABLE V. RESULTS OF EVALUATION RESPONSIBILITY Controller PI FLC Torque responses Shape Same as isq current response Same as isq current response Torque ripple 8% 3% Speed responses Accelerated setting time 2.2 (s) 2.2 (s) Over-adjustment 0% 0% TABLE V. RESULTS OF EVALUATION RESPONSIBILITY Controller PI FLC Torque responses Shape Same as isq current response Same as isq current response Torque ripple 8% 3% Speed responses Accelerated setting time 2.2 (s) 2.2 (s) Over-adjustment 0% 0% Fig. 8. Torque responses of the PI controller. (b) Fig. 6. Stator current responses (a) isd and (b) isq for the PI controller. (a) (b) Fig. 7. Stator current responses (a) isd and (b) isq for the FLC controller. (a) Fig. 8. Torque responses of the PI controller. Fig. 9. Torque responses of the FCL controller. (a) (b) Fig. 9. Torque responses of the FCL controller. Figure 10 shows the speed responses of an electric car transmission system using the PI controller and the FLC. The actual speed response of an electric car in both cases is in line Fig. 7. Stator current responses (a) isd and (b) isq for the FLC controller. Ha: Torque Control of an In-Wheel Axial Flux Permanent Magnet Synchronous Motor using a Fuzzy … Ha: Torque Control of an In-Wheel Axial Flux Permanent Magnet Synchronous Motor using a Fuzzy … www.etasr.com Vol. 13, No. 2, 2023, 10357-10362 Engineering, Technology & Applied Science Research 10362 Research, vol. 12, no. 2, pp. 8316–8320, Apr. 2022, https://doi.org/ 10.48084/etasr.4762. with the set requirements, and the speed response does not have too much speed adjustment at starting, accelerating, and decelerating. [7] M. Yildirim, M. Polat, and H. 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The proposed controller was compared with the PI controller and its efficiency was demonstrated through MATLAB simulations. The proposed controller provides better torque and speed response results than the PI controller in terms of steady-state time, over-adjustment, and torque pulsation. However, this controller has a complicated design, so it is necessary to study more simple but intelligent control solutions in the future. [13] A. Darba, M. Esmalifalak, and E. S. Barazandeh, "Implementing SVPWM technique to axial flux permanent magnet synchronous motor drive with internal model current controller," in 2010 4th International Power Engineering and Optimization Conference (PEOCO), Shah Alam, Malaysia, Jun. 2010, pp. 126–131, https://doi.org/10.1109/ PEOCO.2010.5559197. [14] R. Krishnan, Electric Motor Drives: Modeling, Analysis, and Control, 1st edition. Upper Saddle River, N.J: Pearson, 2001. [15] P. T. Giang, V. T. Ha, and V. H. 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https://openalex.org/W2899370105	https://europepmc.org/articles/pmc6220289?pdf=render	English	null	A Deep Neural Network Model using Random Forest to Extract Feature Representation for Gene Expression Data Classification	Scientific reports	2,018	cc-by	8,057	A Deep Neural Network Model using Random Forest to Extract Feature Representation for Gene Expression Data Classification 8 2018 OPEN Received: 6 April 2018 Accepted: 6 October 2018 Published: xx xx xxxx Received: 6 April 2018 Accepted: 6 October 2018 Published: xx xx xxxx Yunchuan Kong & Tianwei Yu In predictive model development, gene expression data is associated with the unique challenge that the number of samples (n) is much smaller than the amount of features (p). This “n ≪ p” property has prevented classification of gene expression data from deep learning techniques, which have been proved powerful under “n > p” scenarios in other application fields, such as image classification. Further, the sparsity of effective features with unknown correlation structures in gene expression profiles brings more challenges for classification tasks. To tackle these problems, we propose a newly developed classifier named Forest Deep Neural Network (fDNN), to integrate the deep neural network architecture with a supervised forest feature detector. Using this built-in feature detector, the method is able to learn sparse feature representations and feed the representations into a neural network to mitigate the overfitting problem. Simulation experiments and real data analyses using two RNA-seq expression datasets are conducted to evaluate fDNN’s capability. The method is demonstrated a useful addition to current predictive models with better classification performance and more meaningful selected features compared to ordinary random forests and deep neural networks. In the field of bioinformatics, the development of computational methods for predicting clinical outcomes using profiling datasets with a large amount of variables has drawn great interest. In such datasets, the sample sizes tend to be very small compared to the number of predictors (genes), hence resulting in the  n p issue. Moreover, existence of complex unknown correlation structures among predictors has brought more difficulty in prediction and feature extraction. Therefore, the prediction task has been formulated as a classification problem combined with feature representations, and related work tried to solve the problem by utilizing machine learning approaches such as random forests1,2, neural networks3, sparse linear models4,5 and support vector machines6. While the primary goal of these methods are to achieve high classification accuracy, efforts have also been put into learning effective feature representations. Literature shows that among the machine learning techniques, random forests7 (RF) have been an excellent tool to learn feature representations8,9, given their robust classification power and easily interpretable learning mechanism. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Department of Biostatistics and Bioinformatics, Emory University, 1518 Clifton Rd, Atlanta, GA, 30322, USA orrespondence and requests for materials should be addressed to T.Y. (email: tianwei.yu@emory.edu) Scientific REPOrTs \| (2018) 8:16477 \| DOI:10.1038/s41598-018-34833-6 Received: 6 April 2018 Accepted: 6 October 2018 Published: xx xx xxxx A Deep Neural Network Model using Random Forest to Extract Feature Representation for Gene Expression Data Classification 8 2018 OPEN This can be useful in building robust predictive models especially when the underlying structures in the feature space are complex and unknown.i Classification methods have been developed considering known functional links between features. For exam- ple, a variant of the Random Forest method has been proposed where the feature sub-sampling was conducted according to spatial information of genes on a known functional network10.Objective functions of the support vector machine and the logistic regression were modified by adding relational penalty terms, again based on known functional information11–13. Very recently, a method embedding protein-protein interaction feature graph directly into the deep neural network structure has also been proposed14. The authors of these methods have demonstrated that incorporating feature relation structures results in better classification performance. However, considering the functional relation structures explicitly requires external information in addition to gene expression values. This requirement cannot always be satisfied as the functional structure can be unknown or incomplete.i p Trying to develop a powerful classifier which can implicitly extract sparse feature relations from an extremely large feature space, we intend to incorporate a forest “feature detector” with deep neural networks (DNN), which Scientific REPOrTs \| (2018) 8:16477 \| DOI:10.1038/s41598-018-34833-6 1 www.nature.com/scientificreports/ is one of the state-of-the-art learning techniques15. Although in recent years, deep learning models have been proved to be powerful tools in classification, their application in bioinformatics is limited due to the  n p issue16. This is because cell populations and clinical subject populations exhibit large heterogeneity and data characteris- tics across various laboratories are inconsistent, resulting in gene expression datasets to have limited numbers of samples compared to the large numbers of features. On the other hand, deep learning usually requires a large amount of training samples such as in image classification17, therefore the contradiction obstructs the use of deep learning techniques in the field of bioinformatics. Based on these facts, modified deep learning models suitable for disease outcome classification using gene expression data with  n p are in need.i i g g p p Building a supervised feature detector on top of DNN classifiers is a natural choice to achieve sparse learning with less parameters compared to the usual DNN, for the following reasons: (1) the detector detects effective features in a supervised manner, i.e. Forest deep neural networks. f Th f In the fDNN model, a forest  is a collection of decision trees F J Θ = Θ = … m M ( ) { ( )}, 1, , , m m where M is the total number of trees in the forest, Θ = {Θ1, …, ΘM} represents the parameters in . In random forests, Θ includes splitting variables and their splitting values. In the feature detection stage,  is fitted by the training data X and y, where ∈ × X n p  is the input data matrix with n samples and p features and  ∈ y n is the outcome vector containing classification labels. Through the fitted forest, for any observation xi, i = 1, …, n, we obtain the prediction from each tree in : where M is the total number of trees in the forest, Θ = {Θ1, …, ΘM} represents the parameters in . In random forests, Θ includes splitting variables and their splitting values. In the feature detection stage,  is fitted by the training data X and y, where ∈ × X n p  is the input data matrix with n samples and p features and  ∈ y n is the outcome vector containing classification labels. Through the fitted forest, for any observation xi, i = 1, …, n, we obtain the prediction from each tree in : Θ = Θ … Θ f T T x x x ( ; ) ( ( ; ), , ( ; )) , i i M i M T 1 1 where Θ = ˆ T y x ( ; ) m i m im is the binary prediction of observation xi given by m . Hence, denote fi: = f(xi;Θ) for sim- plicity, for an observation xi, fi is a binary vector summarizing the signal detected from the forest and later on serves as the new input features to be fed into the DNN. where Θ = ˆ T y x ( ; ) m i m im is the binary prediction of observation xi given by m . Hence, denote fi: = f(xi;Θ) for sim- plicity, for an observation xi, fi is a binary vector summarizing the signal detected from the forest and later on serves as the new input features to be fed into the DNN. A Deep Neural Network Model using Random Forest to Extract Feature Representation for Gene Expression Data Classification 8 2018 OPEN using the information of training outcomes, resulting in accurate feature representations; (2) the input of the downstream DNN, which is the output of the feature detector, has a much smaller dimension compared to the original feature sets. Also, the rationale of employing random forests over other models lies in two aspects: (1) as an ensemble model, RF is able to output prediction results from all its base learners rather than a single predicted probability score; (2) the importance of features in each base learner can be easily obtained. The first aspect allows us to build downstream DNN following the feature detector, which cannot be achieved if the detector only outputs a single prediction such as in support vector machines and logistic regres- sions. The second aspect facilitates feature evaluation process for the entire integrated model, while other classifi- ers such as kernel based methods may not naturally embrace feature selection mechanism. To the best of our knowledge, no work has been done along this track for gene expression data. In the field of traditional machine learning research such as computer vision, the idea of stacking classifiers18 has been implemented and is now very popular in Kaggle data science competitions (https://www.kaggle.com). Nevertheless, stacking methods are mainly intended to cross-validate a large amount of multi-level models, and consequently require much larger number of instances (samples) than the number of features with no exception. In contrast, our new fDNN classi- fier with supervised forest feature detector is developed for  n p sparse learning. In this paper, we justify our approach by demonstrating the classification performance on both synthetic data and real RNA-seq datasets. Methods and Materials Forest deep neural networks. Our newly proposed forest deep neural network (fDNN) model consists of two parts. The forest part serves as a feature detector to learn sparse representations from raw inputs with the supervision of training outcomes, and the DNN part serves as a learner to predict outcomes with the new feature representations. In the forest part, independent decision trees19 are constructed, and the forest is then an ensemble of the trees. Therefore, a natural choice of building the forest is the Random Forest model7. Other forest constructions are also possible. For example, one can use the network-guided forests10 if the feature space is structured and known, or the forest can be simply built through bagging trees20. In this paper, we only employ random forests as the feature detector. Forest deep neural networks. f Th f µ = = + µ µ µ p g e e e ( ) i i1 i i i 1 0 1 where µ µ = = \| = + = + p Pr y f z w b z w b : ( 1 ) : [ ] : [ ] , i i i i i out T out i out i i out T out i out 0 ( ) 0 ( ) ( ) 1 ( ) 1 ( ) ( ) where i = 1, …, n.h The parameters to be estimated in the DNN are thus all the weights and biases. The model can be trained using a stochastic gradient decent (SGD) based algorithm21 by minimizing the cross-entropy loss function ∑ Ψ = − + − − = ˆ ˆ n ylog p y log p ( ) 1 { ( ) (1 ) (1 )}, i n i i i i 1  where again Ψ denotes all the model parameters, and ˆpi is the fitted value of pi. More details about DNN can be found in standard deep learning reviews21. The entire architecture of the fDNN model is visualized in Fig. 1. where again Ψ denotes all the model parameters, and ˆpi is the fitted value of pi. More details about DNN can be found in standard deep learning reviews21. The entire architecture of the fDNN model is visualized in Fig. 1. Details of model training. The training of fDNN classifier consists of two stages. In the first stage, training data including labels are used to fit the forest, and predictions from each tree in the forest for all instances are then fed into the fully-connected DNN, for training in the second stage. After the two-stage training, given a testing instance, the testing prediction is calculated through the entire model by the fitted forest and DNN. Note that for implementation purpose, the forest prediction feature fi, ∀i is one-hot encoded as shown in Fig. 1. This is the same operation as with the label vectors yi, since the final output dimension from DNN is two. Forest deep neural networks. f Th f Consequently, the actual input for the DNN in our implementation is an n × M × 2 tensor rather than an n × M matrix F.i p p For the DNN model, the activation functions are the rectified linear unit (ReLU)22 with the form (in scalar case) σ = . x max x ( ) ( , 0) ReLU Forest deep neural networks. f Th f p Following the new feature representations provided by the forest, the deep neural network with l hidden layers has a standard architecture σ σ σ \| Ψ = + = + … = + … = + + Pr g y F Z W b Z Z W b Z Z W b Z FW b ( , ) ( ) ( ) ( ) ( ), out out out out l l l k k k k in in 1 1 where F = (fi, …, fM)T is the forest matrix with n samples and M tree predictions, y again is the classification outcome vector, Ψ denotes all the parameters in the DNN model, Zout and Zk, k = 1, …, l − 1 are hidden neurons with corresponding weight matrices Wout, Wk and bias vectors bout, bk. The dimensions of Z and W depend on the number of hidden neurons hin and hk, k = 1, …, l, as well as the input dimension M and the number of classes hout. For binary classification problems, hout ≡ 2 since the elements of y are binary. Usually, the number of hidden neurons decreases from the input layer, namely hin = M > h1 > h2 … > hout. σ(⋅) is the activation function such as sigmoid, hyperbolic tangent or rectifiers. g(⋅) is the softmax function converting values of the output layer into probability prediction i.e. Scientific REPOrTs \| (2018) 8:16477 \| DOI:10.1038/s41598-018-34833-6 2 www.nature.com/scientificreports/ Instance xi …… Tree1 Tree2 Tree M Forest …… Forest prediction fi (one-hot encoded) First hidden layer + ReLU Output layer Second hidden layer + ReLU 1 0 …… 1 0 1 …… 0 Figure 1. Visualization of the architecture of the fDNN model. Instance xi Forest Tree1 Forest prediction fi (one-hot encoded) Second hidden layer + ReLU Figure 1. Visualization of the architecture of the fDNN model. σ = . x max x ( ) ( , 0) ReLU Robustness is also tested as we simulate datasets that do not fully satisfy the correlated feature assumption, and apply the new method to examine whether it can still achieve a reasonable performance.i p For a given number of features p, we first generate a latent feature network using the preferential attach- ment algorithm28. Each node of the network represents one feature. The resulting network is scale-free with a power-law degree distribution. That means only a few features in this network have relatively large number of “neighbors”. Defining the distance between two features in the network as the shortest path between them, a p × p distance matrix D recording pairwise distances among features is then calculated. Next, the distance matrix is transformed into a covariance matrix Σ by letting Σ = . = … . i j p 0 7 , , 1, , ij Dij Here by convention the diagonal elements of D are all zeros meaning the distance between a feature to itself is zero, and thus the diagonal elements of Σ are all ones. Since only a few features have high connections in the feature network, this fact is reflected in the Σ matrix. Utilizing Σ as the covariance matrix for generating sample instances, we are then able to achieve the goal that features have sparse and correlated structures. n multivariate Gaussian samples are simulated forming the data matrix X = (x1, …, xn)T i.e.  Σ = … ~ i n x 0 ( , ), 1, , i here  n p for imitating real gene expression situations T bl l b f To generate outcome variables, we select a subset of all features to be the “true” predictors. The selection is conducted as follows: in the generated feature network mentioned above, we randomly select part of the high-degree features as “cores”, and a portion of their neighbors are also randomly selected. In this way, the true predictors satisfy: (1) sparsity, since only a few of all are high-degree features and only part of the neighbors are selected. (2) correlated structure, since the “core” features have much higher correlation with their neighbors than other distant features. Denoting the number of true predictors as p0, we sample a set of parameters β β β = … ( , , ) p T 1 0 and an intercept β0 within a certain range. σ = . x max x ( ) ( , 0) ReLU This activation has an advantage over sigmoid and hyperbolic tangent as it can avoid the vanishing gradient problem23 during optimization. For the optimization algorithm, We choose the Adam optimizer24 as it is the most widely used variant of traditional gradient descent algorithms in deep learning nowadays. Also, we use the mini-batch training strategy by which the optimizer randomly trains a small proportion of the samples in each iteration. Details about the Adam optimizer and mini-batch training can be seen in deep learning literature21,24. Scientific REPOrTs \| (2018) 8:16477 \| DOI:10.1038/s41598-018-34833-6 3 www.nature.com/scientificreports/ The classification performance of the fDNN model is associated with both hyper-parameters for the forest and for the DNN. Forest hyper-parameters include number of trees in the forest, and tree related parameters such as tree depth, minimum splitting sample size etc. DNN hyper-parameters are architecture related parameters such as the number of layers and the number of hidden neurons in each layer, regularization related parameters such as the dropout proportion and the penalty scale of regularizers, model training related parameters such as the learning rate and the batch size. Those hyper-parameters can be fine-tuned using advanced hyper-parameter optimizing algorithm such as Bayesian Optimization25. However, in this work, our primary interest is to examine the performance of fDNN compared to ordinary classifiers under same or similar settings, instead of fine tuning a “best-of-all” model for specific datasets. Therefore, the hyper-parameters are simply chosen by convention or tuned using grid search with synthetic validation datasets in a feasible hyper-parameter space. The method is implemented in Python with packages Scikit-learn26 and Tensorflow27. Synthetic data generation. The goal of simulation experiments is to mimic disease outcome classification using gene expression data, where  n p and effective features are extremely sparse and correlated, and explore the performance of our new model compared to ordinary classification methods. We compare our fDNN method with usual random forests and DNN, which account for the two parts of fDNN respectively. Through the numer- ical experiments, we are intended to show that fDNN is able to improve the classification performance of pure random forests or DNN, and the better performance cannot be achieved simply by increasing the complexities of the two ordinary classifiers. Results and Discussion Simulation results. In our simulation studies, the fDNN had 300 trees in the forest part and three hidden layers in the DNN part, with 256, 64 and 16 hidden neurons respectively. Note the 300-tree forest served as a supervised feature detector for the downstream DNN, and the feature space was shrunk from p = 5000 to M = 300, mediating intractable  n p situation since n and the input dimension M are now at the same magni- tude. We also observed that adding more hidden layers to DNN resulted in similar prediction performance, hence the three-hidden layer architecture was finalized as a parsimonious choice. To compare, we also recorded the prediction performance from the 300-tree forests (RF_300) in fDNN and experimented with a DNN classifier (DNN_3_256) with the same architecture as the one in fDNN. Moreover, we tested additional random forests with 500 trees (RF_500) and DNN with one more hidden layer (1024 neurons) at the top (DNN_4_1024), for the reason mentioned in the Methods section. For each of the data generation settings, 10 datasets were generated, and all methods mentioned above were applied on the data. For each simulated dataset, we randomly split the dataset into training and testing sets at a 4:1 ratio. The final testing classification performances were then averaged across the ten datasets. All the classification results were evaluated by the area under the receiver operating char- acteristic (ROC) curve (AUC). ( ) ( ) Table 1 and Fig. 2 show the results of the simulation experiments. Corresponding to the “clustered” case, Fig. 2(a) shows the fDNN method outperformed RF_300 and DNN methods, and performed better than RF_500 in most cases. As the number of true predictors increased, there were increasing trends for all of the methods, with a few exceptions due to the randomness of data generation. The trends for fDNN and RF_300 are quite par- allel. However, the downstream DNN in fDNN always improved the prediction from the forests. Note that DNN_4_1024 was actually worse than DNN_3_256, and this makes sense because under  n p the deeper neu- ral network is more affected by the pitfall of the overfitting phenomenon. Hence, it in turn demonstrated the necessity for constructing a model with reduced feature dimension as in fDNN to get around this issue.i As for the “scattered” case (Fig. σ = . x max x ( ) ( , 0) ReLU Scientific REPOrTs \| (2018) 8:16477 \| DOI:10.1038/s41598-018-34833-6 4 www.nature.com/scientificreports/ Case Clustered Scattered # true predictors 10 20 30 40 50 10 20 30 40 50 fDNN 0.79 0.828 0.832 0.872 0.892 0.775 0.781 0.829 0.861 0.851 DNN_3_256 0.762 0.791 0.809 0.829 0.865 0.75 0.727 0.822 0.823 0.836 DNN_4_1024 0.76 0.754 0.76 0.836 0.833 0.742 0.724 0.774 0.846 0.805 RF_300 0.783 0.82 0.823 0.862 0.887 0.772 0.76 0.825 0.858 0.831 RF_500 0.765 0.826 0.824 0.86 0.904 0.765 0.738 0.818 0.843 0.852 Table 1. Classification comparison of the forest Deep Neural Network (fDNN) method, deep neural networks (DNN) and random forests (RF). Statistics are the classification accuracies measured by AUC. Table 1. Classification comparison of the forest Deep Neural Network (fDNN) method, deep neural networks (DNN) and random forests (RF). Statistics are the classification accuracies measured by AUC. Real datasets. We apply the fDNN method to two real datasets. The first is the single-cell RNA-seq data on bone marrow cells29 (GSE99095). The dataset consists of a gene expression matrix with 17,258 genes in 391 control cells from healthy donors, and 588 cells from 5 patients with bone marrow failure. The original study has found the cell populations are diverse both in patients and in healthy donors, with patient cells showing higher diversity due to the existence of aneuploid cells29. We obtain the normalized expression matrix from the Gene Expression Omnibus (GEO). Our interest is to test the method’s capability to classify the source of the cells, i.e. healthy/diseased, despite the presence of cell diversity within each class.hi y p p y The other dataset we study is GSE106291, which contains the RNA-seq expression profiles of 23,368 genes from 250 acute myeloid leukemia patients under intensive treatment30. The primary clinical outcome is treatment resistance. Each patient was labeled as either resistant or sensitive to the treatment. We aim at classifying the two responses with the gene expression data. From the original normalized expression matrix, we delete genes with more than 10% zero measurements, resulting in the final data matrix with 11,068 features and 250 columns. For each feature, the expression value is Z-score transformed, i.e. the expression value minus the mean across all patients and then divided by the standard deviation. σ = . x max x ( ) ( , 0) ReLU p y The two datasets are suitable for testing our method, because the datasets fall into the  n p category, the features are correlated due to their functional relations, and only a small portion of the features are expected to contribute to the biological mechanism that generated the class membership. σ = . x max x ( ) ( , 0) ReLU In our experiments, we first sample β’s from the interval (0.05, 0.1), and some of the parameters are randomly turned into negative, so that we accommodate both positive and negative coefficients. Finally, the outcome variable y is generated through a logistic regression model β β = \| = + = = \| > = … − Pr y logit y Pr y t i n x x x ( 1 ) ( ) ( ( 1 ) ), 1, , i T i i i i i 1 0  = = \| > = … y Pr y t i n x ( ( 1 ) ), 1, , i i i  where (·) is the indicator function, t is a threshold and logit(⋅) is the logit function where (·) is the indicator function, t is a threshold and logit(⋅) is the logit function =   −  . logit x log x x ( ) 1 The inverse logit−1 is equivalent to a binary class softmax function. ht Following the above procedure, we simulate a set of synthetic datasets with 5,000 features and 400 samples. Since we are considering cases with extremely low signal-to-noise ratio, we examine different numbers, i.e. 10, 20, 30 40, and 50 of true predictors, corresponding to 1–5 cores among all the high-degree features. Also, in reality, the true predictors may not be only distributed at the high-degree nodes and their neighbors in the latent feature network. Instead, a few of the true predictors can be quite scattered. To test model robustness in this possible circumstance, in addition to generating datasets following the above procedure, we also simulate another series of datasets where 50% of the true predictors are randomly selected among the entire feature network rather than from high-degree features and their neighbors. We call these two sets of data “clustered” case and “scattered” case respectively, according to the property of predictor structures. Scientific REPOrTs \| (2018) 8:16477 \| DOI:10.1038/s41598-018-34833-6 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 2. Plots of the classification comparison in Table 1. Cases: (a) clustered (b) scattered. Figure 2. Plots of the classification comparison in Table 1. Cases: (a) clustered (b) scattered. Method Architecture Testing AUC fDNN 400Trees + 256 + 64 + 16 0.986 DNN 1024 + 512 + 128 + 16 0.949 RF 1000Trees 0.897 Table 2. Testing results for the GSE99095 dataset. Numbers in the architecture column denote the number of trees in Random Forest and the number of hidden neurons in neural network methods. Method Architecture Testing AUC fDNN 400Trees + 256 + 64 + 16 0.986 DNN 1024 + 512 + 128 + 16 0.949 RF 1000Trees 0.897 Table 2. Testing results for the GSE99095 dataset. Numbers in the architecture column denote the number of trees in Random Forest and the number of hidden neurons in neural network methods. Table 2. Testing results for the GSE99095 dataset. Numbers in the architecture column denote the number of trees in Random Forest and the number of hidden neurons in neural network methods. Method Architecture Testing AUC fDNN 500Trees + 256 + 64 + 16 0.778 DNN 1024 + 256 + 16 0.751 RF 1000Trees 0.716 Table 3. Testing results for the GSE106291 dataset. Numbers in the architecture column denote the number of trees in Random Forest and the number of hidden neurons in neural network methods. Method Architecture Testing AUC fDNN 500Trees + 256 + 64 + 16 0.778 DNN 1024 + 256 + 16 0.751 RF 1000Trees 0.716 Table 3. Testing results for the GSE106291 dataset. Numbers in the architecture column denote the number of trees in Random Forest and the number of hidden neurons in neural network methods. simply increasing the model complexities for random forests and DNN. The method was also robust as it outper- formed other methods in both of the clustered and scattered cases. simply increasing the model complexities for random forests and DNN. The method was also robust as it outper- formed other methods in both of the clustered and scattered cases. Real data results. For each of the two datasets, we again randomly divided all samples into training and testing sets. For GSE99095, we had 700 training and 279 testing samples; for GSE106291, the numbers were 200 and 50 respectively. The three classifiers, fDNN, DNN, and RF were trained on the training samples. Scientific REPOrTs \| (2018) 8:16477 \| DOI:10.1038/s41598-018-34833-6 Results and Discussion 2(b)), fDNN was still the best among the five, while overall AUC’s slightly decreased compared to Fig. 2(a). This is because for neural network methods, DNN inherently tackles correlated features. When the correlation among features decreased, the performance of DNN_3_256 and DNN_4_1024 in (b) became worse than that in Fig. 2(a). At the same time, although not as directly affected as DNN meth- ods, decreased feature correlation also deteriorated the performance of RF_300 and RF_500. Recall we only selected a proportion of high-degree feature neighbors as the true predictors. The remaining neighbors could also be informative when constructing decision trees, due to their high correlation with the true predictors. Consequently, compared to the scattered case where half of the selected true predictors could hardly be connected by others in the feature network, the clustered case is easier for random forests as the chance of selecting “rele- vant” predictors is higher.i p g In summary, the simulation experiments demonstrated that our newly proposed fDNN classifier had better classification performance compared to ordinary random forests or deep neural networks alone, in the situation that  n p and signals are sparse and correlated. Moreover, the improved performance could not be achieved by Scientific REPOrTs \| (2018) 8:16477 \| DOI:10.1038/s41598-018-34833-6 5 www.nature.com/scientificreports/ which combined the variable importance calculation in ordinary random forests and the Connection Weights (CW) method31 introduced in neural networks, to calculate a score for each gene as the variable importance in fDNN.i which combined the variable importance calculation in ordinary random forests and the Connection Weights (CW) method31 introduced in neural networks, to calculate a score for each gene as the variable importance in fDNN.i In random forests, variable importance is quantified by cumulating the decrease of impurity caused by split- ting at a certain feature across all the trees. Based on this fact, the forest  in fDNN is also able to record feature importance during fitting. Moreover, importance scores of features in each tree  are also available, resulting in a p × M tree importance matrix S, where again p is the number of features and M is the number of trees in . p p g p For an ordinary DNN, the CW method tries to quantify the contribution of an input variable by summing over all the absolute values of the weights connecting the variable and the first hidden layers, assuming all input data are standardized. Mathematically, we have ∑ = \| \| = u w , j k p jk in 1 ( ) where uj is the importance score for feature j, w(in) denotes weights between the input and first hidden layers. The same logic applies in fDNN, but instead of calculating the importance of each feature, the CW method helps compute the importance of each tree output by ∑ = \| \| = v w , j k M jk in 1 ( ) and here vj is the importance score for j. Now we have both quantified the feature importance in the forest part and the tree importance in the DNN part. Denoting = … ⁎ ⁎ ⁎ v v v ( , ) M T 1 as the normalized importance scores for all the trees in DNN with ∑ = = ⁎ v 1 i M i 1 , we finally combine the two parts of fDNN with λ = ⁎ Sv , where λ = (λ1, … λp)T is the final importance of the original features. where λ = (λ1, … λp)T is the final importance of the original features. www.nature.com/scientificreports/ Hyper-parameters were chosen by cross-validation using the training datasets. The classification performance was again evaluated by the testing AUC of ROC. The computation times of fDNN training plus testing were 69.9 seconds for GSE99095 and 40.8 seconds for GSE106291 respectively, on a workstation with dual Xeon Gold 6136 processors, 192 GB RAM, and a single Nvidia Quadro P5000 GPU. Tables 2 and 3 list the detailed architec- tures for each classification methods and summarize the testing results for GSE90995 and GSE106291 respec- tively. Corresponding ROC plots are shown in Fig. 3(a,b).i y p g p g From the tables and the ROC plots, we see that fDNN was able to obtain better classification results in terms of ROC, compared to traditional DNN and RF classifiers. All three methods performed reasonably well on GSE99095, which contained close to 1000 samples. Although RF performed better than DNN in simulations, in the real dataset DNN achieved slightly better testing results. Our fDNN method, by learning sparse representation using RF as a feature detector, improved over the two methods in terms of testing data classification. GSE106291 had a smaller sample size of 250, which tested the limits of the methods. The small sample size may be the reason why all three methods performed worse. Still the fDNN achieved slightly better testing data classification error rate, indicating its applicability on gene expression datasets with relatively small sample size. In real analysis of gene expression data, one may not only be concerned about the prediction results, but also be interested in features with major contribution to the classification, as those significant genes can reveal biolog- ical mechanisms. After fitting the fDNN model, we employed a newly developed variable ranking mechanism, Scientific REPOrTs \| (2018) 8:16477 \| DOI:10.1038/s41598-018-34833-6 6 www.nature.com/scientificreports/ Figure 3. ROC plots for (a) GSE99095 and (b) GSE106291. Figure 3. ROC plots for (a) GSE99095 and (b) GSE106291. Conclusion d We presented a new forest - deep neural network classifier aimed at  n p classification problems for clinical outcome prediction using gene expression data. Its machinery relies on supervised learning feature representa- tions from a forest and training classifiers in a deep neural network. Simulation experiments have shown its rela- tively higher classification accuracy compared to existing methods, and the real data application demonstrated the utility of the new model. www.nature.com/scientificreports/ The top 10 overrepresented GO biological processes by the top 1% genes selected in fDNN from GSE99095 data, after manual removal of redundant GO terms. Table 4. The top 10 overrepresented GO biological processes by the top 1% genes selected in fDNN from GSE99095 data, after manual removal of redundant GO terms. GOBPID Pvalue Term Significant in RF selected genes (p < 0.01) Significant in genes uniquely selected by fDNN (p < 0.01) GO:0006935 0.000640609 chemotaxis Y GO:0002274 0.001091917 myeloid leukocyte activation Y GO:0062014 0.001389434 negative regulation of small molecule metabolic process Y GO:0016477 0.001567641 cell migration Y GO:0045055 0.002003684 regulated exocytosis Y GO:0060078 0.002129227 regulation of postsynaptic membrane potential GO:0030334 0.00244581 regulation of cell migration Y GO:0030501 0.002766529 positive regulation of bone mineralization GO:0061045 0.002925766 negative regulation of wound healing GO:0071320 0.003321428 cellular response to cAMP Y Table 5. The top 10 overrepresented GO biological processes by the top 2% genes selected in fDNN from GSE106291 data, after manual removal of redundant GO terms. Table 5. The top 10 overrepresented GO biological processes by the top 2% genes selected in fDNN from GSE106291 data, after manual removal of redundant GO terms. “myeloid leukocyte activation”, was not found by RF. Given the nature of the disease, and the clinical response under study, i.e. treatment resistance, it is expected that the myeloid activation pathways play a critical role. The fDNN also selected small molecule metabolism, exocytosis, and cAMP response as important processes in the resistance to the treatment, which have been implicated in chemotherapy response in other types of cancer. Overall, fDNN selected different important genes from RF, and the biological functions overrepresented by the fDNN selected genes are plausible. “myeloid leukocyte activation”, was not found by RF. Given the nature of the disease, and the clinical response under study, i.e. treatment resistance, it is expected that the myeloid activation pathways play a critical role. The fDNN also selected small molecule metabolism, exocytosis, and cAMP response as important processes in the resistance to the treatment, which have been implicated in chemotherapy response in other types of cancer. Overall, fDNN selected different important genes from RF, and the biological functions overrepresented by the fDNN selected genes are plausible. www.nature.com/scientificreports/ GOBPID Pvalue Term Significant in RF selected genes (p < 0.01) Significant in genes uniquely selected by fDNN (p < 0.01) GO:0006935 0.000640609 chemotaxis Y GO:0002274 0.001091917 myeloid leukocyte activation Y GO:0062014 0.001389434 negative regulation of small molecule metabolic process Y GO:0016477 0.001567641 cell migration Y GO:0045055 0.002003684 regulated exocytosis Y GO:0060078 0.002129227 regulation of postsynaptic membrane potential GO:0030334 0.00244581 regulation of cell migration Y GO:0030501 0.002766529 positive regulation of bone mineralization GO:0061045 0.002925766 negative regulation of wound healing GO:0071320 0.003321428 cellular response to cAMP Y Table 5. The top 10 overrepresented GO biological processes by the top 2% genes selected in fDNN from GSE106291 data, after manual removal of redundant GO terms. GOBPID Pvalue Term Significant in RF selected genes (p < 0.01) Significant in genes uniquely selected by fDNN (p < 0.01) GO:0070125 0.000319438 mitochondrial translational elongation Y GO:1990542 0.000319438 mitochondrial transmembrane transport Y GO:0006119 0.000431138 oxidative phosphorylation Y Y GO:0006412 0.000524598 translation Y GO:0048534 0.000553723 hematopoietic or lymphoid organ development Y GO:0007229 0.00166512 integrin-mediated signaling pathway Y GO:0098754 0.00166512 detoxification Y GO:0016073 0.002434088 snRNA metabolic process GO:0007599 0.004203111 hemostasis GO:1903018 0.00560232 regulation of glycoprotein metabolic process Table 4. The top 10 overrepresented GO biological processes by the top 1% genes selected in fDNN from GSE99095 data, after manual removal of redundant GO terms. GOBPID Pvalue Term Significant in RF selected genes (p < 0.01) Significant in genes uniquely selected by fDNN (p < 0.01) GO:0070125 0.000319438 mitochondrial translational elongation Y GO:1990542 0.000319438 mitochondrial transmembrane transport Y GO:0006119 0.000431138 oxidative phosphorylation Y Y GO:0006412 0.000524598 translation Y GO:0048534 0.000553723 hematopoietic or lymphoid organ development Y GO:0007229 0.00166512 integrin-mediated signaling pathway Y GO:0098754 0.00166512 detoxification Y GO:0016073 0.002434088 snRNA metabolic process GO:0007599 0.004203111 hemostasis GO:1903018 0.00560232 regulation of glycoprotein metabolic process Table 4. The top 10 overrepresented GO biological processes by the top 1% genes selected in fDNN from GSE99095 data, after manual removal of redundant GO terms. GOBPID Pvalue Term Significant in RF selected genes (p < 0.01) Significant in genes uniquely selected by fDNN (p < 0.01) GO:0070125 0.000319438 mitochondrial translational elongation Y GO:1990542 0.000319438 mitochondrial transmembrane transport Y GO:0006119 0.000431138 oxidative phosphorylation Y Y GO:0006412 0.000524598 translation Y GO:0048534 0.000553723 hematopoietic or lymphoid organ development Y GO:0007229 0.00166512 integrin-mediated signaling pathway Y GO:0098754 0.00166512 detoxification Y GO:0016073 0.002434088 snRNA metabolic process GO:0007599 0.004203111 hemostasis GO:1903018 0.00560232 regulation of glycoprotein metabolic process Table 4. www.nature.com/scientificreports/ A l i thi f t l ti d t l d t l pi Applying this feature evaluation procedure to our real data examples, we obtained a ranked gene importance lists for GSE99095 and GSE106291 respectively. For GSE99095, we analyzed top 1% ranked genes from both fDNN and RF for comparison purpose. The reason for comparing RF is that it is commonly used as a variable importance ranking tool. Among the top 1% (172) genes selected by the two methods, 52 genes overlap. GO enrichment results are shown in Table 4.h Among the top 10 fDNN selected pathways, one of the major themes is related to the mitochondria. The top three biological processes include the synthesis of mitochondrial proteins, mitochondrial transport, and the pro- cess of energy generation through oxidative phosphorylation. The results indicate that at the cellular level, mito- chondria biogenesis and energy production is associated with the bone marrow failure outcome. Comparatively, RF also identified the oxidative phosphorylation as a significant process, but not the mitochondrial protein bio- synthesis and transport processes. Both fDNN and RF found the hematopoietic process and the integrin pathway, which are integral parts of blood cell development. The fDNN selected the hemostasis pathway, which is another important part of blood cell regulations.fi For the GSE106291 data, as the total number of genes under study is smaller, to maintain sufficient statisti- cal power in gene set enrichment analysis, we compared the top 2% most important genes from fDNN and RF. Between the two lists of top 221 genes, 81 genes overlap. As shown in the enrichment analysis results (Table 5), both fDNN and RF selected chemotaxis as the top GO term. However, the second term selected by fDNN, Scientific REPOrTs \| (2018) 8:16477 \| DOI:10.1038/s41598-018-34833-6 7 www.nature.com/scientificreports/ GOBPID Pvalue Term Significant in RF selected genes (p < 0.01) Significant in genes uniquely selected by fDNN (p < 0.01) GO:0070125 0.000319438 mitochondrial translational elongation Y GO:1990542 0.000319438 mitochondrial transmembrane transport Y GO:0006119 0.000431138 oxidative phosphorylation Y Y GO:0006412 0.000524598 translation Y GO:0048534 0.000553723 hematopoietic or lymphoid organ development Y GO:0007229 0.00166512 integrin-mediated signaling pathway Y GO:0098754 0.00166512 detoxification Y GO:0016073 0.002434088 snRNA metabolic process GO:0007599 0.004203111 hemostasis GO:1903018 0.00560232 regulation of glycoprotein metabolic process Table 4. The top 10 overrepresented GO biological processes by the top 1% genes selected in fDNN from GSE99095 data, after manual removal of redundant GO terms. g p 7. Breiman, L. Random forests. 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Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps an institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre- ative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not per- mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. 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